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MacAlpine, David Michael

Overview:

Our laboratory is interested in understanding the mechanisms by which the molecular architecture of the chromosome regulates fundamental biological processes such as replication and transcription. Specifically, how are replication, transcription and chromatin modification coordinated on a genomic scale to maintain genomic stability? We are addressing this question by using genomic, computational and biochemical approaches in the model organism Drosophila melanogaster.

DNA replication is an essential cell cycle event required for the timely and accurate duplication of chromosomes. Replication initiates at multiple sites (called origins of replication) distributed across each chromosome. The failure to properly regulate origin selection and activation may result in catastrophic genomic instability and potentially tumorigenesis. Recent metazoan genomic studies have demonstrated a correlation between time of DNA replication and transcriptional activity, with actively transcribed regions of the genome being replicated early. However, the underlying mechanism of this correlation remains unclear. By systematically characterizing the replication dynamics of multiple cell types, each with distinct transcriptional programs, we will be in a position to understand how these processes are coordinated.

Another goal of the laboratory is to identify the chromosomal features that direct and regulate metazoan DNA replication. Origins of DNA replication are marked by the formation of multi-protein complex, called the preRC. Despite conservation of the proteins that comprise the preRC in all eukaryotes, very little is known about the sequence elements required for the selection and regulation of metazoan origins. We are using genomic tiling microarrays to systematically map all the sites of preRC assembly in the Drosophila genome. The high resolution mapping of thousands of replication origins will provide an unprecedented opportunity to use both computational approaches and comparative genomics to identify cis-acting elements that may regulate replication.

Positions:

Associate Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 2001

Ph.D. — University of Texas Southwestern Medical Center at Dallas Southwestern Medical School

Grants:

Novel tissue injury regulation at an organ-organ junction

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
April 01, 2016
End Date
March 31, 2021

Genetics Training Grant

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 1979
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Exploring the Role of Dynamic Chromatin Occupancy in Transcriptional Regulation - Non-competing renewal I

Administered By
Computer Science
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
April 01, 2016
End Date
March 31, 2020

Pharmacology Industry Internships for Ph.D. Students

Administered By
Pharmacology & Cancer Biology
AwardedBy
American Society for Pharmacology and Experimental Therapeutics
Role
Participating Faculty Member
Start Date
January 01, 2017
End Date
December 31, 2019

RalA signal transduction

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
February 01, 2002
End Date
March 31, 2019

Defining chromatin architecture and maturation at sites of DNA replication in the Drosophila melanogaster genome

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2015
End Date
March 31, 2018

Non-Canonical Responses to DNA damage in Drosophila Polyploid Cells

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
March 01, 2015
End Date
February 28, 2018

The role of H3K27M-induced aberrant PRC2 activity in brainstem gliomagenesis

Administered By
Pediatrics, Hematology-Oncology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
September 29, 2015
End Date
September 28, 2017

Chromatin architecture defines DNA replication origins

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 09, 2013
End Date
July 31, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

Defining RAS isoform- and mutation-specific roles in oncogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of North Carolina - Chapel Hill
Role
Collaborating Investigator
Start Date
June 22, 2016
End Date
May 31, 2017

Bioinformatics and Computational Biology Training Program

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2005
End Date
June 30, 2016

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Defining The Human DNA Replication Program-First Resubmission

Administered By
Pharmacology & Cancer Biology
AwardedBy
American Cancer Society, Inc.
Role
Principal Investigator
Start Date
January 01, 2011
End Date
December 31, 2015

Illumina Hi-Seq 2000 Sequencing System

Administered By
Institutes and Centers
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 07, 2012
End Date
May 06, 2013

The Systematic Identification and Analysis of Replication Origins in Drosophila

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 04, 2007
End Date
March 31, 2013

High-performance Computing System for Bioinformatics

Administered By
Institutes and Centers
AwardedBy
National Institutes of Health
Role
Major User
Start Date
June 01, 2009
End Date
May 31, 2010
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Publications:

Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains in Drosophila.

The methylation state of lysine 20 on histone H4 (H4K20) has been linked to chromatin compaction, transcription, DNA repair and DNA replication. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7. PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which has been partially attributed to defects in origin selection and activation. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. We employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and genome stability in Drosophila cells. We find that deregulation of H4K20 methylation had no impact on origin activation throughout the genome. Instead, depletion of PR-Set7 and loss of H4K20me1 results in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. Coincident with the ATR-dependent cell cycle arrest, we find increased DNA damage that is specifically limited to late replicating regions of the Drosophila genome, suggesting that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains.

Authors
Li, Y; Armstrong, RL; Duronio, RJ; MacAlpine, DM
MLA Citation
Li, Y, Armstrong, RL, Duronio, RJ, and MacAlpine, DM. "Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains in Drosophila." Nucleic acids research 44.15 (September 2016): 7204-7218.
PMID
27131378
Source
epmc
Published In
Nucleic Acids Research
Volume
44
Issue
15
Publish Date
2016
Start Page
7204
End Page
7218
DOI
10.1093/nar/gkw333

ORChestrating the human DNA replication program.

Authors
MacAlpine, DM
MLA Citation
MacAlpine, DM. "ORChestrating the human DNA replication program." Proceedings of the National Academy of Sciences of the United States of America 113.33 (August 5, 2016): 9136-9138.
PMID
27496327
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
33
Publish Date
2016
Start Page
9136
End Page
9138
DOI
10.1073/pnas.1610336113

DNA replication origins—Where do we begin?

© 2016 Prioleau and MacAlpine.For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages.

Authors
Prioleau, MN; MacAlpine, DM
MLA Citation
Prioleau, MN, and MacAlpine, DM. "DNA replication origins—Where do we begin?." Genes and Development 30.15 (August 1, 2016): 1683-1697. (Review)
Source
scopus
Published In
Genes & development
Volume
30
Issue
15
Publish Date
2016
Start Page
1683
End Page
1697
DOI
10.1101/gad.285114

Noncoding Transcription Is a Driving Force for Nucleosome Instability in spt16 Mutant Cells.

FACT (facilitates chromatin transcription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation of spt16 results in a global loss of nucleosomes as well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037 Spt16-suppressed noncoding transcripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion.

Authors
Feng, J; Gan, H; Eaton, ML; Zhou, H; Li, S; Belsky, JA; MacAlpine, DM; Zhang, Z; Li, Q
MLA Citation
Feng, J, Gan, H, Eaton, ML, Zhou, H, Li, S, Belsky, JA, MacAlpine, DM, Zhang, Z, and Li, Q. "Noncoding Transcription Is a Driving Force for Nucleosome Instability in spt16 Mutant Cells." Molecular and cellular biology 36.13 (July 2016): 1856-1867.
PMID
27141053
Source
epmc
Published In
Molecular and Cellular Biology
Volume
36
Issue
13
Publish Date
2016
Start Page
1856
End Page
1867
DOI
10.1128/mcb.00152-16

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate.

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.

Authors
Tsai, F-L; Vijayraghavan, S; Prinz, J; MacAlpine, HK; MacAlpine, DM; Schwacha, A
MLA Citation
Tsai, F-L, Vijayraghavan, S, Prinz, J, MacAlpine, HK, MacAlpine, DM, and Schwacha, A. "Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate." Molecular and cellular biology 35.12 (June 2015): 2131-2143.
PMID
25870112
Source
epmc
Published In
Molecular and Cellular Biology
Volume
35
Issue
12
Publish Date
2015
Start Page
2131
End Page
2143
DOI
10.1128/mcb.01357-14

SnapShot: Origins of DNA replication.

The fundamental unit of DNA replication, the replicon, is governed by a cis-acting replicator sequence and a trans-activating initiator factor. Despite the increased size and complexity of eukaryotic genomes, eukaryotic DNA replication continues to be guided by the fundamental principles and concepts established in the replicon model.

