Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. In this study we demonstrate that mutation of Chd2 renders cells susceptible to inefficient DNA repair and genomic instability. Homozygous and heterozygous Chd2 mutant mouse embryonic fibroblast accumulates higher levels of gamma-H2AX after DNA damage. Chd2 mutant cells show inefficiency in DNA repair of DNA lesions induced by X-rays and UV irradiation as assessed by single cell gel electrophoresis assays. These cells also exhibit increased chromosomal aberrations after treatment with low doses of X-ray irradiation (2 Gy) and show increased radiosensitivity in a clonogenic survival assay. At the molecular level, endogenous CHD2 protein level is induced after exposure to X-ray radiation. In addition, we have also demonstrated in this study that CHD2 is phosphorylated after DNA damage and is a potential substrate for phosphoinositide 3-kinase-related kinases (PIKK) - ATM/ATR. Additionally, mass spectrometric analysis showed possible association of CHD2 with the paraspeckle family of proteins known to be involved in an array of cellular processes specifically in RNA processing and DNA repair. An in vivo splicing assay demonstrated that CHD2 played a role in modulation of pre-mRNA splicing event. Collectively, our findings suggest that CHD2 is a multi-functional protein working with the paraspeckle protein complex to facilitate both the pre-mRNA splicing process and the initial DNA repair process. CHD2 may also be involved in the later stages of DNA damage response pathway by influencing p53’s transcriptional activity.

Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition. We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing. By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

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La synthèse des ARN messagers est un processus hautement régulé. Pendant l’initiation de la transcription, un nombre important de protéines est recruté au niveau du promoteur des gènes, comprenant l’ARN polymérase II, les facteurs généraux de transcription comme TFIIH, des co-activateurs et des remodeleurs de la chromatine. L’assemblage du complexe de pré-initiation sur les promoteurs est suivi par leur ouverture. Les modèles acceptés à ce jour suggéraient que la transcription des gènes de classe II nécessite les activités ATPase et hélicase de la sous-unité XPB de TFIIH afin d’ouvrir le promoteur. Or nous avons observé que l’expression des ARNm s’accommode de l’absence de XPB mais nécessite son activité ATPase. Ces observations concordent avec un modèle alternatif dans lequel l’activité ATPase de XPB est utilisée pour transloquer la protéine en amont du site d’initiation et lever un blocage, imposé par la présence de XPB, de l’ouverture du promoteur. De plus, nous avons découvert un nouveau rôle de TFIIH dans le remodelage de la chromatine lors de l’initiation de la transcription. Nous avons mis en évidence un lien étroit entre TFIIH et l’histone acétyltransférase KAT2A, permettant le contrôle de la structure de la chromatine et l’expression des gènes, apportant en outre de nouvelles informations sur le Xeroderma pigmentosum combiné au syndrome de Cockayne, une maladie de la réparation avec une prédisposition au cancer. / The synthesis of messenger RNA is a highly regulated process. During transcription initiation, a large number of proteins are recruited to gene promoter including RNA polymerase II, general transcription factors like TFIIH, co-activators and chromatin remodelers. The assembly of pre-initiation complex on promoters is followed by their opening. Accepted models to date suggested that transcription of class II genes requires TFIIH XPB subunit ATPase and helicase activities to actively open the promoter. However, we have observed that mRNA expression is compatible with the absence of XPB but requires its ATPase activity. These observations are consistent with an alternative model in which the ATPase activity of XPB is used to translocate the protein upstream of the initiation site, alleviating a block, imposed by the presence of XPB, of the promoter opening. Moreover, we found a new role for TFIIH in chromatin remodeling during transcription initiation. We highlighted a tight connection between TFIIH and the histone acetyltransferase KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in combined Xeroderma Pigmentosum and Cockayne syndrome, a cancer-prone DNA repair-deficient disorder.

<div> <p>Mammalian embryos undergo a dramatic amount of epigenetic remodeling during the first week of development to establish the correct epigenetic status to support the developmental program. SWI/SNF chromatin remodeling complexes are multi-subunits complexes and utilize energy from ATP hydrolysis to modify chromatin structure non-covalently. The collection of subunits determines the identity of a given SWI/SNF chromatin-remodeling complex, directs its activity, and dictate where that complex will act. The aims of this study were to 1) determine the requirement of SNF5, a SWI/SNF core subunit found in BAF and PBAF complexes during preimplantation porcine embryo development, 2) determine the requirement of BRD7, a PBAF complex-specific subunit during preimplantation porcine embryo development, and 3) investigate the role of <i>CDH1</i>, a downstream gene regulated by ARID1A, another subunit found exclusively in BAF complexes, in cleavage stage porcine embryos. Our results indicate that the differential requirement for each subunit during early embryo development. Depletion of different subunits results in embryo arrest at distinct developmental stage. Together, our data suggest the SWI/SNF chromatin remodeling complexes are necessary for proper porcine embryo development and this requirement is associated with the composition of the complex.</p> </div> <br>

Indiana University-Purdue University Indianapolis (IUPUI) / Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.

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