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AN ABSTRACT OF THE DISSERTATION OF SOLOMON G. HAILU, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry, presented on August 22, 2017, at Southern Illinois University, School of Medicine. TITLE: DISTINCT GENOME WIDE FUNCTIONS OF CHROMATIN REMODELERS IN NUCLEOSOME ORGANIZATION AND TRANSCRIPTION REGULATION MAJOR PROFESSOR: Dr. Blaine Bartholomew Chromatin remodelers are conserved from yeast to humans and are the gatekeepers of chromatin. They regulate transcription by occluding or exposing DNA regulatory elements globally. They are crucial for DNA processes such as DNA replication, repair and recombination. In addition, they are critical in developmental processes and differentiation. Chromatin remodelers are categorized into several families based on their conserved ATPase domain, an essential component required for their DNA translocation ability. In this study, we investigated the role yeast ISWI and SWI/SNF family of chromatin remodelers play on nucleosome rearrangement and transcription regulation by targeted mutagenesis of domains in accessory subunits and at the C-terminus of the catalytic subunit. All members of the ISWI family (ISW1a, ISW1b, ISW2) share a conserved C-terminal HAND, SANT and SLIDE domains, which are important for sensing linker DNA. We find an auto-regulation of ISWI complexes by the SLIDE domain, independent of the histone H4 Nterminal tail. Our protein-protein chemical crosslinking and mass spectrometry (CX-MS) analysis indicate that the SLIDE domain regulates the ATPase core through N terminal domains of the accessory subunit Itc1. Moreover, we show that the accessory subunits of ISWI modulate the ATPase activity and specificity of ISWI complexes. The DNA sensing ability of the SLIDE domain is required for the in vivo nucleosome spacing and transcription regulation by ISWI. We find that while ISW2 primarily regulates transcription at the 5’ end of genes, ISW1a is important in transcription elongation by rearranging nucleosomes starting at the +2 nucleosome and through the rest of the body of genes towards the 3’ end. ISW1b on the other hand rearrange nucleosomes in the gene body to facilitate suppression of cryptic transcription. For the first time, we show the potential division of labor between ISW1a and ISW1b during transcription elongation. On the other hand, SWI/SNF chromatin remodelers are essential epigenetic factors that are frequently mutated in cancer and neurological disorders. They harbor a C-terminal SnAC and AT hook domains that positively regulate their DNA dependent ATPase activity and nucleosome mobilizing capabilities. By deleting the AT hook motifs, we have identified the role of SWI/SNF in organizing the -1 and +1 nucleosomes at transcription start sites flanking the nucleosome free region (NFR). Our RNA-seq analysis shows SWI/SNF positively regulates the bi-directional transcription of non-coding RNA (ncRNA) which are activated when the AT hook motifs are deleted. Moreover, AT hooks regulate such activities of SWI/SNF through direct protein-protein interactions with the ATPase core as evidenced by our chemical crosslinking and mass spectrometry (CX-MS) analysis.

There is an increasing evidence that alterations in chromatin remodeling play an important role in tumorigenesis. The SWI/SNF chromatin remodeling complexes contribute to the regulation of gene expression by altering the local chromatin structure. Depending on the context, they can act as either transcriptional activators or repressors. All SWI/SNF subcomplexes contain one of two ATPase subunits, Brm (Brahma) or Brg1 (Brahma related gene 1), which provide the energy for remodeling. Malignant melanoma is an aggressive cancer and is known for its notorious resistance to conventional anticancer therapies. MITF (microphthalmia-associated transcription factor) plays an essential role in melanoma biology and is placed on the central crossroad in the regulation of melanocyte development, differentation, maintenance of lineage identity, and survival of both normal and malignant melanocytes. Our results show that the active SWI/SNF complex is strictly required for the expression of MITF. This complex is also required for expression of some transcriptional MITF targets. The survival of melanoma cells is absolutely dependent on functional SWI/SNF complex and all subunits of this complex are expressed at high levels in melanoma cell lines. Primarily, Brg1-containing subcomplexes are more important for MITF...

The packaging of genomic DNA into nucleosomes creates a barrier to transcription which can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex. The SWI/SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI/SNF complex does not possess intrinsic DNA binding ability, and therefore its recruitment to target loci requires interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcription factors (Cdx1, Cdx2 and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI/SNF complex and make direct contact with Brg1, a catalytic member of SWI/SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at certain of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI/SNF chromatin remodeling activity.

