Role Of Chromatin Remodeling Research Articles

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

The Development and Characterization of Antibodies to Site‐specific O‐GlcNAc modified Histones for Epigenetic Research

O‐glycosylation of nuclear and cytoplasmic proteins by a single β‐N‐acetyl‐D‐glucosamine moiety (O‐GlcNAc) is a common post‐translational modification that is highly dynamic and fluctuates in response to cellular stimuli. This type of glycosylation has been found on approximately a thousand human proteins to date and is thought to be nearly as widespread and abundant as protein phosphorylation. Many of the first proteins identified carrying this modification were transcription factors, and it became apparent in the last several years that O‐GlcNAc plays a significant role in chromatin remodeling and gene expression.Given the complexity of the histone code, site‐specific antibodies are needed to begin to elucidate the hierarchy of modifications as well as the biological significance of single and multiple post‐translational modifications of the histones. We have developed and approach to provide high‐affinity site‐specific O‐GlcNAc antibodies that appear to overcome issues related to small carbohydrates being poor antigens and the O‐GlcNAc modified epitopes being self‐antigens. These site‐specific antibodies require a more elaborate characterization scheme than other antibodies as they need to demonstrate both a preference for the O‐GlcNAcylated species over the native peptide sequence and a specificity to the amino acid sequence. We use a multistep evaluation process to characterize our antibodies. 1) ELISA assay, to determine the preference of the antibody to the O‐GlcNAcylated peptide over the unmodified form; 2) Cross‐reactivity of the antibody to other peptide sequences modified with O‐GlcNAc; 3) Immunoprecipitation increasing wash stringencies and 4) Evaluation of the site‐specific O‐GlcNAc histone antibodies for their potential as ChIP‐Seq reagents. Using this process, we have authenticated our site‐specific O‐GlcNAc antibodies and have demonstrated that they possess the preference and specificity needed for epigenetic research.Support or Funding InformationResearch reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers 1R43GM110887‐01 and R44GM110887‐02.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LSD1, a double-edged sword, confers dynamic chromatin regulation but commonly promotes aberrant cell growth.

Histone-modifying enzymes play a critical role in chromatin remodeling and are essential for influencing several genome processes such as gene expression and DNA repair, replication, and recombination. The discovery of lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, dramatically revolutionized research in the field of epigenetics. LSD1 plays a pivotal role in a wide range of biological operations, including development, cellular differentiation, embryonic pluripotency, and disease (for example, cancer). This mini-review focuses on the role of LSD1 in chromatin regulatory complexes, its involvement in epigenetic changes throughout development, and its importance in physiological and pathological processes.

The interplay between ventro striatal BDNF levels and the effects of valproic acid on the acquisition of ethanol-induced conditioned place preference in mice.

Alcohol addiction is a chronic, relapsing and progressive brain disease with serious consequences for health. Compulsive use of alcohol is associated with the capacity to change brain structures involved with the reward pathway, such as ventral striatum. Recent evidence suggests a role of chromatin remodeling in the pathophysiology of alcohol dependence and addictive-like behaviors. In addition, neuroadaptive changes mediated by the brain-derived neurotrophic factor (BDNF) seems to be an interesting pharmacological target for alcoholism treatment. In the present study, we evaluated the effects of the deacetylase inhibitor valproic acid (VPA) (300mg/kg) on the conditioned rewarding effects of ethanol using conditioned place preference (CPP) (15% v/v; 2g/kg). Ethanol rewarding effect was investigated using a biased protocol of CPP. BDNF levels were measured in the ventral striatum. Ethanol administration induced CPP. VPA pretreatment did not reduce ethanol-CPP acquisition. VPA pretreatment increased BDNF levels when compared to ethanol induced-CPP. VPA pretreatment increased BDNF levels even in saline conditioned mice. Taken together, our results indicate a modulatory effect of VPA on the BDNF levels in the ventral striatum. Overall, this study brings initial insights into the involvement of neurotrophic mechanisms in the ventral striatum in ethanol-induced addictive-like behavior.

