Chromatin Remodeling Machinery Research Articles

Dissecting Musashi1 role in chromatin remodeling and its consequences to DNA repair and radio‐resistance

Musashi1 (Msi1) is an evolutionarily conserved RBP with well establish roles in neuronal stem cell self‐renewal and differentiation. In the context of tumorigenesis, Msi1 has been described to be highly expressed in gliomas, medulloblastoma, lung, colon and breast cancers and linked to a poor prognosis. Its expression is required for tumor maintenance and possibly initiation. We recently established that a decrease in Msi1 expression makes glioblastoma cells more radio‐sensitive: Msi1 KD cells subjected to ionizing radiation showed reduced proliferation as evident from colony formation assays; moreover, an increase in DNA damage was observed via comet assay, γ‐H2AX expression levels and 53BP1 ionizing radiation induced foci.Msi1 has been always defined as a translation regulator. However, our recent data shows that Msi1 is often present in the nucleus of tumor samples and cell lines. Moreover, CLIP assays identified a large number of Msi1 interacting sites in intronic regions. We suggest then that Msi1 displays additional regulatory functions in the nucleus that could equally contribute to its role in tumorigenesis. Supporting this idea, among Msi1′s protein partners identified by yeast two‐hybrid and mass spectrometry are several chromatin remodeling proteins including Ino80C. The Ino80 complex has been associated with transcription factors involved in cell proliferation, differentiation and embryonic development. Moreover, Ino80 interacts directly with γH2AX during DNA double‐strand break repair. We are currently conducting experiments to explore the hypothesis that Msi1 influences radio‐resistance and DSB repair via its association with the chromatin remodeling machinery.

The Zinc Finger Transcription Factor Mds1‐Evi1 Forms a Novel Protein Complex in MLL leukemia

The dual transcription factor‐methyltransferase Mds1‐Evi1 is frequently overexpressed in high‐risk leukemias. Mds1‐Evi1 contributes to leukemias induced by the mixed lineage leukemia (MLL) fusion protein MLL‐AF9, although the mechanism is unknown. MLL contains chromatin‐targeting and histone lysine methyltransferase domains, and fusion to AF9 can cause aberrant protein recruitment to target genes. Although Mds1‐Evi1 contains a methyltransferase domain, it does not appear to act on histones. Therefore, we are interested in how protein‐protein interactions involving Mds1‐Evi1 contribute to leukemogenesis. Many known interactors of MLL and Mds1‐Evi1 include chromatin modifying enzymes, suggesting that coordination between epigenetic modulators is key in leukemia pathogenesis.Here, we report the biochemical purification of a novel Mds1‐Evi1‐containing protein complex using a unique murine model of leukemia. To isolate the complex, we induced acute MLL‐AF9 leukemia in mice containing a tandem affinity purification (TAP) tag knocked into the endogenous Mds1‐Evi1 gene. Leukemic spleen tissue from these mice was used to purify the TAP‐tagged protein complex. Proteomic analysis revealed several members of the SWI/SNF chromatin remodeling complex, which is involved in epigenetic control of a variety of gene programs. Association between Mds1‐Evi1 and SWI/SNF may allow the activation of cell‐cycle and differentiation programs, contributing to transformation. SWI/SNF also coordinates with MLL fusion proteins to activate the Hox family of leukemia oncogenes. Our results provide a link between Mds1‐Evi1 and the chromatin remodeling machinery, where abnormal coordination of Mds1‐Evi1, MLL‐AF9 and SWI/SNF may lead to gene activation and the development of leukemia.

How to make neurons--thoughts on the molecular logic of neurogenesis in the central nervous system.

Neuronal differentiation relies on a set of interconnected molecular events to achieve the differentiation of pan-neuronal hallmarks, together with neuronal subtype-specific features. Here, we propose a conceptual framework for these events, based on recent findings. This framework encompasses a dimension in time during development, progressing from early master regulators to later expressed effector genes and terminal selector genes. As a horizontal intersection, we propose the action of permissive fate determinants that are critical in allowing progression through the above transcriptional phases. Typically, these are widely expressed and often interact with the chromatin remodeling machinery. We conclude by discussing this model in the context of the direct fate conversion of various somatic cells into neurons.

Role of Brg1 in progression of esophageal squamous cell carcinoma.

