P68 is a prototypical member of the DEAD box RNA helicase family. Implicated in numerous functions such as cell proliferation, cancer metastasis, transcription regulation and pre-mRNA spllicing, p68 is a multifunctional protein whose roles are still not completely understood. In the studies presented, we found that p68 was an important regulator of numerous cancer related genes. This study focuses on the cancer related genes Snail and hTERT. We show that p68 binds to the promoter and a downstream region within each gene, suggesting that p68 operates via the same mechanism with both genes. We also show that tyrosine phosphorylated p68 is the major player in transcription regulation. p68 was also discovered to recruit CREB-binding protein to the promoters of these genes as well as aid in the removal of HDAC1 from the promoters; these findings are consistent with chromatin remodeling and active transcription. Also, we found that p68 phosphorylation level correlates with the expression level of these genes. Finally, we describe other genes that are potentially regulated by p68 in the same manner, through the use of ChiP-on-chip technology.

How chromatin-remodeling complexes modulate gene networks to control organ-specific properties is not well understood. For example, Baf60c (Smarcd3) encodes a cardiac-enriched subunit of the SWI/SNF-like BAF chromatin complex, but its role in heart development is not fully understood. We found that constitutive loss of Baf60c leads to embryonic cardiac hypoplasia and pronounced cardiac dysfunction. Conditional deletion of Baf60c in cardiomyocytes resulted in postnatal dilated cardiomyopathy with impaired contractile function. Baf60c regulates a gene expression program that includes genes encoding contractile proteins, modulators of sarcomere function, and cardiac metabolic genes. Many of the genes deregulated in Baf60c null embryos are targets of the MEF2/SRF co-factor Myocardin (MYOCD). In a yeast two-hybrid screen, we identified MYOCD as a BAF60c interacting factor; we showed that BAF60c and MYOCD directly and functionally interact. We conclude that Baf60c is essential for coordinating a program of gene expression that regulates the fundamental functional properties of cardiomyocytes.

Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

DNA damage from both normal metabolic activities and environmental factors such as UV and radiation can cause as many as 1 million individual lesions to the DNA per cell per day (Lodish et al 2004). Cells respond to this continuous damage by employing many, highly efficient DNA repair mechanisms and undergo apoptosis when normal DNA repair fails. Of the many types of DNA damage that can occur, double strand breaks (DSBs) are the most toxic (Featherstone & Jackson 1999). A single unrepaired DSB is enough to induce cellular apoptosis and several mechanisms have developed to repair DSBs. The recognition, signalling and repair of DSBs involve large multi-­‐subunit complexes that bind to both the DNA and modified histone tails, which require modification of the chromatin in order to access their bind sites and function effectively (Allard et al 2004). Consequently several chromatin-­‐remodelling proteins have been implicated in DSB repair (van Attikum et al 2004, Chai et al 2005). LSH (Lymphoid specific helicase) is a putative chromatin-­‐remodelling enzyme that interacts with DNA methyltransferases and has been connected to DNA methylation (Myant & Stancheva, 2008). Knockouts of LSH or its homologues in A. thaliana and M. musculus show a reduction in DNA methylation of 60-­‐70% (Jeddeloh et al 1999, Dennis et al 2001). However in addition to this phenotype, knockout A. thaliana also have an increased sensitivity to DNA damage (Shaked et al 2006). A homologue of LSH has also been identified in S. cerevisiae, which interacts with known repair proteins (Collins et al 2007) and may be involved in DSB repair. Although the majority of Lsh-­‐/-­‐ mice die shortly after birth, 40% of the line produced by Sun et al survive and show unexplained premature aging (Sun et al 2004). As premature aging is a hallmark of increased acquisition of DNA damage there is the possibility of a conserved role for LSH in mammalian DNA damage repair. Here I show that LSH depleted mammalian cells have an increased sensitivity specifically to DSB inducing agents and show increased levels of apoptosis. Further analysis shows that cells lacking LSH repair DSBs slower, indicating a novel role for LSH in mammalian repair of DSB. I performed an in depth analysis of the DSB defects in LSH depleted cells in an attempt to elucidate the function of LSH in DSB repair. I found that LSH depleted cells can correctly recognise DSBs but recruit downstream signalling and repair factors, such as γH2AX, less efficiently. I show that reduced recruitment of downstream DSB repair factors is not accompanied by extended cell cycle checkpoint signalling. This suggests that LSH depleted cells continue through the mitosis with unrepaired DSBs, which most likely leads to apoptosis and the increased sensitivity to DSB inducing agents. These experiments also showed that recruitment of DSB signalling and repair factors is not impaired equally at all breaks, and I present a model system created to quantitatively compare individually breaks between WT and LSH depleted cells to identify DSB that require LSH for efficient repair. I also preformed an analysis of Lsh-­/-­ MEFs containing WT or catalytic null mutant LSH rescue constructs and I show that WT but not catalytic null LSH can restore efficient DSB repair. These studies identify a novel role for LSH in mammalian DSB repair and demonstrate the importance of its catalytic activity.

Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

SWI/SNF is a chromatin-remodeling complex and Brahma (BRM) is the ATPase subunit of SWI/SNF. BRM regulates transcription by remodeling the nucleosomes at promoter regions. BRM is also associated with RNA and affects pre-mRNA processing together with other SWI/SNF subunits. In this thesis, I will discuss the roles of BRM in both transcription and pre-mRNA processing. In Paper I, we showed that BRM, as well as other SWI/SNF subunits SNR1 and MOR, affects the alternative processing of a subset of pre-mRNAs, as shown by microarray analysis. This observation was validated by RNAi experiments both in Drosophila S2 cells and in vivo. In Paper II, we characterized the trans-splicing of transcripts derived from the mod(mdg4) gene. RNA interference (RNAi) and overexpression experiments revealed that BRM regulates the trans-splicing of mod(mdg4)-RX in an ATPase independent manner. In Paper III, we analyzed the expression of two BRM-target genes identified in Paper I, CG44250 and CG44251. RNAi and overexpression experiments showed that the expression levels of these two genes were affected by BRM in a manner that is independent of its ATPase activity. Transcriptome analysis further proved that BRM affects gene expression both in ATPase dependent and independent manners. In Paper IV, we showed that BRM is present at the 3’-end of two analyzed genes, CG5174 and CG2051. BRM facilitates the recruitment of the cleavage and polyadenylation machinery to the cleavage sites through protein-protein interactions that do not require the ATPase activity of BRM. Morevoer, BRM promotes the cleavage of the CG5174 and CG2051 pre-mRNAs. To sum up, SWI/SNF plays important roles not only in transcription but also in pre-mRNA processing. To regulate transcription, BRM can either act as an ATPase-dependent chromatin remodeler or in a manner that does not involve ATPase activity. Additionally, BRM interacts with RNA-binding proteins to regulate the processing of a subset of pre-mRNAs, and this function of BRM is independent of its chromatin remodeling activity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Chromatin remodeling enzymes are instrumental for neural development as evidenced by their identification as disease genes underlying human disorders characterized by intellectual-disability. In this regard, the murine Snf2h and Snf2l genes show differential expression patterns during embryonic development, with a unique pattern in the brain where Snf2h is predominant in neural progenitors, while Snf2l expression peaks at the onset of differentiation. These observations led me to investigate the role of Snf2h and Snf2l in brain development by using conditionally targeted Snf2h and Snf2l mice. I selectively ablated Snf2h expression in cortical progenitors, cerebellar progenitors, or postmitotic Purkinje neurons of the cerebellum, while Snf2l was deleted in the germline. I found that Snf2h plays diverse roles in neural progenitor expansion and postmitotic gene expression control, while Snf2l is involved in the precise timing of neural differentiation onset. Gene expression studies revealed that Snf2h and Snf2l co-modulate the FoxG1 and En1 transcription factors during cortical and cerebellar neurogenesis, respectively, to precisely control the transition from a progenitor to a differentiated neuron. Moreover, Snf2h is essential for the postmitotic neural activation of the clustered protocadherin genes, and does so by functionally interacting with the matrix-attachment region protein Satb2. My neurobehavioral studies also provided insight into how Snf2h loss in cerebellar progenitors results in cerebellar ataxia, while Snf2h loss in cortical progenitors, or in postmitotic Purkinje neurons of the cerebellum, resulted in learning and memory deficits, and hyperactive-like behavior. Molecularly, Snf2h plays an important role in linker histone H1e dynamics and higher order chromatin packaging, as evidenced by loss of chromatin ultrastructure upon Snf2h deletion in progenitor and postmitotic neurons. I further demonstrated that Snf2h loss in a neuronal cell culture model results in reduced H1e deposition, and that overexpression of human SNF2H or SNF2L upon Snf2h knockdown rescues this biochemical dysfunction. My experiments suggest that Snf2h and Snf2l are regulatory nucleosome remodeling engines that co-modulate the gene expression programs necessary for proper brain development, maturation and function.