Abstract 637: Role of Nuclear Smooth Muscle Alpha-Actin in the Differentiation of Vascular Smooth Muscle Cells

Although the function of the cytoplasmic smooth muscle (SM) α-actin in contractile units has been well characterized in vascular SMCs, whether the SM α-actin is present in the nucleus and its nuclear function are yet to be determined. Our previous study discovered the mutations in ACTA2 , which encodes SM α-actin, predispose to both thoracic aortic disease and occlusive vascular diseases (stroke and coronary artery disease). We hypothesize that nuclear SM α-actin is critical for differentiation of SMCs. The cytoplasmic and nuclear fractions of mouse SMCs were isolated and the SM α-actin was detected in both cytosol and nucleus by immunoblotting. Moreover, both cytoplasmic and nuclear SM α-actin were dramatically upregulated during the differentiation of SMCs stimulated with TGF-β. Two-dimensional gel electrophoresis, which can separate actin isoforms based on distinct isoelectric points, was used to show that the ratio of SM α-actin to β-actin was increased in nuclear fraction when compared to cytoplasmic fraction with differentiation of SMCs, driven by serum starvation and TGF-β treatment. Furthermore, differentiation was associated with a significantly higher nuclear to cytoplasmic ratio of SM α-actin, indicating SM α-actin accumulates in the nucleus during differentiation. Co-immunoprecipitation experiments identified that nuclear SM α-actin is a component of the chromatin remodeling complex, Ino80, and the immunofluorescence confirmed co-localization of SM α-actin and Ino80 in the nucleus. To investigate the transcriptional function of nuclear SM α-actin, we performed the chromatin immunoprecipitation quantitative PCR, which revealed that the SM α-actin, but not β-actin, accumulated to the promoter region of SMC differentiation markers, including Myh11 , Cnn1 , and Tagln , during the differentiation of SMC. As expected, the Acta2 mutant SMCs (pArg149Cys) disrupt the differentiation of SMCs based on decreased contractile protein levels and increased proliferation. Furthermore, specific Acta2 missense mutations disrupt the ability for SM α-actin to localize to the nucleus. Taken together, our study found the SM α-actin concentrates into the nucleus of SMCs and may play a role in chromatin remodeling required for differentiation of SMCs.

Role of chromatin remodeling and spacing factor 1 in histone H2A ubiquitination mediated gene silencing

Posttranslational modification of histones plays important regulatory roles in chromatin‐based nuclear processes. Histone H2A ubiquitination (H2Aub) is a predominate modification and has been primarily linked to gene silencing; however, the mechanism by which H2Aub achieves the gene silencing function remains unclear. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF (remodeling and spacing factor) complex, as a novel H2Aub binding protein which mediates the repressive function of H2Aub on gene expression. RSF1 preferentially associates with H2Aub‐enriched nucleosomes through a previously uncharacterized region designated as the ubiquitinated H2A binding (UAB) motif. RSF1 specifically interacts with H2Aub nucleosomes and the UAB motif is required for RSF1 to interact with H2Aub nucleosomes in vitro and in vivo. Genes regulated by RSF1 overlap significantly with genes regulated by RNF2 or Ring1B, the catalytic subunit of Polycomb repressive complex 1 (PRC1), in both human and mouse cells. Strikingly, virtually all overlapped genes show changes in the same direction. RSF1 binds to gene promoter regions, and over 85% of H2Aub enriched genes, including the classic PRC1‐target Hox genes, are co‐bound by RSF1. Reduction H2Aub levels by Ring1B knockout resulted in a significant reduction of RSF1 binding. RSF1 knockout did not affect RNF2/Ring1B or H2Aub levels but resulted in re‐expression of H2Aub target genes, accompanied by a reduction in the spacing and stability of H2Aub‐containing nucleosomes. Finally, the direct role of RSF1 in H2Aub‐mediated gene silencing was demonstrated by chromatin based in vitro transcription assay. Therefore, this study identifies RSF1 as a novel H2Aub binding protein and reveals that H2Aub represses gene expression by maintaining a regular spaced, stable nucleosome array at promoter regions through RSF1.Support or Funding InformationHengbin Wang is a Leukemia and Lymphoma Scholar.