Authors
Creager, RL; Li, Y; MacAlpine, DM
MLA Citation
Creager, RL, Li, Y, and MacAlpine, DM. "SnapShot: Origins of DNA replication." Cell 161.2 (April 2015): 418-418.e1.
PMID
25860614
Source
epmc
Published In
Cell
Volume
161
Issue
2
Publish Date
2015
Start Page
418
End Page
418.e1
DOI
10.1016/j.cell.2015.03.043

Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle

© 2015 The Authors.Eukaryotic replication origins are defined by the ORC-dependent loading of the Mcm2-7 helicase complex onto chromatin in G1. Paradoxically, there is a vast excess of Mcm2-7 relative to ORC assembled onto chromatin in G1. These excess Mcm2-7 complexes exhibit little co-localization with ORC or replication foci and can function as dormant origins. We dissected the mechanisms regulating the assembly and distribution of the Mcm2-7 complex in the Drosophila genome. We found that in the absence of cyclin E/Cdk2 activity, there was a 10-fold decrease in chromatin-associated Mcm2-7 relative to the levels found at the G1/S transition. The minimal amounts of Mcm2-7 loaded in the absence of cyclin E/Cdk2 activity were strictly localized to ORC binding sites. In contrast, cyclin E/Cdk2 activity was required for maximal loading of Mcm2-7 and a dramatic genome-wide reorganization of the distribution of Mcm2-7 that is shaped by active transcription. Thus, increasing cyclin E/Cdk2 activity over the course of G1 is not only critical for Mcm2-7 loading, but also for the distribution of the Mcm2-7 helicase prior to S-phase entry.

Authors
Powell, SK; MacAlpine, HK; Prinz, JA; Li, Y; Belsky, JA; MacAlpine, DM
MLA Citation
Powell, SK, MacAlpine, HK, Prinz, JA, Li, Y, Belsky, JA, and MacAlpine, DM. "Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle." EMBO Journal 34.4 (February 12, 2015): 531-543.
Source
scopus
Published In
EMBO Journal
Volume
34
Issue
4
Publish Date
2015
Start Page
531
End Page
543
DOI
10.15252/embj.201488307

Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle.

Eukaryotic replication origins are defined by the ORC-dependent loading of the Mcm2-7 helicase complex onto chromatin in G1. Paradoxically, there is a vast excess of Mcm2-7 relative to ORC assembled onto chromatin in G1. These excess Mcm2-7 complexes exhibit little co-localization with ORC or replication foci and can function as dormant origins. We dissected the mechanisms regulating the assembly and distribution of the Mcm2-7 complex in the Drosophila genome. We found that in the absence of cyclin E/Cdk2 activity, there was a 10-fold decrease in chromatin-associated Mcm2-7 relative to the levels found at the G1/S transition. The minimal amounts of Mcm2-7 loaded in the absence of cyclin E/Cdk2 activity were strictly localized to ORC binding sites. In contrast, cyclin E/Cdk2 activity was required for maximal loading of Mcm2-7 and a dramatic genome-wide reorganization of the distribution of Mcm2-7 that is shaped by active transcription. Thus, increasing cyclin E/Cdk2 activity over the course of G1 is not only critical for Mcm2-7 loading, but also for the distribution of the Mcm2-7 helicase prior to S-phase entry.

Authors
Powell, SK; MacAlpine, HK; Prinz, JA; Li, Y; Belsky, JA; MacAlpine, DM
MLA Citation
Powell, SK, MacAlpine, HK, Prinz, JA, Li, Y, Belsky, JA, and MacAlpine, DM. "Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle." The EMBO journal 34.4 (February 2015): 531-543.
PMID
25555795
Source
epmc
Published In
EMBO Journal
Volume
34
Issue
4
Publish Date
2015
Start Page
531
End Page
543
DOI
10.15252/embj.201488307

Rare codons capacitate Kras-driven de novo tumorigenesis.

The KRAS gene is commonly mutated in human cancers, rendering the encoded small GTPase constitutively active and oncogenic. This gene has the unusual feature of being enriched for rare codons, which limit protein expression. Here, to determine the effect of the rare codon bias of the KRAS gene on de novo tumorigenesis, we introduced synonymous mutations that converted rare codons into common codons in exon 3 of the Kras gene in mice. Compared with control animals, mice with at least 1 copy of this Kras(ex3op) allele had fewer tumors following carcinogen exposure, and this allele was mutated less often, with weaker oncogenic mutations in these tumors. This reduction in tumorigenesis was attributable to higher expression of the Kras(ex3op) allele, which induced growth arrest when oncogenic and exhibited tumor-suppressive activity when not mutated. Together, our data indicate that the inherent rare codon bias of KRAS plays an integral role in tumorigenesis.

Authors
Pershing, NLK; Lampson, BL; Belsky, JA; Kaltenbrun, E; MacAlpine, DM; Counter, CM
MLA Citation
Pershing, NLK, Lampson, BL, Belsky, JA, Kaltenbrun, E, MacAlpine, DM, and Counter, CM. "Rare codons capacitate Kras-driven de novo tumorigenesis." The Journal of clinical investigation 125.1 (January 2015): 222-233.
PMID
25437878
Source
epmc
Published In
Journal of Clinical Investigation
Volume
125
Issue
1
Publish Date
2015
Start Page
222
End Page
233
DOI
10.1172/jci77627

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly.

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).

Authors
Belsky, JA; MacAlpine, HK; Lubelsky, Y; Hartemink, AJ; MacAlpine, DM
MLA Citation
Belsky, JA, MacAlpine, HK, Lubelsky, Y, Hartemink, AJ, and MacAlpine, DM. "Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly." Genes & development 29.2 (January 2015): 212-224.
PMID
25593310
Source
epmc
Published In
Genes & development
Volume
29
Issue
2
Publish Date
2015
Start Page
212
End Page
224
DOI
10.1101/gad.247924.114

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.

Authors
Lujan, SA; Clausen, AR; Clark, AB; MacAlpine, HK; MacAlpine, DM; Malc, EP; Mieczkowski, PA; Burkholder, AB; Fargo, DC; Gordenin, DA; Kunkel, TA
MLA Citation
Lujan, SA, Clausen, AR, Clark, AB, MacAlpine, HK, MacAlpine, DM, Malc, EP, Mieczkowski, PA, Burkholder, AB, Fargo, DC, Gordenin, DA, and Kunkel, TA. "Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition." Genome research 24.11 (November 2014): 1751-1764.
PMID
25217194
Source
epmc
Published In
Genome research
Volume
24
Issue
11
Publish Date
2014
Start Page
1751
End Page
1764
DOI
10.1101/gr.178335.114

DNA copy number evolution in Drosophila cell lines.

Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Authors
Lee, H; McManus, CJ; Cho, D-Y; Eaton, M; Renda, F; Somma, MP; Cherbas, L; May, G; Powell, S; Zhang, D; Zhan, L; Resch, A; Andrews, J; Celniker, SE; Cherbas, P; Przytycka, TM; Gatti, M; Oliver, B; Graveley, B; MacAlpine, D
MLA Citation
Lee, H, McManus, CJ, Cho, D-Y, Eaton, M, Renda, F, Somma, MP, Cherbas, L, May, G, Powell, S, Zhang, D, Zhan, L, Resch, A, Andrews, J, Celniker, SE, Cherbas, P, Przytycka, TM, Gatti, M, Oliver, B, Graveley, B, and MacAlpine, D. "DNA copy number evolution in Drosophila cell lines." Genome biology 15.8 (August 28, 2014): R70-.
PMID
25262759
Source
epmc
Published In
Genome Biology: biology for the post-genomic era
Volume
15
Issue
8
Publish Date
2014
Start Page
R70
DOI
10.1186/gb-2014-15-8-r70

