Date of Award

12-1-2019

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Davie, Judy

Abstract

Eukaryotic DNA is packaged into highly ordered structures knows as chromatin that further packs the DNA into higher-order structures, limiting the accessibility of the underlying genetic information for the processes like transcription, replication, and repair. However, eukaryotic cells have evolved proteins called chromatin regulators that regulate the accessibility of the genetic information when needed. This dissertation aims to characterize the role of two such proteins, JARID2 and the polycomb repressive complex-2 (PRC2), during skeletal muscle proliferation and differentiation.JARID2 is an inactive yet evolutionarily conserved histone demethylase that is shown to be a sub-stoichiometric component of the PRC2 complex. The PRC2 complex represses gene expression through the trimethylation of lysine 27 of histone 3 (H3K27me) tails. H3K27 methylation leads to chromatin compaction. JARID2 helps in targeting of the PRC2 complex to its target loci. JARID2 is shown to be required for the normal development of mice, as loss of Jarid2 leads to lethality in utero. We, for the first time, show that JARID2 is required for the normal skeletal muscle differentiation. We show that the JARID2 regulates the expression of myogenic regulatory factor, Myod1, both through direct repression and activation through the modulation of canonical Wnt signaling pathway. JARID2, in association with the PRC2 complex, represses Wnt antagonist Sfrp1 to modulate the activity of the canonical Wnt signaling pathway. The translocation of Wnt effector protein, b-catenin, from the cytoplasm to the nucleus modulates the activity of the canonical Wnt signaling pathway during activation. We also show that b-catenin directly regulates the expression of Myod1 gene through its direct binding in the distal regulatory region.We further extend the role of JARID2 during skeletal muscle proliferation. We show that JARID2 also plays an essential role in restraining the skeletal muscle proliferation through its direct repression of positive cell cycle regulators cyclin D1 (Ccnd1) and cyclin E1 (Ccne1). Furthermore, we show that retinoblastoma protein 1 (Rb1), a negative regulator of cell proliferation that promotes cell cycle exit and differentiation, is also directly regulated by JARID2 in PRC2 dependent manner. Together, we show that JARID2 precisely controls cell proliferation and differentiation during skeletal muscle differentiation.Further, we show that the regulation of cell proliferation by JARID2 is PRC2 complex dependent. When the PRC2 complex was depleted or inhibited to a modest level, the cells have an increased cell proliferation ability compared to severe loss or inhibition of EZH2, the catalytic subunit of the PRC2 complex, that leads to the apoptosis of the cells. It is also marked by increased expression of known PRC2 targets genes. We show that the increased proliferation upon modest inhibition or depletion of EZH2 is through direct de-repression of positive cell cycle genes, Ccnd1, and Ccne1. It is the first work that shows a context-dependent role of the PRC2 complex during skeletal muscle proliferation and differentiation.My dissertation also makes an extraordinary discovery as to why myogenin is required for the proper function of MyoD during skeletal muscle differentiation, even though both proteins share a large set of overlapping target genes. We show that myogenin is required for the nucleosome disassembly and reassembly at the target genes through recruitment of the FACT complex, a histone chaperone. We also show that myogenin is required for the assembly of the basic transcription machinery and RNA polymerase II to the target muscle genes during differentiation. Surprisingly, we also show that myogenin reinforces its own expression through the activation of Myod1 expression during skeletal muscle differentiation. Myogenin is a known target of MyoD.Taken together, this dissertation provides a molecular mechanism for the crosstalk between a signaling pathway with chromatin regulatory proteins, JARID2, and the PRC2 complex in regulating skeletal muscle differentiation. It also extends the role of JARID2 and the PRC2 complex - known oncogenes, in precise, context-dependent control of cell proliferation and differentiation in skeletal muscle.

Available for download on Tuesday, February 23, 2021

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