Epigenetic regulation of gene expression can be carried out through chromatin remodeling enzymes such as SWI/SNF. Brg1 also known as SMARCA4 is a catalytic subunit of SWI/SNF, which is necessary for MMPs expression. Matrix metalloproteinases (MMPs) are known as important player enzymes during tumor progression and metastasis. Aberrant epigenetic modification of chromatin should be precisely clarified to reveal probable unknown pathways in ESCC progression. Probable role of Brg1 in ESCC tumorigenesis and metastasis was studied through the assessment of Brg1 mRNA expression in KYSE30, and further evaluation about the biology of Brg1 was performed through the Brg1 silencing. Level of Brg1 mRNA expression in KYSE30 was compared to normal tissues using the real time polymerase chain reaction (PCR). Moreover, KYSE30 cells were transfected with Brg1-siRNA to silence the Brg1. Our results showed for the first time that Brg1 mRNA expression was increased in KYSE30 cell line (ESCC cell line) compared with normal esophageal tissue of ESCC patients. Rate of transfection in KYSE30 was also between 40 to 50%, using the pSilencer-Brg1shRNA (1:1 ratio). Our data indicated that chromatin remodeling machinery is a novel aspect in tumor biology of ESCC, and overexpression of Brg1 as an important member of SWI/SNF might be involved in the migration and invasion of ESCC tumoral cells.

42IN - Molecular Classification of Kidney Cancer

ABSTRACT Insight into the somatic genetics of clear cell renal cell carcinoma (ccRCC) has seen considerable advance in the past few years. Leveraging DNA sequencing approaches we have now begun to elaborate the picture of ccRCC, expanding from that dominated by VHL mutation into a more nuanced and complex view of this disease. Perhaps most striking is the contribution of mutations in the chromatin machinery. We and others have documented recurrent, frequent mutations in histone demethylases/methylases (SETD2, JARID1C, KDM6A) and a major role of chromatin remodeling machinery mutations (PBRM1, BAP1). Further, we have demonstrated marked intra-tumor heterogeneity for this disease, lending further insight into its development. This talk will provide an overview of these key insights and put them into context of a more comprehensive view of ccRCC. Disclosure: The author has declared no conflicts of interest.

Chromosome positioning from activity-based segregation

Chromosomes within eukaryotic cell nuclei at interphase are not positioned at random, since gene-rich chromosomes are predominantly found towards the interior of the cell nucleus across a number of cell types. The physical mechanisms that could drive and maintain the spatial segregation of chromosomes based on gene density are unknown. Here, we identify a mechanism for such segregation, showing that the territorial organization of chromosomes, another central feature of nuclear organization, emerges naturally from our model. Our computer simulations indicate that gene density-dependent radial segregation of chromosomes arises as a robust consequence of differences in non-equilibrium activity across chromosomes. Arguing that such differences originate in the inhomogeneous distribution of ATP-dependent chromatin remodeling and transcription machinery on each chromosome, we show that a variety of non-random positional distributions emerge through the interplay of such activity, nuclear shape and specific interactions of chromosomes with the nuclear envelope. Results from our model are in reasonable agreement with experimental data and we make a number of predictions that can be tested in experiments.

Conformational Allostery in Nuclear Receptor/Coregulator Transcriptional Complexes

By recruiting chromatin remodeling machinery to promoter regions of target genes, nuclear receptors (NRs) act as ligand-dependent transcriptional protein scaffolds, exerting powerful influences on all aspects of physiology. The critical mediators that bridge NRs to the transcriptional machinery are called NR coregulators. NR coregulators are broadly classified into coactivators or corepressors depending on their ability to promote or repress gene transcription. Coactivators contain a cluster of LXXLL motifs in a flexible/disordered region called the Receptor Interaction Domain (RID) that mediates their interactions with NRs. Because there is very little atomic level structural information on disordered coactivator RIDs, the physical mechanism driving the interaction between coregulator RIDs and NR transcriptional complexes is poorly understood. We are studying the interaction of Steroid Receptor Coactivator-2 (SRC-2) with the PPARγ-RXR transcriptional complex as a model system for understanding NR/coregulator interactions on the atomic level. SRC-2 is a critical coactivator in the PPARγ-driven differentiation of mesenchymal stem cells into fat cells, a pathway that is a target for therapeutic intervention of metabolic syndrome and type 2 diabetes. Using biochemical, biophysical and solution NMR approaches, we have begun to probe the interaction of the SRC-2 RID with the full-length PPARγ-RXR complex bound to DNA (∼150 kDa complex). Our studies reveal that the binding of DNA to PPARγ-RXR imparts a long-range conformational change that impacts the binding and conformation of SRC-2 RID. Importantly, our work extends the recent proposition that DNA can act as an allosteric effector of NR activity, indicating that DNA binding can affect the conformation of a coregulator bound to a NR transcriptional complex.

