The compact structure of the eukaryotic genome dictates the accessibility to genes, and therefore adds an additional layer of regulation for gene expression. A specialized class of proteins called "chromatin remodelers" facilitates this process in the cell. The imitation switch (ISWI) subfamily of chromatin remodelers is a well studied class of proteins affecting gene expression. Its member ISW2 was recently shown to behave differently from other chromatin remodeling proteins. For instance, the ISW2 complex has been shown to be stimulated by ~5-6 fold in its ATPase activity when bound to a nucleosome rather than to a DNA molecule. Nucleosome remodeling by ISW2 has even been shown to depend on the N-terminal tail of histone H4 and therefore, the octamer of a nucleosome might be playing a significant role in nucleosome remodeling by the ISW2 complex. The aim in this investigation was to delineate the protein-protein interactions that the ISW2 complex establishes with the octamer upon binding to a nucleosome. Several histones with unique cysteines engineered at specific positions were refolded with other wild type histones to produce histone octamers with a single cysteine in one of the four histones. Based on previous reports from site-specific DNA photoaffinity cross-linking and hydroxyl-radical footprinting experiments, it was inferred that the SHL2, entry-exit position and the extranucleosomal linker DNA were contacted by the ISW2 complex on a nucleosome1. Considering these critical regions and taking into account the accessibility of residues in close proximity to these regions, five discrete positions were selected on the octamer surface for scanning the face of the nucleosome. The five sites were residues 19, 89 and 113 of histone H2A (H2A-19, H2A-89, H2A-113), residue 109 of histone H2B (H2B-109), and residue 80 of histone H3 (H3-80). Initially, octamers with cysteine at one position in one of the four histone proteins were reconstituted on a 0N70 DNA (where `N' represents the 147bp 601 DNA sequence, and the lengths of the linker DNA is represented by the numbers 0 and 70). Nucleosomes were modified with the protein-protein cross-linker- MAB (methanethiosulfonate-tetrafluorophenylazide-biotin) reagent. This reagent makes a disulfide bond with the cysteines in the octamer of a nucleosome. The MAB reagent had a distance of ~11.1Aº between its photoreactive tetrafluorophenylazide group and the disulfide forming methanethiosulfonate group. The ISW2 complex was bound to the modified mononucleosomes and cross-linked by irradiating with UV-light. Under reducing conditions the biotin moiety was transferred from the nucleosome to the ISW2 complex. The subunit of the remodeler that was photocross-linked at these positions on the nucleosome was blotted onto nitrocellulose and detected with streptavidin conjugated to horseradish peroxidase (HRP). The catalytic subunit-Isw2 of the ISW2 complex was cross-linked at all five positions but with the following order of intensity from most to least- H2A-89, H3-80, H2B-109, H2A-19, and H2A113. Mass spectrometry was used to decipher these residues, motifs or domains of the catalytic subunit- Isw2 that interacted with the octamer at each position. The ISW2 complex was digested with trypsin, and the biotinylated peptides were enriched using monomeric avidin affinity chromatography. The largest subunit of the ISW2 complex, Itc1, did not get cross-linked at any of the positions.

