Zhong Chen

Knowledge gaps remain on how nucleosome organization and dynamic reorganization are governed by specific pioneer factors in a genome-wide manner. In this study, we generate over three billons of multi-omics sequencing data to exploit dynamic nucleosome landscape governed by pioneer factors (PFs), FOXA1 and GATA2. We quantitatively define nine functional nucleosome states each with specific characteristic nucleosome footprints in LNCaP prostate cancer cells. Interestingly, we observe dynamic switches among nucleosome states upon androgen stimulation, accompanied by distinct differential (gained or lost) binding of FOXA1, GATA2, H1 as well as many other coregulators. Intriguingly, we reveal a noncanonical pioneer model of GATA2 that it initially functions as a PF binding at the edge of a nucleosome in an inaccessible crowding array. Upon androgen stimulation, GATA2 re-configures an inaccessible to accessible nucleosome state and subsequently acts as a master transcription factor either directly or recruits signaling specific transcription factors to enhance WNT signaling in an androgen receptor (AR)-independent manner. Our data elicit a pioneer and master dual role of GATA2 in mediating nucleosome dynamics and enhancing downstream signaling pathways. Our work offers structural and mechanistic insight into the dynamics of pioneer factors governing nucleosome reorganization.

AIM: To express the recombinant fusion protein of hemangiopoietin (HAPO) and prepare the rabbit-anti-human HAPO polyclonal antibody. METHODS: The sequence encoding HAPO was amplified by PCR and cloned into plasmid pET32c to construct recombinant prokaryotic expression system. The recombinant expression vectors were identified by enzyme digestion analysis and transformed into E. coli. The HAPO protein was purified by affinity chromatography. Rabbits were immunized with the HAPO protein, and the immune sera of rabbits were collected. Antibodies (IgG) obtained from the immune sera were purified. RESULTS: The purified HAPO protein was successfully obtained. The purified polyclonal antibody of rabbit-anti-human HAPO was also obtained from the immune sera of rabbits, and could response to human HAPO. CONCLUSION: A prokaryotic expression system of human HAPO has been prepared and the polyclonal antibody against HAPO has been prepared, which can be used to determine HAPO protein.

Mediator activates RNA polymerase II (Pol II) function during transcription, but it remains unclear whether Mediator is able to travel with Pol II and regulate Pol II transcription beyond the initiation and early elongation steps. By using in vitro and in vivo transcription recycling assays, we find that human Mediator 1 (MED1), when phosphorylated at the mammal-specific threonine 1032 by cyclin-dependent kinase 9 (CDK9), dynamically moves along with Pol II throughout the transcribed genes to drive Pol II recycling after the initial round of transcription. Mechanistically, MED31 mediates the recycling of phosphorylated MED1 and Pol II, enhancing mRNA output during the transcription recycling process. Importantly, MED1 phosphorylation increases during prostate cancer progression to the lethal phase, and pharmacological inhibition of CDK9 decreases prostate tumor growth by decreasing MED1 phosphorylation and Pol II recycling. Our results reveal a novel role of MED1 in Pol II transcription and identify phosphorylated MED1 as a targetable driver of dysregulated Pol II recycling in cancer.

OBJECTIVE: To explore the role of DNA methylation in pathogenesis of adult idiopathic thrombocytopenic purpura (ITP). METHODS: We measured DNA methyltransferases (DNMTs) 1, 3A and 3B mRNA expression in peripheral blood mononuclear cells of 48 adult ITP patients and 36 normal controls using real-time polymerase chain reaction, as well as the plasma levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) with reversed phase high performance liquid chromatography (HPLC). Furthermore, we determined the expression of CD(11a)/LFA-1 on CD(4)(+) or CD(8)(+) T cells by three-color flow cytometry. RESULTS: The mRNA expression of two DNA methyltransferases (DNMT3A and DNMT3B) was significantly lower when comparing ITP patients with healthy controls (P < 0.01). Although there were decreased tendency when comparing expression of DNMT1 of ITP patients with healthy controls, no significant differences were found (P > 0.05). The SAH concentration in the plasma of ITP patients was significantly elevated than that in the healthy controls (P < 0.05). However we found significant differences of SAM and SAM/SAH ratio in the plasma of ITP patients when compared with the healthy subjects (both P > 0.05). In addition, CD(11a)/LFA-1 expression on CD(8)(+) T lymphocytes increased in ITP patients compared with the control group (P < 0.01), whereas CD(11a)/LFA-1 expression on CD(4)(+) T lymphocytes and CD(4)(+)CD(11a)(+)/CD(8)(+)CD(11a)(+) ratio was significantly decreased in ITP patients than that in control group (both P < 0.01). CONCLUSION: Aberrant DNA methylation in peripheral blood and CD(11a)/LFA-1 expression on T cell surface may play an important role in the pathogenesis of ITP, although the underlying mechanisms still await further investigation.

OBJECTIVE: DNA methylation is known to play an important role in gene transcription and alterations of methylation contribute to the development of certain disorders such as cancer and immunodeficiency. Recent years have found an increasing interest in the role of epigenetic modifications in the etiology of human autoimmune diseases, such as systemic lupus erythromatosus (SLE) and rheumatoid arthritis (RA). DNA methyltransferases (DNMTs) are involved in the epigenetic control of DNA methylation processes. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, have important indicator action of cellular methylation status. The aim of this study is to explore if DNA methylation plays a role in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: DNMT1, DNMT3A, and DNMT3B mRNA expression in peripheral blood mononuclear cells (PBMCs) of adult ITP patients were analyzed by real-time quantitative polymerase chain reaction. Plasma SAM and SAH levels were assayed with reversed-phase high performance liquid chromatography (HPLC). RESULTS: DNMT3A and DNMT3B mRNA expressions were significantly lower in ITP patients than in healthy controls (p < 0.001), while DNMT1 mRNA expression was not significantly different between the two groups (p = 0.774). Plasma SAH concentration was significantly elevated in ITP patients than in healthy controls (p < 0.05), while the plasma SAM and SAM/SAH were not significantly different between the two groups (p = 0.133, p = 0.624 respectively). CONCLUSIONS: Our observations suggest that aberrant DNA methylation status reflected by increased plasma SAH concentration and decreased mRNA expression levels of DNMT3A and 3B are possibly involved in the pathogenesis of ITP although the precise mechanisms need further study.