Abstract B095: CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression

Abstract CARM1 belongs to the type I protein arginine (R) methyltransferase (PRMT) family and asymmetrically di-methylates proteins on arginines. Because CARM1 is highly expressed in high grade breast tumors and was shown to transactivate cancer-relevant transcription factors including ERa, NF-kb, p53 and E2F1, it has been strongly implicated in tumorigenesis. To fully characterize functions of CARM1 in breast cancer, we knocked-out CARM1 from several breast cancer cell lines using Zinc-Finger Nuclease technology. Western blot using an asymmetric di-methylated R antibody revealed striking differences between CARM1 KO and parental MCF7 cell lysates, indicating that CARM1 methylates a significant number of substrates in breast cancer cells. BAF155, a core subunit of SWI/SNF chromatin remodeling complex, was identified as a CARM1 substrate by mass spectrometry. Further biochemical analyses validated that BAF155 was methylated by CARM1 in vivo and in vitro and methylation was mapped to a single site, R1064. We next generated BAF155R1064K knock-in MCF7 and MDA-mb-231 cells and showed that methylation of BAF155 is essential for breast cancer cell growth, colony formation and migration. Further, we developed an antibody that selectively recognized methylated form of BAF155 (me-BAF155) and observed strong nuclear me-BAF155 staining in invasive ductal carcinoma but not in the normal breast tissue. Importantly, me-BAF155 immunostaining positively correlates with tumor stage and metastasis. To understand the molecular mechanism by which BAF155 methylation affects tumorigenesis, we performed ChIP-seq using ChIP-validated BAF155 antibody in the BAF155WT or BAF155R1064K knock-in MCF7 cells. Interestingly, C-Myc pathway genes were only identified in WT but not mutant BAF155 regulated categories. Individual ChIP assays validated differential binding of BAF155WT or BAF155R1064K to specific target genes and importantly, the mRNA levels of these genes were affected correspondingly. This result suggests that BAF155 methylation affects gene expression by altering genomic association of BAF155, and perhaps the SWI/SNF complex, to chromatin regions. Collectively, our study revealed novel mechanisms by which CARM1 perturbs the chromatin remodeling machinery to enhance tumor progression. Citation Format: Lu Wang. CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B095.

Proteomics identifies ubiquitin–proteasome targets and new roles for chromatin-remodeling in the Arabidopsis response to phosphate starvation

In order to identify new regulators of the phosphate (Pi) starvation signaling pathway in plants, we analyzed variation in the abundance of nuclear-enriched proteins isolated from Arabidopsis roots that depends on Pi supply. We used 2-D fluorescence difference gel electrophoresis and MALDI-TOF/TOF techniques for proteome separation, visualization and relative protein abundance quantification and identification. Pi-controlled proteins identified in our analysis included components of the chromatin remodeling, DNA replication, and mRNA splicing machineries. In addition, by combining Pi starvation conditions with proteasome inhibitor treatments, we characterized the role of the ubiquitin-proteasome system, a major mechanism for targeted protein degradation in eukaryotes, in the control of the stability of Pi-responsive proteins. Among Pi-responsive proteins, the histone chaperone NAP1;2 was selected for further characterization, and was shown to display differential nucleo-cytoplasmic accumulation in response to Pi deprivation. We also found that mutants affecting three members of the NAP1 family accumulate lower Pi levels and display reduced expression of Pi starvation-inducible genes, reflecting a potential regulatory role for these chromatin-remodeling proteins in Pi homeostasis. In this study, we explore the feasibility of nuclear proteomics to identify regulatory proteins and ubiquitin-proteasome targets within a specific stress signaling pathway in plants, in our case phosphate starvation signaling in Arabidopsis. It will be of interest for researchers involved in the dissection of any signaling pathway in plants, in particular those with an interest in the ubiquitin-proteasome functions, and for the plant nutrition community.

