David Valle-Garcia

MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Here, using fibroblasts derived from macroH2A double knockout mice we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild type fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A double knockout induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation ‘lock’ at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.

Role of microRNAs in central nervous system development and pathology

Gene expression regulation is essential for correct functioning of the cell. Complex processes such as development, apoptosis, cell differentiation, and cell cycling require a fine tuning of gene expression. MicroRNAs (miRNAs) are small RNAs that have been recognized as key components of the gene expression regulatory machinery. By sequence complementarity, miRNAs recognize target mRNAs and inhibit their function through degradation or by repressing their translation. The development of the central nervous system (CNS) requires precise and exquisitely regulated gene expression patterns. It is now widely recognized that miRNAs have the capacity to provide such fine regulation both in time and in space. High‐throughput analyses as well as classical molecular biology approaches have allowed the identification of essential miRNAs for CNS development and function. Moreover, recent studies in several model organisms are beginning to show intricate regulatory networks involving miRNAs, transcription factors, and epigenetic regulators during CNS development. Here we review recent findings on the role that miRNAs play in the development of the CNS as well as in neuropathologies such as schizophrenia, Parkinson disease, and Alzheimers disease, among others.

Signaling Epigenetics: Novel Insights on Cell Signaling and Epigenetic Regulation

Cells must be able to respond rapidly and precisely not only to changes in their external environment but also to developmental and differentiation cues to determine when to divide, die, or acquire a particular cell fate. Signal transduction pathways are responsible for the integration and interpretation of most of such signals into specific transcriptional states. Those states are achieved by the modulation of chromatin structure that activates or represses transcription at particular loci. Although a large variety of signal transduction pathways have already been described, much less is known about the crosstalk between signal transduction and its consequent changes in chromatin structure and, therefore, gene expression. Here we present some examples of the relationship between chromatin‐associated proteins and important signal transduction pathways during critical processes like development, differentiation, and disease. There is a great diversity of epigenetic mechanisms that have unexpected interactions with signaling pathways to establish transcriptional programs. Moreover, there are also particular cases where signaling pathways directly affect important components of the epigenetic machinery. Based on such examples, we further propose future research directions linking cell signaling and epigenetics. It is foreseeable that analyzing the relationship between cell signaling and epigenetics will be a huge area for future development that will help us understand the complex process by which a cell is able to induce transcriptional changes in response to external and internal signals.

ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment

ABSTRACT ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3′ exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3′ exonic regions encode the zinc finger motifs, which can range from 1–40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3′ exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3′ exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3′ exons of ZNFs to maintain their genomic stability through preservation of H3K9me3.

Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a transmembrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

Decreased Expression of the Chromatin Remodeler ATRX Associates with Melanoma Progression