Coordination of gene expression within the cell requires the integrated actions of various multi-protein, gene regulatory complexes. The Mediator and BRG1 Associate Factor (BAF) complexes are large, dynamic regulatory cofactors (COF) that are made up of multiple different submodules, and play key roles in regulating gene expression. Gene-specific regulation requires that transcription factors (TFs) recruit these COF complexes to gene promoters. How separate subdomains in each complex interact with distinct sets of TFs in each cell remains an important question. In this study, to address this question, we sought to apply the nuclear extract protein-binding microarray (nextPBM) technology being developed in our lab to study interactions between TFs and subunits of the Mediator and BAF complexes. To facilitate this, we cloned, expressed and purified subdomains of proteins from the Mediator and BAF complexes. We then used the nextPBM technology to study the interactions of our subdomains with TFs in human macrophages. We identified several new interactions with TFs, and demonstrate the utility of this approach to student TF-COF interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21645号 / 医博第4451号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 羽賀 博典, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / There are many clinical diseases involving abnormal differentiation of smooth muscle, such as atherosclerosis, hypertension and asthma. In these diseases, one important pathological process is the disruption of the balance between differentiation and proliferation of smooth muscle cells. Serum Response Factor (SRF) has been shown to be a key regulator of smooth muscle differentiation, proliferation and migration through its interaction with various accessory proteins. Myocardin Related Transcrition Factors (MRTFs) are important co-activators of SRF that induce smooth muscle differentiation. Elucidating the mechanism of how MRTFs and SRF discriminate between genes required to regulate smooth muscle differentiation and those regulating proliferation will be a significant step toward finding a cure for these diseases. We hypothesized that SWI/SNF ATPdependent chromatin remodeling complexes, containing Brg1 and Brm, may play a role in this process. Results from western blotting and quantitative reverse transcription - polymerase chain reaction (qRT-PCR) analysis demonstrated that expression of dominant negative Brg1 or knockdown of Brg1 with silence ribonucleic acid (siRNA) attenuated expression of SRF/MRTF dependent smooth muscle-specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and that Brg1 directly binds MRTFs, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated SRF binding and the ability of MRTFs to increase SRF binding to the promoters of smooth muscle-specific genes, but not proliferation-related early response genes. The above data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in differentially regulating expression of SRF/MRTF-dependent genes through controlling the accessibility of SRF/MRTF to their target gene promoters. To examine the role of SWI/SNF in smooth muscle cells in vivo, we have generated mice harboring a smooth muscle-specific knockout of Brg1. Preliminary analysis of these mice revealed defects in gastrointestinal (GI) development, including a significantly shorter gut in Brg1 knockout mice. These data suggest that Brg1-containing SWI/SNF complexes play an important role in the development of the GI tract.

There is an increasing evidence that alterations in chromatin remodeling play an important role in tumorigenesis. The SWI/SNF chromatin remodeling complexes contribute to the regulation of gene expression by altering the local chromatin structure. Depending on the context, they can act as either transcriptional activators or repressors. All SWI/SNF subcomplexes contain one of two ATPase subunits, Brm (Brahma) or Brg1 (Brahma related gene 1), which provide the energy for remodeling. Malignant melanoma is an aggressive cancer and is known for its notorious resistance to conventional anticancer therapies. MITF (microphthalmia-associated transcription factor) plays an essential role in melanoma biology and is placed on the central crossroad in the regulation of melanocyte development, differentation, maintenance of lineage identity, and survival of both normal and malignant melanocytes. Our results show that the active SWI/SNF complex is strictly required for the expression of MITF. This complex is also required for expression of some transcriptional MITF targets. The survival of melanoma cells is absolutely dependent on functional SWI/SNF complex and all subunits of this complex are expressed at high levels in melanoma cell lines. Primarily, Brg1-containing subcomplexes are more important for MITF...

Indiana University-Purdue University Indianapolis (IUPUI) / Understanding β-cell development and function is essential for generating more effective treatment options for individuals with diabetes. A key player in pancreatogenesis, islet development, and mature β-cell function is the Pdx1 transcription factor (TF). Pdx1 activity is modulated through interactions with various coregulators, including the Swi/Snf chromatin remodeling and Nucleosome Remodeling and Deacetylase (NuRD) complexes. Loss of one Swi/Snf ATPase subunit, Brg1, in early pancreatogenesis reduces final pancreas mass, and β-cell-specific deletion of both subunits, Brg1 and Brm, leads to glucose intolerance and loss of insulin production in the β-cell. Here, we hypothesized Swi/Snf governs endocrine progenitor cell development and postnatal islet function. To test this, we generated conditional murine knockouts of Brg1 (Brg1Δendo;Brm+/-), Brm (Brg1Δendo/+;Brm-/-), or both subunits (DKOΔendo) during endocrine cell development. No DKOΔendo mice were recovered at weaning, and loss of Brg1 but not Brm led to severe glucose intolerance, ad-lib fed hyperglycemia, and reduced insulin levels by four weeks of age. Brg1Δendo;Brm+/- mice had fewer islets and compromised insulin secretion. Together, these data suggest that loss of Brg1 during endocrine cell development has negative impacts on postnatal islet function, with loss of both Brg1 and Brm being early postnatal lethal. Pdx1 has been shown to also interact with the Chd4 helicase subunit of the NuRD complex. Here, we demonstrate Pdx1:Chd4 interactions are increased under stimulatory conditions and hypothesize that Chd4 modulates expression of Pdx1-bound genes critical for β-cell function. To test this, we generated a tamoxifen inducible, β-cell-specific Chd4 knockout mouse model (Chd4Δβ). Four weeks following Chd4 removal, Chd4Δβ mutants were glucose intolerant with severe insulin secretion defects. Additionally, Chd4Δβ islets contained fewer mature insulin granules and secreted more proinsulin. RNA-sequencing from Chd4Δβ β-cells identified numerous upregulated (eg Hk2, Mycl) and downregulated genes (eg MafA, Chga, Chgb, Slc2a2). Through ATAC-sequencing, we discovered several differentially accessible genomic regions, including Chd4-bound and Pdx1-controlled MafA Region 3, which had reduced accessibility in Chd4Δβ β-cells. Lastly, we demonstrate that CHD4 impacts human β-cell function and PDX1:CHD4 interactions were reduced in human donor β-cells with type 2 diabetes, demonstrating loss of these interactions is a significant feature of diabetes pathogenesis.

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