Epigenetic regulation in Autism spectrum disorder

AbstractAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by an impaired social communication skill and often results in repetitive, stereotyped behavior which is observed in children during the first few years of life. Other characteristic of this disorder includes language disabilities, difficulties in sensory integration, lack of reciprocal interactions and in some cases, cognitive delays. One percentage of the general population is affected by ASD and is four times more common in boys than girls. There are hundreds of genes, which has been identified to be associated with ASD etiology. However it remains difficult to comprehend our understanding in defining the genetic architecture necessary for complete exposition of its pathophysiology. Seeing the complexity of the disease, it is important to adopt a multidisciplinary approach which should not only focus on the “genetics” of autism but also on epigenetics, transcriptomics, immune system disruption and environmental factors that could all impact the pathogenesis of the disease. As environmental factors also play a key role in regulating the trigger of ASD, the role of chromatin remodeling and DNA methylation has started to emerge. Such epigenetic modifications directly link molecular regulatory pathways and environmental factors, which might be able to explain some aspects of complex disorders like ASD. The present review will focus on the role of epigenetic regulation in defining the underlying cause for ASD.

Antibodies to H1 histone from the sera of HIV-infected patients recognize and catalyze site-specific degradation of this histone.

Histones and their posttranslational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive feature of some autoimmune and viral diseases. Electrophoretically and immunologically homogeneous IgGs containing no canonical enzymes were isolated from sera of human immunodeficiency virus-infected patients by chromatography on several affinity sorbents. In contrast to canonical proteases (trypsin, chymotrypsin, and proteinase K), IgGs from human immunodeficiency virus-infected patients purified by affinity chromatography on Sepharose containing immobilized histones specifically recognized and hydrolyzed only histones but not many other tested globular proteins. Using matrix-assisted laser desorption/ionization mass spectrometry, the sites of H1 histone (193 amino acids [AAs]) cleavage by anti-H1 histone IgGs were determined for the first time. It was shown that 1 cluster of 2 major and 4 moderate sites of cleavage is located at the beginning (106-112 AAs) of the known antigenic determinants disposed at the long C-terminal sequence of H1. Two clusters of minor and very weak sites of the protein cleavage correspond to middle (8 sites, 138-158 AAs) and terminal (5 sites, 166-176 AAs) parts of the antigenic determinants. It was shown that in contrast to canonical proteases, N-terminal part of H1 histone (1-136 AAs) containing no antigenic determinants is an unpredictably very resistant against hydrolysis by abzymes, while it can be easily cleavage by canonical proteases. Because histones act as damage-associated molecules, abzymes against H1 and other histones can play important role in pathogenesis of acquired immune deficiency syndrome and probably other different diseases.

Histone Acetylation and the Regulation of Major Histocompatibility Class II Gene Expression.

Major histocompatibility complex (MHC) class II molecules are essential for processing and presenting exogenous pathogen antigens to activate CD4+ T cells. Given their central role in adaptive immune responses, MHC class II genes are tightly regulated in a tissue- and activation-specific manner. The regulation of MHC class II gene expression involves various transcription factors that interact with conserved proximal cis-acting regulatory promoter elements, as well as MHC class II transactivator that interacts with a variety of chromatin remodeling machineries. Recent studies also identified distal regulatory elements within MHC class II gene locus that provide enormous insight into the long-range coordination of MHC class II gene expression. Novel therapeutic modalities that can modify MHC class II genes at the epigenetic level are emerging and are currently in preclinical and clinical trials. This review will focus on the role of chromatin remodeling, particularly remodeling that involves histone acetylation, in the constitutive and inducible regulation of MHC class II gene expression.