To the Editor ATRX is a member of the SWI/SNF family of chromatin remodelers, originally identified as mutated in patients with Alpha Thalassemia/Mental Retardation, X-linked syndrome. The protein product contains several highly conserved domains, including an ADD (ATRX-DNMT3-DNMT3L) domain that binds methylated histone H3 at lysine 9 and an ATPase domain responsible for its remodeling activities (Ratnakumar & Bernstein, 2013). Recently, whole genome sequencing studies identified ATRX mutations in multiple tumors, including those of neural crest cell origin: neuroblastoma, low-grade glioma and glioblastoma (Cheung et al., 2012; Heaphy et al., 2011a; Jiao et al., 2011; Kannan et al., 2012; Schwartzentruber et al., 2012). ATRX alterations encompass point mutations throughout the coding region as well as large N terminal deletions. While mechanistically unclear, ATRX mutations result in loss of protein as assessed by immunohistochemistry (IHC) and often correlate with alternative lengthening of telomeres (ALT) (Cheung et al., 2012; Heaphy et al., 2011a; Kannan et al., 2012; Schwartzentruber et al., 2012). To our knowledge, an investigation of ATRX in cutaneous melanoma is currently lacking. Our previous studies have demonstrated that decreased expression of histone variant macroH2A drives melanoma cell proliferation and metastasis (Kapoor et al., 2010), and that ATRX interacts with macroH2A to negatively regulate its association with chromatin (Ratnakumar et al., 2012). Taken together with recent reports of decreased ATRX protein in neural crest cell-derived tumors, we hypothesized that ATRX function might be compromised in melanoma. In order to test this hypothesis, we performed IHC on a panel of 23 benign nevi, 33 primary melanoma (≥1.0 mm deep) and 25 metastatic melanoma specimens that were formalin fixed paraffin embedded (FFPE) (Figure 1a, c). Slides were evaluated by two dermatopathologists in a blinded fashion using a scoring system based on number of positive nuclei and staining intensity (inter-rater correlation r=0.693, p<0.0001; see Supplemental Methods for details). As depicted in Figure 1a, ATRX protein expression is appreciably reduced with increased malignancy. Benign nevi showed a higher proportion and intensity of nuclear staining when compared to metastatic lesions (Figure 1a, b; p<0.0001). Furthermore, ATRX protein expression was reduced between benign nevi and primary melanoma, with heterogeneous staining observed in the latter (p=0.0026; Figure 1a, b), and between primary and metastatic melanoma (p=0.0113; Figure 1b). This suggests a potential step-wise loss of ATRX expression during melanoma progression. Figure 1 Loss of ATRX protein expression is associated with melanoma progression We further examined whether ATRX levels in primary melanoma correlated with clinicopathologic predictors of prognosis. ATRX staining did not correlate with depth of the lesion (data not shown), however the primary melanomas examined in our cohort were of Breslow thickness greater than 1.0mm (average depth 5.6 mm), and thus quite aggressive. We did however find an inverse correlation with the presence of ulceration, a poor prognostic factor (Figure 1d). Because our study is retrospective with a small sample size, we note that any correlations, or lack thereof, are preliminary. Because structural variations of ATRX exist in neuroblastoma and osteosarcoma (Cheung et al., 2012; Lovejoy et al., 2012), we determined whether such alterations are present in metastatic melanoma. Using a technique to detect structural variations of ATRX, we performed qualitative reverse transcriptase (RT)-PCR of cDNA derived from a cohort of fresh frozen metastatic melanoma samples (n=7). Due to the large ATRX coding region, we amplified the cDNA into five fragments ranging from 1.5-2 kilobase pairs. Because ATRX is located on the X chromosome, we analyzed both male and female patients for potential effects due to gene dosage. Our analysis shows that the ATRX gene product is intact in all metastatic melanomas assayed, as evidenced by appropriately sized bands within each sample (Figure 2a). The cell line WM266-4 derived from a melanoma metastasis served as a positive control for PCR amplicons, as it is devoid of ATRX mutations (Cancer Cell Line Encyclopedia at http://cbioportal.org). The osteosarcoma cell line U2OS, which has large deletions of the ATRX locus (Lovejoy et al., 2012) was used to ensure our assay worked effectively. This analysis suggested that decreased ATRX protein level in metastatic melanoma is unlikely the result of large genomic alterations. Figure 2 ATRX mRNA levels are decreased in metastatic melanoma We next queried whether diminished ATRX protein in metastatic disease was due to transcriptional regulation. We performed qPCR analysis on a cohort of 18 fresh frozen benign nevi and 20 metastatic melanoma tumors, including those samples analyzed for deletions (Figure 2a; indicated in red in Figure 2b). Using both N- and C- terminal primers for ATRX, we found a statistically significant loss of ATRX mRNA levels in metastatic melanoma as compared to benign tissue (p<0.0001; Figure 2b). We next performed IHC on a subset of these tumors, for which FFPE tissue was available (indicated in blue in Figure 2b). The level of ATRX protein indeed corroborated our qPCR findings (Figure 2c). Collectively, these results indicate that ATRX loss occurs, at least in part, by transcriptional repression resulting in loss of protein expression in late stage disease. Collectively, we demonstrate that ATRX loss correlates with melanoma progression. Using two independent cohorts (FFPE and fresh frozen; total of 119 tissues), we found a significant decrease of both mRNA and protein levels of ATRX in metastatic melanoma. While it remains to be tested in a prospective study, ATRX may serve as a biomarker to predict prognosis of disease. Though we did not find evidence of large genomic alterations in a subset of melanoma patients, we do not exclude the possibility of ATRX mutations in melanoma. In fact, a 4-7.5% rate of mutation in cutaneous melanoma is reported by TCGA, Broad and Yale studies (http://www.cbioportal.org). Interestingly, these mutations are distributed throughout the ATRX coding region and do not correlate with decreased mRNA levels (Supplemental Figure S1). This suggests that multiple mechanisms underlie ATRX dysregulation in melanoma – transcriptional regulation as described here and point mutations that may result in loss of protein expression, as reported for other tumor types (Cheung et al., 2012; Kannan et al., 2012; Schwartzentruber et al., 2012). While ATRX staining did not anti-correlate with macroH2A levels (data not shown), we previously showed that macroH2A is transcriptionally silenced by DNA methylation in malignant melanoma and thus might not be regulated at the level of chromatin deposition (Kapoor et al., 2010). The mechanism by which ATRX transcription is suppressed in melanoma may also be through epigenetic silencing (e.g. DNA methylation or histone modifications), or by microRNA mediated regulation (Pacurari et al., 2013). Finally, we posit that investigating the chromatin landscape of tumors that have lost ATRX expression should provide insight into the mechanism(s) by which ATRX loss drives melanoma progression.