Comparative analysis of metazoan chromatin organization.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Authors
Ho, JWK; Jung, YL; Liu, T; Alver, BH; Lee, S; Ikegami, K; Sohn, K-A; Minoda, A; Tolstorukov, MY; Appert, A; Parker, SCJ; Gu, T; Kundaje, A; Riddle, NC; Bishop, E; Egelhofer, TA; Hu, SS; Alekseyenko, AA; Rechtsteiner, A; Asker, D; Belsky, JA; Bowman, SK; Chen, QB; Chen, RA-J; Day, DS; Dong, Y; Dose, AC; Duan, X; Epstein, CB; Ercan, S; Feingold, EA; Ferrari, F; Garrigues, JM; Gehlenborg, N; Good, PJ; Haseley, P; He, D; Herrmann, M; Hoffman, MM; Jeffers, TE; Kharchenko, PV; Kolasinska-Zwierz, P et al.
MLA Citation
Ho, JWK, Jung, YL, Liu, T, Alver, BH, Lee, S, Ikegami, K, Sohn, K-A, Minoda, A, Tolstorukov, MY, Appert, A, Parker, SCJ, Gu, T, Kundaje, A, Riddle, NC, Bishop, E, Egelhofer, TA, Hu, SS, Alekseyenko, AA, Rechtsteiner, A, Asker, D, Belsky, JA, Bowman, SK, Chen, QB, Chen, RA-J, Day, DS, Dong, Y, Dose, AC, Duan, X, Epstein, CB, Ercan, S, Feingold, EA, Ferrari, F, Garrigues, JM, Gehlenborg, N, Good, PJ, Haseley, P, He, D, Herrmann, M, Hoffman, MM, Jeffers, TE, Kharchenko, PV, and Kolasinska-Zwierz, P et al. "Comparative analysis of metazoan chromatin organization." Nature 512.7515 (August 2014): 449-452.
PMID
25164756
Source
epmc
Published In
Nature
Volume
512
Issue
7515
Publish Date
2014
Start Page
449
End Page
452
DOI
10.1038/nature13415

DNA replication and transcription programs respond to the same chromatin cues.

DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.

Authors
Lubelsky, Y; Prinz, JA; DeNapoli, L; Li, Y; Belsky, JA; MacAlpine, DM
MLA Citation
Lubelsky, Y, Prinz, JA, DeNapoli, L, Li, Y, Belsky, JA, and MacAlpine, DM. "DNA replication and transcription programs respond to the same chromatin cues." Genome research 24.7 (July 2014): 1102-1114.
PMID
24985913
Source
epmc
Published In
Genome research
Volume
24
Issue
7
Publish Date
2014
Start Page
1102
End Page
1114
DOI
10.1101/gr.160010.113

Chromatin and DNA replication.

The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

Authors
MacAlpine, DM; Almouzni, G
MLA Citation
MacAlpine, DM, and Almouzni, G. "Chromatin and DNA replication. (Published online)" Cold Spring Harb Perspect Biol 5.8 (August 1, 2013): a010207-. (Review)
PMID
23751185
Source
pubmed
Published In
Cold Spring Harbor perspectives in biology
Volume
5
Issue
8
Publish Date
2013
Start Page
a010207
DOI
10.1101/cshperspect.a010207

Rare codons regulate KRas oncogenesis.

Oncogenic mutations in the small Ras GTPases KRas, HRas, and NRas render the proteins constitutively GTP bound and active, a state that promotes cancer. Ras proteins share ~85% amino acid identity, are activated by and signal through the same proteins, and can exhibit functional redundancy. Nevertheless, manipulating expression or activation of each isoform yields different cellular responses and tumorigenic phenotypes, even when different ras genes are expressed from the same locus. We now report a novel regulatory mechanism hardwired into the very sequence of RAS genes that underlies how such similar proteins impact tumorigenesis differently. Specifically, despite their high sequence similarity, KRAS is poorly translated compared to HRAS due to enrichment in genomically underrepresented or rare codons. Converting rare to common codons increases KRas expression and tumorigenicity to mirror that of HRas. Furthermore, in a genome-wide survey, similar gene pairs with opposing codon bias were identified that not only manifest dichotomous protein expression but also are enriched in key signaling protein classes and pathways. Thus, synonymous nucleotide differences affecting codon usage account for differences between HRas and KRas expression and function and may represent a broader regulation strategy in cell signaling.

Authors
Lampson, BL; Pershing, NLK; Prinz, JA; Lacsina, JR; Marzluff, WF; Nicchitta, CV; MacAlpine, DM; Counter, CM
MLA Citation
Lampson, BL, Pershing, NLK, Prinz, JA, Lacsina, JR, Marzluff, WF, Nicchitta, CV, MacAlpine, DM, and Counter, CM. "Rare codons regulate KRas oncogenesis." Curr Biol 23.1 (January 7, 2013): 70-75.
PMID
23246410
Source
pubmed
Published In
Current Biology
Volume
23
Issue
1
Publish Date
2013
Start Page
70
End Page
75
DOI
10.1016/j.cub.2012.11.031

Rare codons regulate KRas oncogenesis

Oncogenic mutations in the small Ras GTPases KRas, HRas, and NRas render the proteins constitutively GTP bound and active, a state that promotes cancer [1]. Ras proteins share ∼85% amino acid identity [2], are activated by [3] and signal through [4] the same proteins, and can exhibit functional redundancy [5, 6]. Nevertheless, manipulating expression or activation of each isoform yields different cellular responses [7-10] and tumorigenic phenotypes [11-13], even when different ras genes are expressed from the same locus [6]. We now report a novel regulatory mechanism hardwired into the very sequence of RAS genes that underlies how such similar proteins impact tumorigenesis differently. Specifically, despite their high sequence similarity, KRAS is poorly translated compared to HRAS due to enrichment in genomically underrepresented or rare codons. Converting rare to common codons increases KRas expression and tumorigenicity to mirror that of HRas. Furthermore, in a genome-wide survey, similar gene pairs with opposing codon bias were identified that not only manifest dichotomous protein expression but also are enriched in key signaling protein classes and pathways. Thus, synonymous nucleotide differences affecting codon usage account for differences between HRas and KRas expression and function and may represent a broader regulation strategy in cell signaling. © 2013 Elsevier Ltd.

Authors
Lampson, BL; Pershing, NLK; Prinz, JA; Lacsina, JR; Marzluff, WF; Nicchitta, CV; MacAlpine, DM; Counter, CM
MLA Citation
Lampson, BL, Pershing, NLK, Prinz, JA, Lacsina, JR, Marzluff, WF, Nicchitta, CV, MacAlpine, DM, and Counter, CM. "Rare codons regulate KRas oncogenesis." Current Biology 23.1 (2013): 70-75.
Source
scival
Published In
Current Biology
Volume
23
Issue
1
Publish Date
2013
Start Page
70
End Page
75
DOI
10.1016/j.cub.2012.11.031

Identification of E2F target genes that are rate limiting for dE2F1-dependent cell proliferation.

BACKGROUND: Microarray studies have shown that the E2F transcription factor influences the expression of many genes but it is unclear how many of these targets are important for E2F-mediated control of cell proliferation. RESULTS: We assembled a collection of mutant alleles of 44 dE2F1-dependent genes and tested whether these could modify visible phenotypes caused by the tissue-specific depletion of dE2F1. More than half of the mutant alleles dominantly enhanced de2f1-dsRNA phenotypes suggesting that the in vivo functions of dE2F1 can be limited by the reduction in the level of expression of many different targets. Unexpectedly, several mutant alleles suppressed de2f1-dsRNA phenotypes. One of the strongest of these suppressors was Orc5. Depletion of ORC5 increased proliferation in cells with reduced dE2F1 and specifically elevated the expression of dE2F1-regulated genes. Importantly, these effects were independent of dE2F1 protein levels, suggesting that reducing the level of ORC5 did not interfere with the general targeting of dE2F1. CONCLUSIONS: We propose that the interaction between ORC5 and dE2F1 may reflect a feedback mechanism between replication initiation proteins and dE2F1 that ensures that proliferating cells maintain a robust level of replication proteins for the next cell cycle.