Abstract C15: Neutrophil elastase (NE) induces epithelial-mesenchymal transition (EMT) via upregulation of inhibitor of DNA binding 1 (ID1)

Abstract Neutrophil elastase is a neutrophil-derived serine protease with broad substrate specificity. We have previously shown that NE promotes lung tumor growth in vitro and in vivo and that NE is capable of entering tumor cell endosomes in a clathrin-dependent fashion. Epithelial-mesenchymal transition (EMT) is a fundamental event for primary tumors to metastasize to distant sites. Altered gene and protein expression causes changes in cell morphology and behavior resulting in the loss of attachment to neighboring cells, intravasation, and migration into distant tissue. However, the entities residing within the tumor microenvironment that drive EMT are poorly understood. We identified NE, expressed and released by tumor-associated neutrophils, as such an entity. We found that NE induces EMT-like morphology and behavior in several cancer cell lines as well as upregulates genes (Snail and Twist) and proteins (N-cadherin, α-SMA, Fibronectin and Vimentin) associated with EMT. Furthermore, NE provokes tumor cell migration and invasion in vitro and is required for metastasis formation in vivo. Besides known EMT markers we identified significant upregulation of inhibitor of DNA binding 1 (ID1) in tumor cells in the presence of NE. Increased ID1 expression was previously shown to correlate with tumor progression and metastasis. Clathrin-dependent NE internalization is required for both ID1 expression and the EMT-like phenotype. We localized NE in different compartments of tumor cells and found it predominantly in the nuclear fraction; more specifically, NE is located in the chromatin-bound fraction, indicating a potential role in chromatin remodeling and transcriptional regulation. Additionally, the proteolytic activity of NE was measured in the nuclear and chromatin-bound fractions and is required for induction of ID1 expression and EMT-like behavior. Taken together, these data suggest the importance of NE-induced ID1 expression in metastasis formation. Citation Format: Julia Kargl, Alyssa Gregory, Hye Youn Yang, Stephanie Busch, Heather Metz, A. McGarry Houghton. Neutrophil elastase (NE) induces epithelial-mesenchymal transition (EMT) via upregulation of inhibitor of DNA binding 1 (ID1). [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C15.

Abstract 993: Identification of estrogen receptor alpha 1 bound lncRNAs in aggressive breast cancer

Abstract Breast cancer (BC) is the second most common newly diagnosed cancer and the second leading cause of cancer death among women in the United States. Around 70% of diagnosed BCs are estrogen receptor positive (ER+). Despite the proven benefits of adjuvant endocrine therapy in women with hormone receptor positive breast cancer, relapses still occur even after initial treatment with endocrine therapy for 5 years, referred to as late stage relapse. While existing studies have focused on the role of protein-coding genes, long non-coding RNAs (lncRNAs) are an emerging and under-characterized class of transcripts that have been shown to be dysregulated in breast cancer. Recently, lncRNAs have been shown to function by interfacing with corresponding RNA binding proteins to play critical regulatory roles in chromatin remodeling and diverse cellular processes by acting as decoys, guides, and scaffolds. As estrogen receptor expression is controlled mostly by epigenetic and post-transcriptional mechanisms, and very rarely at the genomic level, we hypothesize that lncRNAs may interact with ER to promote aggressive disease. To address this, we aimed to identify lncRNAs bound to the estrogen receptor alpha 1 (ESR1) that promote late stage breast cancer. To accomplish this, we first used transcriptome sequencing to identify altered expression levels of lncRNAs between primary tumors and late-stage relapse breast cancer patients. We detected 2086 altered lncRNAs when comparing the metastatic to the primary samples with an FDR <0.05, of which 202 were novel. As expected, Gene Set Enrichment Analysis of differentially expressed protein-coding genes revealed an enrichment of biological concepts associated with breast cancer and metastasis. Next, in order to identify ESR1 bound lncRNAs associated with aggressive disease, we conducted RNA Immunoprecipitation Sequencing (RIP-Seq) of all transcripts bound to ESR1 as compared to an IgG control in the ER+ T47D cell line in triplicate. We identified 210 transcripts bound to ESR1, which we term ESR1 bound lncRNAs (ESRlncs). The identified ESRlncs consisted of both novel (unannotated) and known (annotated) lncRNAs. Interestingly we found ∼50% of ESRlncs had significantly altered expression in the metastatic samples, with 96% (105) having more than a two fold increase in expression. Further characterization of these ESRlncs is ongoing to decipher how they interact with ESR1 to promote aggressive and metastatic disease. Overall, this is the first study to discover ESR1 bound lncRNAs that may be contributing to late stage relapse in breast cancer patients. Citation Format: Jessica M. Silva-Fisher, Abdallah M. Eteleeb, Torsten O. Nielsen, Charles M. Perou, Jorge S. Reis-Filho, Mathew J. Ellis, Elaine R. Mardis, Christopher A. Maher. Identification of estrogen receptor alpha 1 bound lncRNAs in aggressive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 993.