Background: The higher-order structure of chromatin changes in response to extracellular and environmental signals. We observed nuclear morphological changes in biopsied cancer tissue after chemotherapy. Since chromatin structure dictates gene expression, and therefore function, further investigation of this phenomenon may increase our understanding of therapeutic responses. I hypothesised that nuclear morphological changes in cancer in response to DNA-damage by chemotherapy are mediated by histone deacetylases (de Ruijter, van Gennip et al.). Methods: Ovarian cancer cell lines PEO1/PEO4 (platinum sensitive/resistant) were selected as in vitro models, and primary ovarian cancer xenografts OV1002 and HOX424 as in vivo models. Expression levels of HDACs, heterochromatin protein 1 (HP1), and DNA damage response (DDR) proteins were profiled by Western blot analysis after treatment with cisplatin. Immunofluorescence imaging was undertaken using confocal microscopy, and nuclear texture and γH2AX foci were measured in Image J. Cell cycle and apoptosis were detected by flow cytometry. Thirty eight different ovarian cancer biopsies and 175 xenograft samples were assessed for HDAC and HP1 expression in response to chemotherapy by quantitative immunofluorescence. HDAC2 expression was modulated by interfering RNAs (siRNA). Results: I demonstrated nuclear morphological changes in clinical tumours, xenografts, and cell lines in response to platinum chemotherapy by robust measurement of nuclear texture. Expression of HDAC2 increased in PEO1 cells treated with cisplatin at 24h, and this was accompanied by high expression of HP1s. Expression of components of both HDACs and DDR pathways (pBRCA1, γH2AX, pATM, pATR) showed time dependent changes after cisplatin treatment. Knockdown of HDAC2 reduced the expression of HP1, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DDR induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, I observed reversible alteration of chromatin patterns during cisplatin treatment to some degree. In clinical ovarian cancer specimens, expression of HDAC4, HDAC8 and HP1γ significantly increased after chemotherapy in sensitive patients, with enhanced heterogeneity in chromatin pattern. HDAC2, HDAC8, and HP1 expression were also increased after carboplatin treatment in carboplatin-sensitive xenografts. Conclusion: These results demonstrate alterations in nuclear morphology after chemotherapy, and implicate HDACs in having a role in higher order chromatin changes and in cellular DNA damage responses in ovarian cancer both in vitro and in vivo.

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

Chromatin remodelers are ATP-dependent multisubunit assemblies that regulate transcription and other processes by altering DNA-histone contacts. The mechanism of action is based on the transduction of energy released by ATP hydrolysis to translocation on DNA and ultimately the movement of histones in cis or trans. Though the critical ATP burning and translocation activities are fulfilled by a conserved ATPase domain in the catalytic subunit, there are accessory domains and subunits that are speculated to regulate these activities. Important questions in the field center around the identification of these domains and subunits, whether they affect complex formation, substrate affinity or a critical step in remodeling. If they do affect remodeling, what is the structural basis of the regulatory activity. In this study, these questions have been addressed using the prototype remodeler SWI/SNF from budding yeast. ySWI/SNF is a 12 subunit complex that includes the catalytic subunit Swi2/Snf2. It affects 6% of the yeast genome being primarily involved in gene activation. We employed a systematic protein or domain deletion strategy and characterized the mutant complexes in vitro and in vivo. A key finding was that SWI/SNF is organized in distinct structural modules and that the Snf2 module regulates most of its activities. Snf2 is a central subunit in this module and the function of conserved regions within Snf2 were studied. The N terminus preceding the HSA and ATPase domain has three major roles - complex assembly, recruitment and regulation of catalytic activity. A novel SnAC domain located C terminal to ATPase domain was identified to play critical role in coupling ATP hydrolysis to nucleosome movement by acting as a histone anchor. Finally the tandem AT-hooks between SnAC and bromodomain serve as DNA binding domains but also affect ATPase activity and nucleosome mobilization independent of its binding activity. Taken together, this study provides a comprehensive overview of the function of regulatory domains in SWI/SNF.