The dynamic nature of eukaryotic chromatin enables the packaging of large amounts of genetic material in a small space. At the same time, it provides controlled access to genomic DNA for a variety of nuclear processes for example, transcription and DNA repair. The transition between open and closed chromatin states is largely governed by ATP-dependent chromatin remodeling complexes, which operate on nucleosomes in concert, to modulate chromatin structure and composition. Exchange of the canonical and variant forms of histones in nucleosomes, and altering the spacing between consecutive nucleosomes, are two major ways which regulate chromatin-based processes and chromatin higher-order organization. The evolutionarily conserved INO80 and ISW1a complexes mediate these two aspects of nucleosome remodeling, respectively. Despite sharing conserved domain architecture of the core remodeling machinery, chromatin remodelers differ significantly in their modes of interaction with nucleosomes, and how they alter histone-DNA contacts. In this study, we have used a site-specific photocrosslinking approach coupled with peptide mapping to determine the interactions of subunits and domains of the S. cerevisiae INO80 and ISW1a complexes with nucleosomes. We find that specific interactions of remodelers with different regions of the nucleosome largely dictate their specialized functions and mechanisms. The ATP-dependent helicase-like (ATPase) domains of remodelers belonging to the ISWI and SWI/SNF families translocate along DNA close to the center of nucleosomes in order to mobilize, space or disassemble nucleosomes. In contrast, we observed that INO80 has a strikingly distinct mechanism, which is different even from its paralog SWR1. INO80 mobilizes nucleosomes as well as catalyzes the exchange of histone variant H2A.Z for the canonical histone H2A, while SWR1 mediates the reverse exchange of H2A for H2A.Z, without being able to mobilize nucleosomes. We have found that INO80, in order to promote H2A-H2B dimer exchange, translocates along DNA at the H2A-H2B interface close to the edge of nucleosomes and persistently displace DNA from H2A-H2B. Blocking either DNA translocation or the accumulation of DNA torsions close to the edge of the nucleosome interferes with this dimer exchange by INO80. SWR1 and other SWI/SNF and ISWI remodeling complexes translocate along DNA at the H3-H4 interface and do not persistently displace DNA from the histone octamer as does INO80. This study shows for the first time an ATP-dependent chromatin remodeler that invades nucleosomes at the DNA entry site instead of the center − a more logical approach for the displacement of H2A-H2B. We also investigated nucleosomal DNA interactions of other INO80 subunits and domains to understand the architecture of INO80 bound to nucleosomes. We found that the HSA (helicase-SANT-associated) domain of Ino80 along with actin-related protein (Arp) subunits Arp8 and Arp4 bind to the extranucleosomal DNA and is potentially involved in a coupling mechanism with the ATPase domain to regulate its activity. We also mapped the DNA binding regions of Arp8 and Arp4, which might be involved in recruiting INO80 to genomic sites. The ISWI remodeler ISW1a regulates the distance (spacing) between nucleosomes in an array by simultaneously interacting with two nucleosomes and directionally remodels one of them. We mapped DNA interactions of ISW1a subunits in mono- and di-nucleosomes. Our results show that the catalytic Isw1 subunit specifically interacts with the region of DNA translocation and DNA entry site of the asymmetrically positioned nucleosome in a di-nucleosome, which is preferentially mobilized. In contrast, the Ioc3 subunit interacts extensively with the linker DNA as well as the extranucleosomal DNA of the un-remodeled nucleosome. This bias in nucleosomal DNA interactions of ISW1a enables directional remodeling, which reveals the molecular basis of nucleosome spacing. We have identified a novel domain within the non-catalytic Ioc3 subunit of ISW1a that regulates nucleosome spacing. We found that when this domain is deleted, the catalytic Isw1 subunit loses its specificity and interacts with both the nucleosomes of a di-nucleosome substrate. This is consistent with the domain-deleted ISW1a mobilizing both nucleosomes efficiently, leading to the loss of its nucleosome spacing activity. In summary, this dissertation explores how different remodeling complexes have customized and regulated modes of nucleosome interaction in order to accomplish specialized remodeling outcomes. INO80 places its ATPase domain for translocation at the H2A-H2B dimer interface and persistently displaces DNA from its surface to promote H2A.Z exchange. Nucleosome spacing by ISW1a requires the catalytic Isw1 subunit to engage with and reposition one out of two consecutive nucleosomes in an array, while the Ioc3 subunit likely monitors the distance between them.