Abstract 1240: Targeting epigenetic regulation in clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell cancer, is highly resistant to traditional radiation and chemotherapy. Recently, targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved; thus, treatment options are scarce for patients bearing this disease. The majority of ccRCC cases (∼90%) are characterized by bi-allelic loss of the von Hippel Lindau (VHL) gene, followed by mutations in the epigenetic regulators PBRM1 (23%), SETD2 (8.4%), BAP1 (7.1%), and KDM5C (4.7%). The latter four genes implicate an integral role for chromatin remodeling in ccRCC, and thus signify a new realm of exploration and therapeutic targeting for this disease. Given the limited number of commercially available ccRCC cell lines, and reports that they may not accurately reflect primary tumors at the molecular level, our lab developed a novel method to generate primary patient-derived ccRCC (VHLmut) cells and matched normal renal proximal tubular epithelial (VHLwt) cells from human surgical specimens with high efficiency. This involved fluorescence-activated cell sorting of primary tumor single cell suspensions using the cell surface marker carbonic anhydrase IX (CA9), a HIF target which is upregulated upon VHL loss, to establish CA9+ VHLmut and CA9− VHLwt cells. Transcriptional profiling of VHLmut and VHLwt pairs found gene signatures that corresponded with patient matched primary ccRCC tumors and adjacent normal tissues in The Cancer Genome Atlas, demonstrating that these cells represent novel models for interrogation of ccRCC biology and testing of therapeutic strategies. Here, in the current study, these newly established cells were utilized to screen a focused library of 30 well-characterized drug-like compounds targeting specific components of chromatin remodeling complexes to determine the effect on cell growth in culture. At Day 0, 786-0 (−VHL) cells and VHLmut cells corresponding to four patient primary ccRCC tumors were plated at 2,500 cells/well in 96-well black clear-bottom plates (n = 5). Cells were then treated with the library of chromatin-targeting drugs on Day 1 and monitored until control-treated cells were 90-95% confluent. At endpoint, cells were fixed and stained with DRAQ5, a DNA intercalating dye that stains DNA stoichiometrically, for visualization on the LI-COR Odyssey CLx Imaging System. Across all five samples, there was a significant reduction in growth observed among cells treated with drugs targeting: i) BET bromodomains (BRD2, BRD3, BRD4 and BRDT), ii) EZH1/2, and iii) JMJD3, UTX and JARID1B. Ongoing work will determine mechanisms of action and whether these targets are also affected in matching VHLwt cells in order to identify compounds that may translate well in the clinic to treat patients with ccRCC. Citation Format: Anthony J. Apostoli, Nazleen Lobo, Panagiotis Prinos, Dalia Barsyte-Lovejoy, Cheryl Arrow Smith, Laurie Ailles. Targeting epigenetic regulation in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1240.

Epigenetic regulation in the brain

Neurobiology The activity of neurons in the brain controls the transcription of genes that influence the pruning of dendritic connections between neurons, and such modifications can influence animal behavior. Yang et al. propose a role for chromatin remodeling by the nucleosome remodeling and deacetylase complex (NuRD) in the inactivation of such activity-dependent transcription in the mouse cerebellum (see the Perspective by Sweatt). Deposition of the histone variant H2A.z at promoters of activity-dependent genes required the NuRD complex. Loss of the NuRD complex function resulted in hypersensitivity of mice to sensory stimuli and excessive neuronal connectivity in animals performing a task on a treadmill. Science , this issue p. [300][1]; see also p. [218][2] [1]: /lookup/doi/10.1126/science.aad4225 [2]: /lookup/doi/10.1126/science.aah4055

Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology.

PBRM1 Regulates the Expression of Genes Involved in Metabolism and Cell Adhesion in Renal Clear Cell Carcinoma.