Authors
Herr, A; Longworth, M; Ji, J-Y; Korenjak, M; Macalpine, DM; Dyson, NJ
MLA Citation
Herr, A, Longworth, M, Ji, J-Y, Korenjak, M, Macalpine, DM, and Dyson, NJ. "Identification of E2F target genes that are rate limiting for dE2F1-dependent cell proliferation." Dev Dyn 241.11 (November 2012): 1695-1707.
PMID
22972499
Source
pubmed
Published In
Developmental Dynamics
Volume
241
Issue
11
Publish Date
2012
Start Page
1695
End Page
1707
DOI
10.1002/dvdy.23857

Genome-wide localization of replication factors.

Chromatin Immunoprecipitation (ChIP) is a powerful tool for the identification and characterization of protein-DNA interactions in vivo. ChIP has been utilized to study diverse nuclear processes such as transcription regulation, chromatin modification, DNA recombination and DNA replication at specific loci and, more recently, across the entire genome. Advances in genomic approaches, and whole genome sequencing in particular, have made it possible and affordable to comprehensively identify specific protein binding sites throughout the genomes of higher eukaryotes. The dynamic nature of the DNA replication program and the transient occupancy of many replication factors throughout the cell cycle present additional challenges that may not pertain to the mapping of site specific transcription factors. Here we discuss the specific considerations that need to be addressed in the application of ChIP to the genome-wide location analysis of protein factors involved in DNA replication.

Authors
Lubelsky, Y; MacAlpine, HK; MacAlpine, DM
MLA Citation
Lubelsky, Y, MacAlpine, HK, and MacAlpine, DM. "Genome-wide localization of replication factors." Methods 57.2 (June 2012): 187-195.
PMID
22465279
Source
pubmed
Published In
Methods
Volume
57
Issue
2
Publish Date
2012
Start Page
187
End Page
195
DOI
10.1016/j.ymeth.2012.03.022

Developmental control of gene copy number by repression of replication initiation and fork progression.

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

Authors
Sher, N; Bell, GW; Li, S; Nordman, J; Eng, T; Eaton, ML; Macalpine, DM; Orr-Weaver, TL
MLA Citation
Sher, N, Bell, GW, Li, S, Nordman, J, Eng, T, Eaton, ML, Macalpine, DM, and Orr-Weaver, TL. "Developmental control of gene copy number by repression of replication initiation and fork progression." Genome Res 22.1 (January 2012): 64-75.
PMID
22090375
Source
pubmed
Published In
Genome research
Volume
22
Issue
1
Publish Date
2012
Start Page
64
End Page
75
DOI
10.1101/gr.126003.111

Epigenome characterization at single base-pair resolution.

We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes. To characterize DNA-binding features of transcription factors, we plotted the length of each fragment versus its position in the genome, which defined the minimal protected region of each factor. This process led to the precise mapping of protected and exposed regions at and around binding sites, and also determination of the degree to which they are flanked by phased nucleosomes and subnucleosome-sized particles. Our protocol and mapping method provide a general strategy for epigenome characterization, including nucleosome phasing and dynamics, ATP-dependent nucleosome remodelers, and transcription factors, from a single-sequenced sample.

Authors
Henikoff, JG; Belsky, JA; Krassovsky, K; MacAlpine, DM; Henikoff, S
MLA Citation
Henikoff, JG, Belsky, JA, Krassovsky, K, MacAlpine, DM, and Henikoff, S. "Epigenome characterization at single base-pair resolution." Proc Natl Acad Sci U S A 108.45 (November 8, 2011): 18318-18323.
PMID
22025700
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
45
Publish Date
2011
Start Page
18318
End Page
18323
DOI
10.1073/pnas.1110731108

Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression.

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.

Authors
Kim, JC; Nordman, J; Xie, F; Kashevsky, H; Eng, T; Li, S; MacAlpine, DM; Orr-Weaver, TL
MLA Citation
Kim, JC, Nordman, J, Xie, F, Kashevsky, H, Eng, T, Li, S, MacAlpine, DM, and Orr-Weaver, TL. "Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression." Genes Dev 25.13 (July 1, 2011): 1384-1398.
PMID
21724831
Source
pubmed
Published In
Genes & development
Volume
25
Issue
13
Publish Date
2011
Start Page
1384
End Page
1398
DOI
10.1101/gad.2043111

Defining the replication program through the chromatin landscape.

DNA replication is an essential cell cycle event required for the accurate and timely duplication of the chromosomes. It is essential that the genome is replicated accurately and completely within the confines of S-phase. Failure to completely copy the genome has the potential to result in catastrophic genomic instability. Replication initiates in a coordinated manner from multiple locations, termed origins of replication, distributed across each of the chromosomes. The selection of these origins of replication is a dynamic process responding to both developmental and tissue-specific signals. In this review, we explore the role of the local chromatin environment in regulating the DNA replication program at the level of origin selection and activation. Finally, there is increasing molecular evidence that the DNA replication program itself affects the chromatin landscape, suggesting that DNA replication is critical for both genetic and epigenetic inheritance.

Authors
Ding, Q; MacAlpine, DM
MLA Citation
Ding, Q, and MacAlpine, DM. "Defining the replication program through the chromatin landscape." Crit Rev Biochem Mol Biol 46.2 (April 2011): 165-179. (Review)
PMID
21417598
Source
pubmed
Published In
Critical Reviews in Biochemistry and Molecular Biology (Informa)
Volume
46
Issue
2
Publish Date
2011
Start Page
165
End Page
179
DOI
10.3109/10409238.2011.560139

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Authors
Kharchenko, PV; Alekseyenko, AA; Schwartz, YB; Minoda, A; Riddle, NC; Ernst, J; Sabo, PJ; Larschan, E; Gorchakov, AA; Gu, T; Linder-Basso, D; Plachetka, A; Shanower, G; Tolstorukov, MY; Luquette, LJ; Xi, R; Jung, YL; Park, RW; Bishop, EP; Canfield, TK; Sandstrom, R; Thurman, RE; MacAlpine, DM; Stamatoyannopoulos, JA; Kellis, M; Elgin, SCR; Kuroda, MI; Pirrotta, V; Karpen, GH; Park, PJ
MLA Citation
Kharchenko, PV, Alekseyenko, AA, Schwartz, YB, Minoda, A, Riddle, NC, Ernst, J, Sabo, PJ, Larschan, E, Gorchakov, AA, Gu, T, Linder-Basso, D, Plachetka, A, Shanower, G, Tolstorukov, MY, Luquette, LJ, Xi, R, Jung, YL, Park, RW, Bishop, EP, Canfield, TK, Sandstrom, R, Thurman, RE, MacAlpine, DM, Stamatoyannopoulos, JA, Kellis, M, Elgin, SCR, Kuroda, MI, Pirrotta, V, Karpen, GH, and Park, PJ. "Comprehensive analysis of the chromatin landscape in Drosophila melanogaster." Nature 471.7339 (March 24, 2011): 480-485.
PMID
21179089
Source
pubmed
Published In
Nature
Volume
471
Issue
7339
Publish Date
2011
Start Page
480
End Page
485
DOI
10.1038/nature09725

A cis-regulatory map of the Drosophila genome.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

Authors
Nègre, N; Brown, CD; Ma, L; Bristow, CA; Miller, SW; Wagner, U; Kheradpour, P; Eaton, ML; Loriaux, P; Sealfon, R; Li, Z; Ishii, H; Spokony, RF; Chen, J; Hwang, L; Cheng, C; Auburn, RP; Davis, MB; Domanus, M; Shah, PK; Morrison, CA; Zieba, J; Suchy, S; Senderowicz, L; Victorsen, A; Bild, NA; Grundstad, AJ; Hanley, D; MacAlpine, DM; Mannervik, M; Venken, K; Bellen, H; White, R; Gerstein, M; Russell, S; Grossman, RL; Ren, B; Posakony, JW; Kellis, M; White, KP
MLA Citation
Nègre, N, Brown, CD, Ma, L, Bristow, CA, Miller, SW, Wagner, U, Kheradpour, P, Eaton, ML, Loriaux, P, Sealfon, R, Li, Z, Ishii, H, Spokony, RF, Chen, J, Hwang, L, Cheng, C, Auburn, RP, Davis, MB, Domanus, M, Shah, PK, Morrison, CA, Zieba, J, Suchy, S, Senderowicz, L, Victorsen, A, Bild, NA, Grundstad, AJ, Hanley, D, MacAlpine, DM, Mannervik, M, Venken, K, Bellen, H, White, R, Gerstein, M, Russell, S, Grossman, RL, Ren, B, Posakony, JW, Kellis, M, and White, KP. "A cis-regulatory map of the Drosophila genome." Nature 471.7339 (March 24, 2011): 527-531.
PMID
21430782
Source
pubmed
Published In
Nature
Volume
471
Issue
7339
Publish Date
2011
Start Page
527
End Page
531
DOI
10.1038/nature09990

Chromatin signatures of the Drosophila replication program.