Functional Dissection of Regulatory Models Using Gene Expression Data of Deletion Mutants

Genome-wide gene expression profiles accumulate at an alarming rate, how to integrate these expression profiles generated by different laboratories to reverse engineer the cellular regulatory network has been a major challenge. To automatically infer gene regulatory pathways from genome-wide mRNA expression profiles before and after genetic perturbations, we introduced a new Bayesian network algorithm: Deletion Mutant Bayesian Network (DM_BN). We applied DM_BN to the expression profiles of 544 yeast single or double deletion mutants of transcription factors, chromatin remodeling machinery components, protein kinases and phosphatases in S. cerevisiae. The network inferred by this method identified causal regulatory and non-causal concurrent interactions among these regulators (genetically perturbed genes) that are strongly supported by the experimental evidence, and generated many new testable hypotheses. Compared to networks reconstructed by routine similarity measures or by alternative Bayesian network algorithms, the network inferred by DM_BN excels in both precision and recall. To facilitate its application in other systems, we packaged the algorithm into a user-friendly analysis tool that can be downloaded at http://www.picb.ac.cn/hanlab/DM_BN.html.

Abstract A47: Investigating Zbtb protein recognition of epigenetically modified DNA

Abstract Methylation of CpG dinucleotides is essential for genomic stability, control of gene expression and the regulation of chromatin structure in normal eukaryotic cells. Aberrant alterations in these genomic methylation patterns have been linked with many diseases, including cancer. This correlation has prompted the need to better understand the regulatory mechanisms of DNA methylation in gene transcription. The Zbtb family, consisting of Zbtb33, Zbtb4 and Zbtb38, is a set of methyl-CpG binding proteins (MBPs), which recognize both methylated as well as sequence-specific non-methylated DNA sites through conserved Cys2His2 zinc fingers. DNA recognition subsequently recruits chromatin remodeling machinery to the target site resulting in chromatin compaction and gene silencing. While sufficient evidence suggests that the Zbtb proteins play a role in cancer, an extensive analysis of the gene targets and signaling pathways regulated by each protein has yet to be investigated. Here we utilize an interdisciplinary approach combining cellular biology, molecular biology and structural biology to determine the epigenetic targets regulated by the Zbtb MBPs and to identify the protein region(s) necessary for high-affinity DNA recognition. Utilizing western blot analysis, we have determined that the three Zbtb proteins exhibit differential endogenous expression levels in a variety of cancer cell lines. Further, ChIP qRT-PCR analysis demonstrated that the occupancy of Zbtb33 at various methylated and sequence-specific genes was highly variable between the different cancer cell lines. Combined, these results suggest that the Zbtb proteins likely mediate differential signaling pathways in different cancers, possibly by targeting specific regulatory gene elements. Mapping of the global gene occupation for Zbtb33 in various cancer cell lines is in progress. Additionally, we have designed multiple protein constructs around the conserved Cys2His2 zinc finger DNA binding region as well as additional zinc fingers located in the C-terminal regions of Zbtb4 and Zbtb38, as the functions of the latter have not yet been identified. The constructs have been analyzed by solution NMR and EMSA to determine whether they are amenable for further structural analysis and to characterize their DNA binding interactions. This multidisciplinary approach provides the means for advancing our understanding of the molecular basis by which these proteins interpret and translate epigenetic signals in the cancerous state. Citation Format: Tommy W. Terooatea, Maya J. Pandya, Alan C. Chugg, Bethany A. Buck-Koehntop. Investigating Zbtb protein recognition of epigenetically modified DNA. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A47.