Polybromo-1 (PBRM1) is a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex and is the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). Mutation of PBRM1 is believed to be an early event in carcinogenesis, however its function as a tumor suppressor is not understood. In this study, we have employed Next Generation Sequencing to profile the differentially expressed genes upon PBRM1 re-expression in a cellular model of ccRCC. PBRM1 re-expression led to upregulation of genes involved in cellular adhesion, carbohydrate metabolism, apoptotic process and response to hypoxia, and a downregulation of genes involved in different stages of cell division. The decrease in cellular proliferation upon PBRM1 re-expression was confirmed, validating the functional role of PBRM1 as a tumor suppressor in a cell-based model. In addition, we identified a role for PBRM1 in regulating metabolic pathways known to be important for driving ccRCC, including the regulation of hypoxia response genes, PI3K signaling, glucose uptake, and cholesterol homeostasis. Of particular novelty is the identification of cell adhesion as a major downstream process uniquely regulated by PBRM1 expression. Cytoskeletal reorganization was induced upon PBRM1 reexpression as evidenced from the increase in the number of cells displaying cortical actin, a hallmark of epithelial cells. Genes involved in cell adhesion featured prominently in our transcriptional dataset and overlapped with genes uniquely regulated by PBRM1 in clinical specimens of ccRCC. Genes involved in cell adhesion serve as tumor suppressor and maybe involved in inhibiting cell migration. Here we report for the first time genes linked to cell adhesion serve as downstream targets of PBRM1, and hope to lay the foundation of future studies focusing on the role of chromatin remodelers in bringing about these alterations during malignancies.

DNA damage response and repair data with pharmacological modulators of Tousled.

Human Tousled kinase 1 (TLK1) plays an important role in chromatin remodeling, replication, and DNA damage response and repair. TLK1 activity is immediately, but transiently, downregulated after genotoxic insult, and its recovery is important for exit from checkpoint arrest and cell survival after radiation. The data in this article compliments research presented in the paper titled, “Tousled kinase activator, gallic acid, promotes DNA repair and suppresses radiation cytotoxicity in salivary gland cells” [1]. The identification of small molecule activators and inhibitors of TLK1 provided an opportunity to pharmacologically alter the protein׳s activity to elucidate its role in DNA damage response pathways. TLK1 effectors, gallic acid (GA) and thioridazine (THD) activate and inhibit the kinase, respectively, and the data report on the impact of these compounds and the significance of TLK1 to DNA break repair and the survival of human salivary acinar cells.

Integrated molecular mechanism directing nucleosome reorganization by human FACT.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Antibodies from the sera of HIV-infected patients efficiently hydrolyze all human histones.

Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Here, using ELISA it was shown that sera of HIV-infected patients and healthy donors contain autoantibodies against histones. Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether antibodies from sera of HIV-infected patients can hydrolyze human histones. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of HIV-infected patients by chromatography on several affinity sorbents. We present first evidence showing that 100% of IgGs purified from the sera of 32 HIV-infected patients efficiently hydrolyze from one to five human histones. Several rigid criteria have been applied to show that the histone-hydrolyzing activity is an intrinsic property of IgGs of HIV-infected patients. The relative efficiency of hydrolysis of histones (H1, H2a, H2b, H3, and H4) significantly varied for IgGs of different patients. IgGs from the sera of 40% of healthy donors also hydrolyze histones but with an average efficiency approximately 16-fold lower than that of HIV-infected patients. Similar to proteolytic abzymes from the sera of patients with several autoimmune diseases, histone-hydrolyzing IgGs from HIV-infected patients were inhibited by specific inhibitors of serine and of metal-dependent proteases, but an unexpected significant inhibition of the activity by specific inhibitor of thiol-like proteases was also observed. Because IgGs can efficiently hydrolyze histones, a negative role of abzymes in development of acquired immune deficiency syndrome cannot be excluded. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract B67: Identifying obesity-linked gene expression changes in prostate cancer