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Authors
Eaton, ML; Prinz, JA; MacAlpine, HK; Tretyakov, G; Kharchenko, PV; MacAlpine, DM
MLA Citation
Eaton, ML, Prinz, JA, MacAlpine, HK, Tretyakov, G, Kharchenko, PV, and MacAlpine, DM. "Chromatin signatures of the Drosophila replication program." Genome Res 21.2 (February 2011): 164-174.
PMID
21177973
Source
pubmed
Published In
Genome research
Volume
21
Issue
2
Publish Date
2011
Start Page
164
End Page
174
DOI
10.1101/gr.116038.110

Developmental control of the DNA replication and transcription programs.

Polyploid or polytene cells, which have more than 2C DNA content, are widespread throughout nature and present in most differentiated Drosophila tissues. These cells also can display differential replication, that is, genomic regions of increased or decreased DNA copy number relative to overall genomic ploidy. How frequently differential replication is used as a developmental strategy remains unclear. Here, we use genome-wide array-based comparative genomic hybridization (aCGH) to profile differential DNA replication in isolated and purified larval fat body and midgut tissues of Drosophila, and we compare them with recent aCGH profiles of the larval salivary gland. We identify sites of euchromatic underreplication that are common to all three tissues and others that are tissue specific. We demonstrate that both common and tissue-specific underreplicated sites are dependent on the Suppressor of Underreplication protein, SUUR. mRNA-seq profiling shows that whereas underreplicated regions are generally transcriptionally silent in the larval midgut and salivary gland, transcriptional silencing and underreplication have been uncoupled in the larval fat body. In addition to revealing the prevalence of differential replication, our results show that transcriptional silencing and underreplication can be mechanistically uncoupled.

Authors
Nordman, J; Li, S; Eng, T; Macalpine, D; Orr-Weaver, TL
MLA Citation
Nordman, J, Li, S, Eng, T, Macalpine, D, and Orr-Weaver, TL. "Developmental control of the DNA replication and transcription programs." Genome Res 21.2 (February 2011): 175-181.
PMID
21177957
Source
pubmed
Published In
Genome research
Volume
21
Issue
2
Publish Date
2011
Start Page
175
End Page
181
DOI
10.1101/gr.114611.110

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Authors
modENCODE Consortium, ; Roy, S; Ernst, J; Kharchenko, PV; Kheradpour, P; Negre, N; Eaton, ML; Landolin, JM; Bristow, CA; Ma, L; Lin, MF; Washietl, S; Arshinoff, BI; Ay, F; Meyer, PE; Robine, N; Washington, NL; Di Stefano, L; Berezikov, E; Brown, CD; Candeias, R; Carlson, JW; Carr, A; Jungreis, I; Marbach, D; Sealfon, R; Tolstorukov, MY; Will, S; Alekseyenko, AA; Artieri, C; Booth, BW; Brooks, AN; Dai, Q; Davis, CA; Duff, MO; Feng, X; Gorchakov, AA; Gu, T; Henikoff, JG; Kapranov, P; Li, R et al.
MLA Citation
modENCODE Consortium, , Roy, S, Ernst, J, Kharchenko, PV, Kheradpour, P, Negre, N, Eaton, ML, Landolin, JM, Bristow, CA, Ma, L, Lin, MF, Washietl, S, Arshinoff, BI, Ay, F, Meyer, PE, Robine, N, Washington, NL, Di Stefano, L, Berezikov, E, Brown, CD, Candeias, R, Carlson, JW, Carr, A, Jungreis, I, Marbach, D, Sealfon, R, Tolstorukov, MY, Will, S, Alekseyenko, AA, Artieri, C, Booth, BW, Brooks, AN, Dai, Q, Davis, CA, Duff, MO, Feng, X, Gorchakov, AA, Gu, T, Henikoff, JG, Kapranov, P, and Li, R et al. "Identification of functional elements and regulatory circuits by Drosophila modENCODE." Science 330.6012 (December 24, 2010): 1787-1797.
PMID
21177974
Source
pubmed
Published In
Science
Volume
330
Issue
6012
Publish Date
2010
Start Page
1787
End Page
1797
DOI
10.1126/science.1198374

Preferential re-replication of Drosophila heterochromatin in the absence of geminin.

To ensure genomic integrity, the genome must be duplicated exactly once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. Cdt1, also known as Double-parked (Dup) in Drosophila, is a key regulator of the assembly of the pre-replicative complex (pre-RC) and its activity is strictly limited to G1 by multiple mechanisms including Cul4-Ddb1 mediated proteolysis and inhibition by geminin. We assayed the genomic consequences of disregulating the replication licensing mechanisms by RNAi depletion of geminin. We found that not all origins of replication were sensitive to geminin depletion and that heterochromatic sequences were preferentially re-replicated in the absence of licensing mechanisms. The preferential re-activation of heterochromatic origins of replication was unexpected because these are typically the last sequences to be duplicated in a normal cell cycle. We found that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather by the formation of the pre-RC. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin by elevated cyclin A-CDK activity. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.

Authors
Ding, Q; MacAlpine, DM
MLA Citation
Ding, Q, and MacAlpine, DM. "Preferential re-replication of Drosophila heterochromatin in the absence of geminin. (Published online)" PLoS Genet 6.9 (September 9, 2010): e1001112-.
PMID
20838463
Source
pubmed
Published In
PLoS genetics
Volume
6
Issue
9
Publish Date
2010
Start Page
e1001112
DOI
10.1371/journal.pgen.1001112

The conserved bromo-adjacent homology domain of yeast Orc1 functions in the selection of DNA replication origins within chromatin.

The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.

Authors
Müller, P; Park, S; Shor, E; Huebert, DJ; Warren, CL; Ansari, AZ; Weinreich, M; Eaton, ML; MacAlpine, DM; Fox, CA
MLA Citation
Müller, P, Park, S, Shor, E, Huebert, DJ, Warren, CL, Ansari, AZ, Weinreich, M, Eaton, ML, MacAlpine, DM, and Fox, CA. "The conserved bromo-adjacent homology domain of yeast Orc1 functions in the selection of DNA replication origins within chromatin." Genes Dev 24.13 (July 1, 2010): 1418-1433.
PMID
20595233
Source
pubmed
Published In
Genes & development
Volume
24
Issue
13
Publish Date
2010
Start Page
1418
End Page
1433
DOI
10.1101/gad.1906410

Conserved nucleosome positioning defines replication origins.

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

Authors
Eaton, ML; Galani, K; Kang, S; Bell, SP; MacAlpine, DM
MLA Citation
Eaton, ML, Galani, K, Kang, S, Bell, SP, and MacAlpine, DM. "Conserved nucleosome positioning defines replication origins." Genes Dev 24.8 (April 15, 2010): 748-753.
PMID
20351051
Source
pubmed
Published In
Genes & development
Volume
24
Issue
8
Publish Date
2010
Start Page
748
End Page
753
DOI
10.1101/gad.1913210

Expression in aneuploid Drosophila S2 cells.

Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression) genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets of autosomes, or 2X;4A). Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system "anticipates" the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal-independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components.

Authors
Zhang, Y; Malone, JH; Powell, SK; Periwal, V; Spana, E; Macalpine, DM; Oliver, B
MLA Citation
Zhang, Y, Malone, JH, Powell, SK, Periwal, V, Spana, E, Macalpine, DM, and Oliver, B. "Expression in aneuploid Drosophila S2 cells. (Published online)" PLoS Biol 8.2 (February 23, 2010): e1000320-.
Website
http://hdl.handle.net/10161/4445
PMID
20186269
Source
pubmed
Published In
PLoS biology
Volume
8
Issue
2
Publish Date
2010
Start Page
e1000320
DOI
10.1371/journal.pbio.1000320

Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading.

The origin recognition complex (ORC) is an essential DNA replication initiation factor conserved in all eukaryotes. In Saccharomyces cerevisiae, ORC binds to specific DNA elements; however, in higher eukaryotes, ORC exhibits little sequence specificity in vitro or in vivo. We investigated the genome-wide distribution of ORC in Drosophila and found that ORC localizes to specific chromosomal locations in the absence of any discernible simple motif. Although no clear sequence motif emerged, we were able to use machine learning approaches to accurately discriminate between ORC-associated sequences and ORC-free sequences based solely on primary sequence. The complex sequence features that define ORC binding sites are highly correlated with nucleosome positioning signals and likely represent a preferred nucleosomal landscape for ORC association. Open chromatin appears to be the underlying feature that is deterministic for ORC binding. ORC-associated sequences are enriched for the histone variant, H3.3, often at transcription start sites, and depleted for bulk nucleosomes. The density of ORC binding along the chromosome is reflected in the time at which a sequence replicates, with early replicating sequences having a high density of ORC binding. Finally, we found a high concordance between sites of ORC binding and cohesin loading, suggesting that, in addition to DNA replication, ORC may be required for the loading of cohesin on DNA in Drosophila.

Authors
MacAlpine, HK; Gordân, R; Powell, SK; Hartemink, AJ; MacAlpine, DM
MLA Citation
MacAlpine, HK, Gordân, R, Powell, SK, Hartemink, AJ, and MacAlpine, DM. "Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading." Genome Res 20.2 (February 2010): 201-211.
PMID
19996087
Source
pubmed
Published In
Genome research
Volume
20
Issue
2
Publish Date
2010
Start Page
201
End Page
211
DOI
10.1101/gr.097873.109

Co-orientation of replication and transcription preserves genome integrity.

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

Authors
Srivatsan, A; Tehranchi, A; MacAlpine, DM; Wang, JD
MLA Citation
Srivatsan, A, Tehranchi, A, MacAlpine, DM, and Wang, JD. "Co-orientation of replication and transcription preserves genome integrity. (Published online)" PLoS Genet 6.1 (January 15, 2010): e1000810-.
Website
http://hdl.handle.net/10161/4458
PMID
20090829
Source
pubmed
Published In
PLoS genetics
Volume
6
Issue
1
Publish Date
2010
Start Page
e1000810
DOI
10.1371/journal.pgen.1000810

Unlocking the secrets of the genome.

Authors
Celniker, SE; Dillon, LAL; Gerstein, MB; Gunsalus, KC; Henikoff, S; Karpen, GH; Kellis, M; Lai, EC; Lieb, JD; MacAlpine, DM; Micklem, G; Piano, F; Snyder, M; Stein, L; White, KP; Waterston, RH; modENCODE Consortium,
MLA Citation
Celniker, SE, Dillon, LAL, Gerstein, MB, Gunsalus, KC, Henikoff, S, Karpen, GH, Kellis, M, Lai, EC, Lieb, JD, MacAlpine, DM, Micklem, G, Piano, F, Snyder, M, Stein, L, White, KP, Waterston, RH, and modENCODE Consortium, . "Unlocking the secrets of the genome." Nature 459.7249 (June 18, 2009): 927-930.
PMID
19536255
Source
pubmed
Published In
Nature
Volume
459
Issue
7249
Publish Date
2009
Start Page
927
End Page
930
DOI
10.1038/459927a

Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila.

Post-translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone-deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes-4, whereas trimethylation accumulates toward the 3' end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di- and trimethylation decreases lysine 16 acetylation. Thus di- and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation.

Authors
Bell, O; Wirbelauer, C; Hild, M; Scharf, AND; Schwaiger, M; MacAlpine, DM; Zilbermann, F; van Leeuwen, F; Bell, SP; Imhof, A; Garza, D; Peters, AHFM; Schübeler, D
MLA Citation
Bell, O, Wirbelauer, C, Hild, M, Scharf, AND, Schwaiger, M, MacAlpine, DM, Zilbermann, F, van Leeuwen, F, Bell, SP, Imhof, A, Garza, D, Peters, AHFM, and Schübeler, D. "Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila." EMBO J 26.24 (December 12, 2007): 4974-4984.
PMID
18007591
Source
pubmed
Published In
EMBO Journal
Volume
26
Issue
24
Publish Date
2007
Start Page
4974
End Page
4984
DOI
10.1038/sj.emboj.7601926

Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells.

Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent on DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants.

Authors
Georlette, D; Ahn, S; MacAlpine, DM; Cheung, E; Lewis, PW; Beall, EL; Bell, SP; Speed, T; Manak, JR; Botchan, MR
MLA Citation
Georlette, D, Ahn, S, MacAlpine, DM, Cheung, E, Lewis, PW, Beall, EL, Bell, SP, Speed, T, Manak, JR, and Botchan, MR. "Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells." Genes Dev 21.22 (November 15, 2007): 2880-2896.
PMID
17978103
Source
pubmed
Published In
Genes & development
Volume
21
Issue
22
Publish Date
2007
Start Page
2880
End Page
2896
DOI
10.1101/gad.1600107

Drosophila follicle cell amplicons as models for metazoan DNA replication: a cyclinE mutant exhibits increased replication fork elongation.

Gene clusters amplified in the ovarian follicle cells of Drosophila serve as powerful models for metazoan DNA replication. In response to developmental signals, specific genomic regions undergo amplification by repeated firing of replication origins and bidirectional movement of replication forks for approximately 50 kb in each direction. Previous work focused on initiation of amplification, defining replication origins, establishing the role of the prereplication complex and origin recognition complex (ORC), and uncovering regulatory functions for the Myb, E2F1, and Rb transcription factors. Here, we exploit follicle cell amplification to investigate the control of DNA replication fork progression and termination, poorly understood processes in metazoans. We identified a mutant in which, during gene amplification, the replication forks move twice as far from the origin compared with wild type. This phenotype is the result of an amino acid substitution mutation in the cyclinE gene, cyclinE(1f36). The rate of oogenesis is normal in cyclinE(1f36)/cyclinE(Pz8) mutant ovaries, indicating that increased replication fork progression is due to increased replication fork speed, possibly from increased processivity. The increased amplification domains observed in the mutant imply that there are not replication fork barriers preventing replication forks from progressing beyond the normal 100-kb amplified region. These results reveal a previously unrecognized role for CyclinE in controlling replication fork movement.

Authors
Park, EA; Macalpine, DM; Orr-Weaver, TL
MLA Citation
Park, EA, Macalpine, DM, and Orr-Weaver, TL. "Drosophila follicle cell amplicons as models for metazoan DNA replication: a cyclinE mutant exhibits increased replication fork elongation." Proc Natl Acad Sci U S A 104.43 (October 23, 2007): 16739-16746.
PMID
17940024
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
43
Publish Date
2007
Start Page
16739
End Page
16746
DOI
10.1073/pnas.0707804104

Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation.

DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome is re-replicated by a subset of origins, some of which are capable of multiple reinitiation events. Translocation experiments demonstrate that origin-proximal sequences are sufficient to predispose an origin to re-replication. Origins that reinitiate are largely limited to those that can recruit Mcm2-7 under re-replicating conditions; however, the formation of a pre-RC is not sufficient for reinitiation. Our findings allow us to categorize origins with respect to their propensity to reinitiate and demonstrate that pre-RC formation is not the only target for the mechanisms that prevent genomic re-replication.

Authors
Tanny, RE; MacAlpine, DM; Blitzblau, HG; Bell, SP
MLA Citation
Tanny, RE, MacAlpine, DM, Blitzblau, HG, and Bell, SP. "Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation." Mol Biol Cell 17.5 (May 2006): 2415-2423.
PMID
16525018
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
17
Issue
5
Publish Date
2006
Start Page
2415
End Page
2423
DOI
10.1091/mbc.E05-11-1037

Temporal profile of replication of human chromosomes.

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

Authors
Jeon, Y; Bekiranov, S; Karnani, N; Kapranov, P; Ghosh, S; MacAlpine, D; Lee, C; Hwang, DS; Gingeras, TR; Dutta, A
MLA Citation
Jeon, Y, Bekiranov, S, Karnani, N, Kapranov, P, Ghosh, S, MacAlpine, D, Lee, C, Hwang, DS, Gingeras, TR, and Dutta, A. "Temporal profile of replication of human chromosomes." Proc Natl Acad Sci U S A 102.18 (May 3, 2005): 6419-6424.
PMID
15845769
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
18
Publish Date
2005
Start Page
6419
End Page
6424
DOI
10.1073/pnas.0405088102

A genomic view of eukaryotic DNA replication.

Recent advances in DNA microarray technology have enabled eukaryotic replication to be studied at whole-chromosome and genome-wide levels. These studies have provided new insights into the mechanisms that influence origin selection and the temporally co-ordinated activation of replication initiation from these sites. Here we describe multiple microarray-based approaches that have been used to study DNA replication in both S. cerevisiae and higher eukaryotes. We have also compiled the data from the yeast microarray-based replication studies to generate a comprehensive list of origins that has been verified in three independent studies. The comprehensive nature of the microarray-based studies has revealed clear connections between chromosome organization and the pattern of replication. For example, in yeast, the centromeric proximal sequences are consistently early replicating and telomeric regions are consistently late replicating. The metazoan studies reveal a recurring theme of gene-dense transcriptionally active regions of the genome replicating before gene-sparse regions. In addition to the insights they have provided already, microarray-based replication assays combined with genetic analysis will provide a powerful new approach to define the mechanisms that regulate replication origin function.

Authors
MacAlpine, DM; Bell, SP
MLA Citation
MacAlpine, DM, and Bell, SP. "A genomic view of eukaryotic DNA replication." Chromosome Res 13.3 (2005): 309-326. (Review)
PMID
15868424
Source
pubmed
Published In
Chromosome Research
Volume
13
Issue
3
Publish Date
2005
Start Page
309
End Page
326
DOI
10.1007/s10577-005-1508-1

Coordination of replication and transcription along a Drosophila chromosome.

The mechanisms by which metazoan origins of DNA replication are defined, regulated, and influenced by chromosomal events remain poorly understood. To gain insights into these mechanisms, we developed a systematic approach using a Drosophila high-resolution genomic microarray to determine replication timing, identify replication origins, and map protein-binding sites along a chromosome arm. We identify a defined temporal pattern of replication that correlates with the density of active transcription. These data indicate that the influence of transcription status on replication timing is exerted over large domains (>100 kb) rather than at the level of individual genes. We identify 62 early activating replication origins across the chromosome by mapping sites of nucleotide incorporation during hydroxyurea arrest. Using genome-wide location analysis, we demonstrate that the origin recognition complex (ORC) is localized to specific chromosomal sites, many of which coincide with early activating origins. The molecular attributes of ORC-binding sites include increased AT-content and association with a subset of RNA Pol II-binding sites. Based on these findings, we suggest that the distribution of transcription along the chromosome acts locally to influence origin selection and globally to regulate origin activation.

Authors
MacAlpine, DM; Rodríguez, HK; Bell, SP
MLA Citation
MacAlpine, DM, Rodríguez, HK, and Bell, SP. "Coordination of replication and transcription along a Drosophila chromosome." Genes Dev 18.24 (December 15, 2004): 3094-3105.
PMID
15601823
Source
pubmed
Published In
Genes & development
Volume
18
Issue
24
Publish Date
2004
Start Page
3094
End Page
3105
DOI
10.1101/gad.1246404

The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote.

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity.

Authors
Schübeler, D; MacAlpine, DM; Scalzo, D; Wirbelauer, C; Kooperberg, C; van Leeuwen, F; Gottschling, DE; O'Neill, LP; Turner, BM; Delrow, J; Bell, SP; Groudine, M
MLA Citation
Schübeler, D, MacAlpine, DM, Scalzo, D, Wirbelauer, C, Kooperberg, C, van Leeuwen, F, Gottschling, DE, O'Neill, LP, Turner, BM, Delrow, J, Bell, SP, and Groudine, M. "The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote." Genes Dev 18.11 (June 1, 2004): 1263-1271.
PMID
15175259
Source
pubmed
Published In
Genes & development
Volume
18
Issue
11
Publish Date
2004
Start Page
1263
End Page
1271
DOI
10.1101/gad.1198204

Visualization of replication initiation and elongation in Drosophila.

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2-7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre-replication complex component, double-parked protein/cell division cycle 10-dependent transcript 1, is not only necessary for proper MCM2-7 localization, but, unexpectedly, is present during elongation.

Authors
Claycomb, JM; MacAlpine, DM; Evans, JG; Bell, SP; Orr-Weaver, TL
MLA Citation
Claycomb, JM, MacAlpine, DM, Evans, JG, Bell, SP, and Orr-Weaver, TL. "Visualization of replication initiation and elongation in Drosophila." J Cell Biol 159.2 (October 28, 2002): 225-236.
PMID
12403810
Source
pubmed
Published In
The Journal of Cell Biology
Volume
159
Issue
2
Publish Date
2002
Start Page
225
End Page
236
DOI
10.1083/jcb.200207046

Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA.

Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.

Authors
MacAlpine, DM; Kolesar, J; Okamoto, K; Butow, RA; Perlman, PS
MLA Citation
MacAlpine, DM, Kolesar, J, Okamoto, K, Butow, RA, and Perlman, PS. "Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA." EMBO J 20.7 (April 2, 2001): 1807-1817.
PMID
11285243
Source
pubmed
Published In
EMBO Journal
Volume
20
Issue
7
Publish Date
2001
Start Page
1807
End Page
1817
DOI
10.1093/emboj/20.7.1807

The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway.

Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid). We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number. In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number. The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination. Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control. Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.

Authors
MacAlpine, DM; Perlman, PS; Butow, RA
MLA Citation
MacAlpine, DM, Perlman, PS, and Butow, RA. "The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway." EMBO J 19.4 (February 15, 2000): 767-775.
PMID
10675346
Source
pubmed
Published In
EMBO Journal
Volume
19
Issue
4
Publish Date
2000
Start Page
767
End Page
775
DOI
10.1093/emboj/19.4.767

The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo.

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.

Authors
MacAlpine, DM; Perlman, PS; Butow, RA
MLA Citation
MacAlpine, DM, Perlman, PS, and Butow, RA. "The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo." Proc Natl Acad Sci U S A 95.12 (June 9, 1998): 6739-6743.
PMID
9618482
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
12
Publish Date
1998
Start Page
6739
End Page
6743

Holliday junctions in yeast mitochondrial DNA are stabilized by the HMG box protein, ABF2p

Authors
MacAlpine, DM; Perlman, PS; Butow, RA
MLA Citation
MacAlpine, DM, Perlman, PS, and Butow, RA. "Holliday junctions in yeast mitochondrial DNA are stabilized by the HMG box protein, ABF2p." MOLECULAR BIOLOGY OF THE CELL 8 (November 1997): 2589-2589.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
8
Publish Date
1997
Start Page
2589
End Page
2589

Developmental regulation of DNA replication: replication fork barriers and programmed gene amplification in Tetrahymena thermophila.

