Li, Chi Han Samson. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 158-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

AN ABSTRACT OF THE DISSERTATION OF Priya Londhe, for the Doctor of Philosophy degree in Biochemistry and Molecular Biology, presented on 30th July, 2013 at Southern Illinois University Carbondale. TITLE: UNRAVELING THE MOLECULAR MECHANISMS OF THE CLASS II TRANSACTIVATOR IN SKELETAL MUSCLE MAJOR PROFESSOR: Dr. Judy Davie The inflammatory cytokine, interferon gamma, IFN-gamma orchestrates a diverse array of fundamental physiological processes and exhibits complex effects on myogenesis. IFN-gamma also induces the class II transactivator, CIITA, which is a critical mediator of IFN-gamma mediated repression and activation. The aims in my dissertation are directed towards understanding the role of IFN-gamma and CIITA in muscle. Stimulation by IFN-gamma in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. We show that the IFN-gamma induced inhibition of myogenesis is mediated by CIITA, which specifically interacts with myogenin. CIITA acts by both, repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. The IFN-gamma mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-gamma. We also show that CIITA is indispensible for the inhibition of myogenesis. To gain a mechanistic insight into the IFN-gamma induced repression of myogenesis, we have discovered that IFN-gamma and CIITA inhibit myogenesis by modifying gene regulation in a muscle cell subject to inflammation. We show that CIITA first interacts with JARID2, a non catalytic subunit of PRC2 complex, which induces a paused RNAPII, phosphorylated at serine 5 and then interacts with the catalytic subunit EZH2, in a JARID2 dependent manner. Our data show that both CIITA and IFN-gamma block myogenesis by the induction and recruitment of the PRC2 complex, which is normally silenced in a differentiating muscle cell. One of my dissertation aims sheds light on the silencing of CIITA in Rhabdomyosarcoma. Silencing of CIITA prevents the expression of MHC Class I and II genes. We have found that IFN-gamma signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells, but shows a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have also observed that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-gamma treatment, is blocked in both types of RMS cells. Further, our studies also impart a novel role for IFN-gamma and CIITA in inhibiting the IGF induced activation of muscle specific genes. Our data show that IFN-gamma does not block the signaling cascade of IGF. However, blocking exogenous IFN-gamma restores IGF activation of muscle specific genes. My dissertation also reveals an important role for the FACT complex in the early steps of gene activation through its histone chaperone activities that serve to open chromatin structure and facilitate transcription promoting muscle differentiation. We show that myogenin interacts with the FACT complex and the recruitment of FACT complex to muscle specific genes is dependent on myogenin. The final aim in my dissertation highlights the distinct binding profiles of the MRFs and E proteins during proliferation and differentiation. Our sequential ChIP assays show that MYOD, MYOG, and MYF5 co-occupy promoters. Taken together, my dissertation provides a comprehensive understanding of the molecular mechanisms during myogenesis and reveals the deleterious effects of chronic inflammation in skeletal muscle.

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.

Development in multicellular organisms relies on establishing and maintaining gene expression profiles that give cells identity. Transcription factors establish gene expression profiles by integrating positional, temporal, and environmental cues to regulate genes essential for a cell's identity. These signals are often short lived while the differentiated state may persist for a long time. Epigenetic factors maintain these gene expression profiles by making heritable chemical alterations to target gene chromatin to stabilize transcriptional patterns. Here we explore the evolution and function of an epigenetic regulator, the Polycomb Repressive Complex 2 (PRC2), and a transcription factor, ASYMMETRIC LEAVES 1 (AS1) , in the lower eudicot Aquilegia. PRC2 is an important and deeply conserved epigenetic regulator, which is critical to many plant developmental processes, including the regulation of major developmental transitions and lateral organ development. We find that Aquilegia has a relatively simple complement of PRC2 genes that are expressed throughout development. Contrary to findings in other plant species, two members of the Aquilegia PRC2, AqSWN and AqCLF, are not imprinted in Aquilegia endosperm. Using virusinduced gene silencing (VIGS), we determined that Aquilegia PRC2 regulates aspects of lateral organ development, including branching within the leaf and lamina expansion, along with caroteinoid production in floral organs. PRC2 targeting of several floral MADS box genes may be conserved in Aquilegia, but other known targets such as the class I KNOX gene are not. AS1 is a transcription factor that plays a conserved role in controlling differentiation and polarity of lateral organs. In species with simple leaves, AS1 promotes cell determination by suppressing the expression of the class I KNOX genes in leaf primordia and regulates abaxial-adaxial polarity in the developing leaf. However, in species with compound leaves, KNOX genes and AS1 often work together to control leaflet initiation and arrangement. In Aquilegia, AqAS1 appears to primarily contribute to proper regulation of class I KNOX genes with a more minor role in leaflet polarity and positioning. Most interestingly, these combined datasets suggest that contrary to the widely held model, class I KNOX genes are neither necessary nor sufficient for leaf complexity in Aquilegia.

Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3, leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cells in mouse embryonic and adult tissues. However, the role of PRC2 in human skin remains largely unknown. This study investigated the role of PRC2 in human epidermal homeostasis and hair growth. The expression of EZH2 was elevated in differentiating suprabasal layers of the human epidermis. Consistently, EZH1/2 expression and enzymatic activity was upregulated in differentiating primary human keratinocytes (NHEKs) in vitro. Inhibition of EZH2 and Embryonic ectoderm development (EED) in NHEKs stimulated the expression of differentiation-associated genes, therefore leading to their premature differentiation; while inhibition of EZH1/2 reduced cell proliferation and promoted apoptosis. Silencing of EZH2 in NHEKs induced complex changes in gene expression programmes, including the upregulation of terminal differentiation genes, such as Filaggrin. EZH2 expression was downregulated in aged keratinocytes accompanied with upregulation of senescence-associated genes, p16INK4A and p19INK4D, suggesting EZH2 involvement in epidermal aging. In human anagen hair follicle (HF), EZH2 was detected in stem and progenitor cells; and hair matrix keratinocytes. Silencing EZH2 in HFs accelerated anagen-catagen transition and retarded hair growth accompanied by decreased proliferation and increased apoptosis. Silencing EZH2 in outer root sheath keratinocytes resulted in upregulation of p14ARF and K15, suggesting EZH2 involvement in regulating proliferation and stem cell activity. Thus, this study demonstrates that PRC2-mediated repression is crucial for epidermal homeostasis and hair growth. Modulating the activities of PRC2 in skin might offer a new therapeutic approach for disorders of epidermal differentiation and hair growth.

No description available.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.