Abstract Background: Obese men are at higher risk of advanced prostate cancer and have higher rates of cancer-specific mortality after diagnosis. The biological mechanisms explaining this association are unknown. Using mRNA expression profiles of tissue from prostate cancer patients, we aimed to identify pathways that are differentially altered among overweight/obese men compared to healthy weight men. Methods: The study includes men with prostate cancer in the Health Professionals Follow-up Study and the Physicians' Health Study, for whom archival tissue and long-term follow-up is available. Whole genome mRNA expression profiling was performed on tumor (N=402) and adjacent normal (N=200) tissue using the Affymetrix GeneChip Human Gene 1.0 ST Array. Body mass index (BMI) was taken from questionnaires before diagnosis. Among 402 men in the study, 113 lethal events (metastasis or death) were recorded. We identified top Gene Ontology biological processes differentially expressed by obesity status using Gene Set Enrichment Analysis (GSEA). Pathway scores were computed for each tumor by averaging the normalized signal intensities of all member genes. Odds ratios for lethal disease were estimated using logistic regression. Results: 48% of men were healthy weight (BMI 18.5 to <25 kg/m2), 31% were overweight (BMI 25 to <27.5 kg/m2), and 21% were very overweight/obese (BMI ≥27.5 kg/m2) at diagnosis. GSEA identified 15 gene sets enriched in the tumor tissue of very overweight/obese vs healthy weight men (FDR < 0.25). Interestingly, 9 of the 15 gene sets were epigenetic processes related to chromatin remodeling and RNA processing. Other biological processes identified were macromolecular complex disassembly, Golgi vesicle transport, and ribonucleotide metabolic process. No pathways reached FDR < 0.25 in adjacent normal tissue indicating these results are tumor-specific. To look further into the role of chromatin remodeling, we calculated a pathway score based on 35 chromatin-related leading edge genes from the obesity GSEA. As expected, the score was significantly associated with BMI. In addition, the score was positively associated with pathologic stage and Gleason grade. After adjustment for age and year at diagnosis, the score was associated with lethal progression, with an odds ratio of 6.78 (95% CI: 3.42, 14.16) for lethal outcome comparing extreme quartiles. After further adjusting for stage and Gleason grade the odds ratio decreased slightly but remained significant. In a mediation analysis, we found that 36% of the BMI effect on lethal outcome was mediated by the score. Conclusion: Genes involved in chromatin regulation were enriched in tumor tissue of obese men. Tumors with high expression of these genes were found to have worse clinical characteristics and poorer outcomes. Our findings support a role for epigenetic regulation in the obesity-lethal prostate cancer relationship. Citation Format: Ericka M. Ebot, Travis Gerke, Svitlana Tyekucheva, David P. Labbé, Giorgia Zadra, Rachel S. Kelly, Michaela Bowden, Jennifer R. Rider, M Kathryn Wilson, Neil E. Martin, Myles Brown, Matthew G. Vander Heiden, Edward L. Giovannucci, Massimo Loda, Lorelei A. Mucci. Identifying obesity-linked gene expression changes in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B67.

Proteomic characterization of histone variants in the mouse testis by mass spectrometry-based top-down analysis.

Various histones, including testis-specific histones, exist during spermatogenesis and some of them have been reported to play a key role in chromatin remodeling. Mass spectrometry (MS)-based characterization has become the important step to understand histone structures. Although individual histones or partial histone variant groups have been characterized, the comprehensive analysis of histone variants has not yet been conducted in the mouse testis. Here, we present the comprehensive separation and characterization of histone variants from mouse testes by a top-down approach using MS. Histone variants were successfully separated on a reversed phase column using high performance liquid chromatography (HPLC) with an ion-pairing reagent. Increasing concentrations of testis-specific histones were observed in the mouse testis and some somatic histones increased in the epididymis. Specifically, the increase of mass abundance in H3.2 in the epididymis was inversely proportional to the decrease in H3t in the testis, which was approximately 80%. The top-down characterization of intact histone variants in the mouse testis was performed using LC-MS/MS. The masses of separated histone variants and their expected post-translation modifications were calculated by performing deconvolution with information taken from the database. TH2A, TH2B and H3t were characterized by MS/MS fragmentation. Our approach provides comprehensive knowledge for identification of histone variants in the mouse testis that will contribute to the structural and functional research of histone variants during spermatogenesis.