Abstract PR06: CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression

Abstract CARM1 belongs to the type I protein arginine (R) methyltransferase (PRMT) family and asymmetrically di-methylates proteins on arginines. Because CARM1 is highly expressed in high grade breast tumors and was shown to transactivate cancer-relevant transcription factors including ERα, NF-κB, p53 and E2F1, it has been strongly implicated in tumorigenesis. To fully characterize functions of CARM1 in breast cancer, we knocked-out CARM1 from several breast cancer cell lines using Zinc-Finger Nuclease technology. Western blot using an asymmetric di-methylated R antibody revealed striking differences between CARM1 KO and parental MCF7 cell lysates, indicating that CARM1 methylates a significant number of substrates in breast cancer cells. BAF155, a core subunit of SWI/SNF chromatin remodeling complex, was identified as a CARM1 substrate by mass spectrometry. Further biochemical analyses validated that BAF155 was methylated by CARM1 in vivo and in vitro and methylation was mapped to a single site, R1064. We next generated BAF155R1064K knock-in MCF7 and MDA-mb-231 cells and showed that methylation of BAF155 is essential for breast cancer cell growth, colony formation and migration. Further, we developed an antibody that selectively recognized methylated form of BAF155 (me-BAF155) and observed strong nuclear me-BAF155 staining in invasive ductal carcinoma but not in the normal breast tissue. Importantly, me-BAF155 immunostaining positively correlates with tumor stage and metastasis. To understand the molecular mechanism by which BAF155 methylation affects tumorigenesis, we performed ChIP-seq using ChIP-validated BAF155 antibody in the BAF155WT or BAF155R1064K knock-in MCF7 cells. Interestingly, C-Myc pathway genes were only identified in WT but not mutant BAF155 regulated categories. Individual ChIP assays validated differential binding of BAF155WT or BAF155R1064K to specific target genes and importantly, the mRNA levels of these genes were affected correspondingly. This result suggests that BAF155 methylation affects gene expression by altering genomic association of BAF155, and perhaps the SWI/SNF complex, to chromatin regions. Collectively, our study revealed novel mechanisms by which CARM1 perturbs the chromatin remodeling machinery to enhance tumor progression. This work is supported by DOD ERA of HOPE Scholar Award and Shaw Scientist Award to W.X. This abstract is also presented as Poster A37. Citation Format: Lu Wang, Zibo Zhao, Mark Meyer, Wesley J. Pike, Wei Xu. CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR06.

Histone variants: emerging players in cancer biology

Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology.

Brg1 Governs a Positive Feedback Circuit in the Hair Follicle for Tissue Regeneration and Repair

Hair follicle stem cells (bulge cells) are essential for hair regeneration and early epidermal repair after wounding. Here we show that Brg1, a key enzyme in the chromatin-remodeling machinery, is dynamically expressed in bulge cells to control tissue regeneration and repair. In mice, sonic hedgehog (Shh) signals Gli to activate Brg1 in bulge cells to begin hair regeneration, whereas Brg1 recruits NF-κB to activate Shh in matrix cells to sustain hair growth. Such reciprocal Brg1-Shh interaction is essential for hair regeneration. Moreover, Brg1 is indispensable for maintaining the bulge cell reservoir. Without Brg1, bulge cells are depleted over time, partly through the ectopic expression of the cell-cycle inhibitor p27(Kip1). Also, bulge Brg1 is activated by skin injury to facilitate early epidermal repair. Our studies demonstrate a molecular circuit that integrates chromatin remodeling (Brg1), transcriptional regulation (NF-κB, Gli), and intercellular signaling (Shh) to control bulge stem cells during tissue regeneration.

The clinical significance of SWI/SNF complex in pancreatic cancer.

149 Background: Chromatin remodeling factors have been the subject of great interest in oncology. However, little is known about their role in pancreatic cancer. The objective of this study is to clarify the clinical significance of SWI/SNF complex, one of the major chromatin remodeling machineries, in patients with pancreatic cancer. Methods: 68 cases with pancreatic cancer who underwent R0, 1 resection were enrolled. Cancer tissues were processed to tissue microarray, then stained immunohistochemically by using antibody of SWI/SNF components; BRM, BRG1, BAF250a, BAF180, and BAF47. The correlation of expression level and clinicopathological outcome including overall survival were analyzed. Results: Expression level of the SWI/SNF components was categorized as low or high according to the median value of Histo score. Statistical analysis revealed that related factors were tumor size, T factor, M factor, lymphatic invasion, and Stage in BRM, histology and Stage in BRG1, tumor size in BAF180, lymphatic invasion in BAF47, respectively. Multivariate Cox propotional hazard analysis showed high-BRM and low-BAF180 expression level were independent predictors of worse survival in patients with pancreatic cancer. The hazard analysis was also examined in the patients treated with adjuvant gemcitabine, indicating that high-BRM, and low-BAF180 were independent prognostic factors for poor survival. Conclusions: These results suggest that the specific cofactors of SWI/SNF chromatin remodeling complex certainly have roles in pancreatic cancer. High-BRM, and low-BAF180 are useful markers for prognosis of pancreatic cancer.