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

INO80, a member of the multi-subunit SWI2/SNF2 superfamily, is involved in transcription regulation, DNA repair and replication. Not much is known about its substrate specificity and remodeling mechanism or how it differs in comparison to SWI/SNF or ISWI. Site-directed mapping of histone-DNA contacts showed that INO80 generally remodels mononucleosomes by moving them to the center of DNA. The length of extranucleosomal DNA was found to play an important role in nucleosome binding as well as remodeling by INO80 much like ISW2 and ISW1a. INO80 preferentially binds to nucleosomes containing >20bp of extranucleosomal DNA. Similarly, INO80 remodeling of mononucleosomes with different lengths of extranucleosomal DNA showed that at least 33bp of extranucleosomal DNA on one side of the nucleosome was required for initiation of remodeling. These data suggest that INO80 behaves much like ISW2 and ISW1a complexes based on their requirement for extranucleosomal DNA. INO80 does not unravel or displace nucleosomes like SWI/SNF. There are several key aspects of how INO80 interacts with and remodels nucleosomes that are quite distinct from SWI/SNF, ISW2, and ISW1a. Previously SWI/SNF and ISW2 were shown to initiate nucleosome movement by translocating along nucleosomal DNA two helical turns from the dyad axis. Nucleosome movement by INO80 instead requires translocation by the complex along nucleosomal DNA near the entry/exit site at the dimer-tetramer interface. Sliding interference of INO80 by the presence of nicks indicated that torsional strain at the site of translocation is required for nucleosome mobilization by INO80. Hydroxyl radical footprinting of the INO80-nucleosome complex shows found that INO80 interactioninteracts with extranucleosomal DNA at, the entry-exit site and to lesser extent at the dyad axis, but it lacks the protection found indoes not contact 2 helical turns from the dyad like ISW2 and SWI/SNF at two helical turns from the dyad axis as determined by photoaffinity cross-linking studies. The catalytic subunit (Ino80) rather than being found associated 2 helical turns from the dyad, was bound to extranucleosomal DNA and nucleosomal DNA near the entry-exit site. Other subunits (Arp8p, Arp5p and Nhp10) were also found to be contacting both nucleosomal and extranucleosomal DNA. Site-specific histone cross-linking studies revealed that Ino80, Arp5 and Arp4 interact extensively with the histone dimer of the nucleosome in comparison to H3-H4 tetramer. Although N-terminal histone tails are often important for chromatin remodeling, INO80 shows no requirement of histone tails for its nucleosome binding and mobilizing activities. The deviation of INO80 from the canonical model of how ATP-dependent remodelers interact and mobilize nucleosome is apparently due to its unique role as a member of the remodeling complexes that promote the exchange of H2A/H2B dimer from core nucleosome particle.

C-terminal Binding Protein (CtBP) is a transcriptional co-repressor that plays an important role in mammalian development and tumorigenesis. CtBP is known to interact with Ikaros, an important transcriptional regulator in the pituitary; however CtBP itself has not been examined in this gland. I examined the role of CtBP in pituitary cell growth and survival. Compared to control pituitary GH4 cells, CtBP1-deficient cells exhibit reduced proliferation and de-regulation of genes involved in cell cycle and growth factor signaling. CtBP1-deficient cells were more susceptible to hypoxia-induced apoptosis and showed a reduction in hypoxia-induced Ikaros expression. Interactions between CtBP and Ikaros isoforms were demonstrated in pituitary tumor cell lines. CtBP and Ikaros also bound a common region of the previously characterized Ikaros target, the LDL-R promoter. These results identify oncogenic properties of CtBP1 in the pituitary and set the groundwork for future studies into regulatory roles of CtBP and Ikaros in the pituitary.

C-terminal Binding Protein (CtBP) is a transcriptional co-repressor that plays an important role in mammalian development and tumorigenesis. CtBP is known to interact with Ikaros, an important transcriptional regulator in the pituitary; however CtBP itself has not been examined in this gland. I examined the role of CtBP in pituitary cell growth and survival. Compared to control pituitary GH4 cells, CtBP1-deficient cells exhibit reduced proliferation and de-regulation of genes involved in cell cycle and growth factor signaling. CtBP1-deficient cells were more susceptible to hypoxia-induced apoptosis and showed a reduction in hypoxia-induced Ikaros expression. Interactions between CtBP and Ikaros isoforms were demonstrated in pituitary tumor cell lines. CtBP and Ikaros also bound a common region of the previously characterized Ikaros target, the LDL-R promoter. These results identify oncogenic properties of CtBP1 in the pituitary and set the groundwork for future studies into regulatory roles of CtBP and Ikaros in the pituitary.