The palindromic Tetrahymena ribosomal DNA (rDNA) minichromosome is amplified 10,000-fold during development. Subsequent vegetative replication is cell cycle regulated. rDNA replication differs fundamentally in cycling vegetative and nondividing amplifying cells. Using two-dimensional gel electrophoresis, we show for the first time that replication origins that direct gene amplification also function in normal dividing cells. Two classes of amplification intermediates were identified. The first class is indistinguishable from vegetative rDNA, initiating in just one of the two 5' nontranscribed spacer (NTS) copies in the rDNA palindrome at either of two closely spaced origins. Thus, these origins are active throughout the life cycle and their regulation changes at different developmental stages. The second, novel class of amplification intermediates is generated by multiple initiation events. Intermediates with mass greater than fully replicated DNA were observed, suggesting that onionskin replication occurs at this stage. Unlike amplified rDNA in Xenopus laevis, the novel Tetrahymena species are not produced by random initiation; replication also initiates in the 5' NTS. Surprisingly, a replication fork barrier which is activated only in these amplifying molecules blocks the progression of forks near the center of the palindrome. Whereas barriers have been previously described, this is the first instance in which programmed regulation of replication fork progression has been demonstrated in a eukaryote.

Authors
Zhang, Z; Macalpine, DM; Kapler, GM
MLA Citation
Zhang, Z, Macalpine, DM, and Kapler, GM. "Developmental regulation of DNA replication: replication fork barriers and programmed gene amplification in Tetrahymena thermophila." Mol Cell Biol 17.10 (October 1997): 6147-6156.
PMID
9315675
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
17
Issue
10
Publish Date
1997
Start Page
6147
End Page
6156

Type I elements mediate replication fork pausing at conserved upstream sites in the Tetrahymena thermophila ribosomal DNA minichromosome.

Two-dimensional gel electrophoresis was used to study replication of the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome. During vegetative growth, the rDNA is replicated exclusively from origins in the 5' nontranscribed spacer (NTS). Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5' NTS is not. Replication forks arrest transiently at three prominent replication fork pausing sites (RFPs) located in or immediately adjacent to nucleosome-free regions of the 5' NTS. Pausing at these sites is dramatically diminished during replication in Escherichia coli, suggesting that chromatin organization or Tetrahymena-specific proteins may be required. A conserved tripartite sequence was identified at each pausing site. Mutations in type I elements diminish pausing at proximal RFPs. Hence, type I elements, previously shown to control replication initiation, also regulate elongation of existing replication forks. Studies with rDNA transformants revealed a strong directional bias for fork pausing. Strong pausing only occurred in forks moving toward the rRNA-coding region. We propose that fork pausing in the 5' NTS evolved to synchronize replication and transcription of the downstream rRNA genes.

Authors
MacAlpine, DM; Zhang, Z; Kapler, GM
MLA Citation
MacAlpine, DM, Zhang, Z, and Kapler, GM. "Type I elements mediate replication fork pausing at conserved upstream sites in the Tetrahymena thermophila ribosomal DNA minichromosome." Mol Cell Biol 17.8 (August 1997): 4517-4525.
PMID
9234709
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
17
Issue
8
Publish Date
1997
Start Page
4517
End Page
4525

HOW MAIZE SEEDS AND SEEDLINGS COPE WITH OXYGEN DEFICIT

Authors
COBB, BG; DREW, MC; ANDREWS, DL; JOHNSON, J; MACALPINE, DM; DANIELSON, TL; TURNBOUGH, MA; DAVIS, R
MLA Citation
COBB, BG, DREW, MC, ANDREWS, DL, JOHNSON, J, MACALPINE, DM, DANIELSON, TL, TURNBOUGH, MA, and DAVIS, R. "HOW MAIZE SEEDS AND SEEDLINGS COPE WITH OXYGEN DEFICIT." HORTSCIENCE 30.6 (October 1995): 1160-1164.
Source
wos-lite
Published In
HortScience : a publication of the American Society for Horticultural Science
Volume
30
Issue
6
Publish Date
1995
Start Page
1160
End Page
1164

STABILITY OF TRANSCRIPTS FOR THE GLYCOLYTIC AND ETHANOLIC FERMENTATIVE PATHWAYS IN MAIZE ROOT-TIPS DURING HYPOXIA AND ANOXIA

Authors
ANDREWS, DL; MACALPINE, DM; COBB, BG; DREW, MC
MLA Citation
ANDREWS, DL, MACALPINE, DM, COBB, BG, and DREW, MC. "STABILITY OF TRANSCRIPTS FOR THE GLYCOLYTIC AND ETHANOLIC FERMENTATIVE PATHWAYS IN MAIZE ROOT-TIPS DURING HYPOXIA AND ANOXIA." PLANT PHYSIOLOGY 108.2 (June 1995): 110-110.
Source
wos-lite
Published In
Plant physiology
Volume
108
Issue
2
Publish Date
1995
Start Page
110
End Page
110

MAIZE INDUCTION OF LDH OCCURS IN THE ROOT AXIS, AND NOT THE ROOT-TIP UNDER LONG-TERM EXPOSURE TO HYPOXIA

Authors
MACALPINE, DM; ANDREWS, DL; DREW, MC; COBB, BG
MLA Citation
MACALPINE, DM, ANDREWS, DL, DREW, MC, and COBB, BG. "MAIZE INDUCTION OF LDH OCCURS IN THE ROOT AXIS, AND NOT THE ROOT-TIP UNDER LONG-TERM EXPOSURE TO HYPOXIA." PLANT PHYSIOLOGY 108.2 (June 1995): 114-114.
Source
wos-lite
Published In
Plant physiology
Volume
108
Issue
2
Publish Date
1995
Start Page
114
End Page
114

Differential induction of mRNAs for the glycolytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings.

Fructose-1,6-biphosphate aldolase (ALD) and enolase (ENO) from the glycolytic pathway and pyruvate decarboxylase (PDC) and alcohol dehydrogenase 2 (ADH2) from the ethanolic fermentative pathway, are enzymes previously identified as among those synthesized selectively in O2-deficient roots of maize (Zea mays L.). The present study measured levels of transcripts representing these two pathways in 5-mm root tips, root axes (the remainder of the primary seminal root), and shoots of maize seedlings to determine how closely both pathways were co-induced and how they were modulated by changes in O2 concentration. In hypoxic seedlings with the roots in solution sparged with 5% (v/v) O2 (balance N2) and the shoots in the same gaseous atmosphere, mRNAs for Pdc1 and Adh2 in root tips both increased about 15-fold during the first 12 h, followed by a decline toward initial levels by 18 to 24h. Message levels for Ald1 and Eno1 showed only small changes during hypoxia. When expression was examined under anoxia, the extent to which all four mRNAs increased in different tissues depended on whether the seedlings had been previously acclimated to hypoxia or were anoxically shocked. The results show that although all the genes examined increased expression during hypoxia and/or anoxia, they differed in the rapidity and magnitude of the response and in the time to reach maximal message levels: there was no common pattern of change of message levels for the glycolytic or for the fermantative enzymes.

Authors
Andrews, DL; MacAlpine, DM; Johnson, JR; Kelley, PM; Cobb, BG; Drew, MC
MLA Citation
Andrews, DL, MacAlpine, DM, Johnson, JR, Kelley, PM, Cobb, BG, and Drew, MC. "Differential induction of mRNAs for the glycolytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings." Plant Physiol 106.4 (December 1994): 1575-1582.
PMID
7846162
Source
pubmed
Published In
Plant physiology
Volume
106
Issue
4
Publish Date
1994
Start Page
1575
End Page
1582
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