Author

Liangqi Xie

Date

April 2012

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

Pluripotent embryonic stem cells (ESCs) are derived from the epiblast of preimplantation embryos and have two defining features: indefinite self-renewal in vitro and pluripotency-- the capacity to differentiate into all somatic cells and the germ lineage. ESCs depend on the leukemia inhibitory factor (LIF) signaling pathways to maintain the naïve pluripotent state and therefore can readily incorporate the inner cell mass (ICM) to resume normal embryonic development. Understanding the molecular mechanisms that establish and maintain the ESCs pluripotency is essential for their application to regenerative medicine. The core pluripotency factor Nanog is proposed to specify and stabilize a naïve pluripotent state in both embryogenesis and induced reprogramming. Nevertheless, the downstream epigenetic mechanisms by which Nanog constructs an epigenetic landscape permissive to the naïve pluripotent state are unclear. Nanog ChIP-Seq and Nanog knockdown RNA-Seq analysis identify histone H3 trimethyl lysine 4 (H3K4me3) demethylase Kdm5b as a direct downstream target of Nanog in mouse ESCs. Kdm5b expression is highly enriched in the ICM of mouse preimplantation embryos and pluripotency-associated cells and tissues. Acute Kdm5b mRNA knockdown impairs ESCs proliferation and self-renewal. Forced expression of Kdm5b can sustain ESCs in an undifferentiated state in the absence of LIF signaling for up to two months. These cells are still pluripotent because they can contribute to teratoma formation. Epistasis experiments demonstrate that Kdm5b overexpression can compensate for loss of Nanog but not Klf4, suggesting that Kdm5b is a major downstream effector of Nanog. Ectopic expression of Kdm5b can also boost the reprogramming efficiency from neural stem cells in conjunction with Oct4 or Oct4/Klf4. These lines of evidence suggest that Kdm5b is an important epigenetic regulator for the ESC naïve pluripotent state. Although KDM5B is believed to function as a promoter-bound repressor, analysis of Kdm5b knockdown RNA-Seq suggests that it paradoxically functions as an activator of a gene network associated with ESC self-renewal. Our ChIP-Seq data reveals that KDM5B predominantly occupies active intragenic regions and significantly correlates with the elongating RNA Pol II and histone H3 trimethyl lysine 36 (H3K36me3). KDM5B is recruited to H3K36me3 via an interaction with the chromodomain protein MRG15. Genome-wide analysis of MRG15 occupancy shows a high degree of colocalization with KDM5B. Mrg15 is an ortholog of eaf3, a component of the yeast Rpd3S complex which functions to repress cryptic intragenic transcription. Likewise, knockdown of Kdm5b or Mrg15 significantly increases cryptic intragenic transcription, promotes unphosphorylated RNA Pol II recruitment and reduces elongation-associated RNA Pol II occupancy selectively at KDM5B target genes. We propose that KDM5B activates a selfrenewal-associated gene network by repressing intragenic cryptic initiation and maintaining a H3K4me3 gradient important for productive elongation. Proteomic analysis of the KDM5B demethylase complex reveals additional H3K36me3 and transcriptional elongation-associated proteins, including H3K36 methylation writer and reader protein NSD3. Our preliminary data demonstrate that NSD3 can mediate the recruitment of KDM5B to intragenic regions at KDM5B target genes. We show that KDM5B can occupy and activate genes involved in cell cycle and DNA synthesis control and that forced expression of genes in the DNA synthesis machinery can partially rescue the proliferation defect after Kdm5b knockdown. KDM5B is also found to interact with the DNA replication machinery, suggesting a positive feedback loop to reinforce KDM5B‘s role in regulating DNA replication. Taken together, these results provide novel insights into the role of Kdm5b in regulating the ESC naïve pluripotency and transcriptional elongation. Kdm5b mRNA is also up-regulated in multiple human cancers and implicated in cancer cell proliferation and self-renewal. Thus, KDM5B may represent an attractive drug target for tumor therapy.

Identifier

doi:10.6083/M4K35RNN

School

School of Medicine

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