ArabidopsisChromatin Remodeling Factor PICKLE Interacts with Transcription Factor HY5 to Regulate Hypocotyl Cell Elongation

Photomorphogenesis is a critical plant developmental process that involves light-mediated transcriptome changes, histone modifications, and inhibition of hypocotyl growth. However, the chromatin-based regulatory mechanism underlying this process remains largely unknown. Here, we identify ENHANCED PHOTOMORPHOGENIC1 (EPP1), previously known as PICKLE (PKL), an ATP-dependent chromatin remodeling factor of the chromodomain/helicase/DNA binding family, as a repressor of photomorphogenesis in Arabidopsis thaliana. We show that PKL/EPP1 expression is repressed by light in the hypocotyls in a photoreceptor-dependent manner. Furthermore, we reveal that the transcription factor ELONGATED HYPOCOTYL5 (HY5) binds to the promoters of cell elongation-related genes and recruits PKL/EPP1 through their physical interaction. PKL/EPP1 in turn negatively regulates HY5 by repressing trimethylation of histone H3 Lys 27 at the target loci, thereby regulating the expression of these genes and, thus, hypocotyl elongation. We also show that HY5 possesses transcriptional repression activity. Our study reveals a crucial role for a chromatin remodeling factor in repressing photomorphogenesis and demonstrates that transcription factor-mediated recruitment of chromatin-remodeling machinery is important for plant development in response to changing light environments.

Akirin

Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development.

Chromatin-Remodeling Factor Brg1 Is Required for Schwann Cell Differentiation and Myelination

Schwann cells produce myelin sheaths and thereby permit rapid saltatory conductance in the vertebrate peripheral nervous system. Their stepwise differentiation from neural crest cells is driven bya defined setof transcription factors. How this is linked to chromatin changes is not well understood. Here we show that the glial transcription factor Sox10 functions in Schwann cells by recruiting Brg1-containingchromatin-remodeling complexes via Baf60a toregulatory regions of Oct6 and Krox20 genes. Itthereby stimulates expression of these transcriptional regulators that then cooperate with Sox10 toconvert immature into myelinating Schwann cells.The functional interaction between Sox10 andBrg1 is evident from gain- and loss-of-function studies, similar neuropathies in the corresponding mouse mutants, and an aggravated neuropathy in compound mutants. Our results demonstrate that the transcription factor-mediated recruitment of thechromatin-remodeling machinery to specific genomic loci is an essential driving force for Schwann cell differentiation and myelination.

Characterization of anti-silencing factor 1 in Leishmania major

Anti-silencing factor 1 (ASF1) is a histone chaperone that contributes to the histone deposition during nucleosome assembly in newly replicated DNA. It is involved in chromatin disassembly, transcription activation and in the cellular response to DNA damage. In Leishmania major the ASF1 gene (LmASF1) is located in chromosome 20 and codes for a protein showing 67% of identity with the Trypanosoma brucei TbASF1a. Compared to orthologous proteins, LmASF1 conserves the main residues relevant for its various biological functions. To study ASF1 in Leishmania we generated a mutant overexpressing LmASF1 in L. major. We observed that the excess of LmASF1 impaired promastigotes growth rates and had no impact on cell cycle progress. Differently from yeast, ASF1 overproduction in Leishmania did not affect expression levels of genes located on telomeres, but led to an upregulation of proteins involved in chromatin remodelling and physiological stress, such as heat shock proteins, oxidoreductase activity and proteolysis. In addition, we observed that LmASF1 mutant is more susceptible to the DNA damaging agent, methyl methane sulphonate, than the control line. Therefore, our study suggests that ASF1 from Leishmania pertains to the chromatin remodelling machinery of the parasite and acts on its response to DNA damage.

Synergy Between Chromatin Remodeling Complexes and pre‐mRNA Splicing

Pre‐messenger RNA splicing plays a critical role in gene expression. In spite of the considerable progress that has been made in understanding the mechanism of splicing, an important question that remains poorly understood is how changes in the chromatin template affect splicing. Observations of enriched nucleosome occupancy and histone modifications in the exonic regions of genes raise the possibility that specific histone marks may be associated with splicing signals. For example, proteins that bind to methylated histones facilitate the recruitment of snRNPs to nascent transcript and influence the efficiency of splicing. Recently, we have shown that GCN5‐dependent histone acetylation is critical for cotranscriptional spliceosome assembly and its rearrangements. To gain further mechanistic insights, we have explored the possibility of communication between the chromatin remodeling machinery that interact with acetylated histones and the splicing machinery. Genetic analyses have identified functional interactions between components of chromatin remodeling complexes and components of the U2 snRNP. Furthermore, in vivo interaction studies demonstrate physical interactions between the Swi/Snf complex and the U2 snRNP. Together, these studies suggest a synergistic relationship between the Swi/Snf complex and the splicing machinery in gene expression.Funding: NIGMS (GM085474 and GM085764)