ATRX and DAXX: Mechanisms and Mutations

Recent genome sequencing efforts in a variety of cancers have revealed mutations and/or structural alterations in ATRX and DAXX, which together encode a complex that deposits histone variant H3.3 into repetitive heterochromatin. These regions include retrotransposons, pericentric heterochromatin, and telomeres, the latter of which show deregulation in ATRX/DAXX-mutant tumors. Interestingly, ATRX and DAXX mutations are often found in pediatric tumors, suggesting a particular developmental context in which these mutations drive disease. Here we review the functions of ATRX and DAXX in chromatin regulation as well as their potential contributions to tumorigenesis. We place emphasis on the chromatin remodeler ATRX, which is mutated in the developmental disorder for which it is named, α-thalassemia, mental retardation, X-linked syndrome, and at high frequency in a number of adult and pediatric tumors.

Identical repeated backbone of the human genome

BackgroundIdentical sequences with a minimal length of about 300 base pairs (bp) have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR) events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB) of the reference human genome.ResultsThe IRB sequences are distributed all over the genome in 66,600 regions, which correspond to ~2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs). By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes.ConclusionsIn general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

Abstract A12: Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma

Malignant melanoma is the most lethal form of skin cancer with rising incidence. Once metastasis occurs, patients have a dismal prognosis, largely due to limited systemic treatment with chemotherapy and resistance to targeted therapies. Thus, effective therapies with long-term responses are currently lacking. Although much effort has focused on characterizing and targeting the genetic alterations in melanoma, the identification of epigenetic players remains poorly understood. Chromatin dynamics have recently been shown to exert a critical function in a number of cancers, including melanoma, and emerging evidence points towards a role of histone variants as key regulatory molecules in cancer. H2A.Z is a highly conserved H2A variant, harboring two different isoforms in vertebrates, H2A.Z.1 and H2A.Z.2. High levels of H2A.Z promote cell proliferation in breast, prostate and bladder cancers, however studies so far have focused primarily on H2A.Z.1 or did not clearly distinguish between the two isoforms. Here, we report a role for the unappreciated isoform H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma. To our knowledge, this is the first evidence to implicate a distinct role for this H2A.Z isoform in any tumor type. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation, which occurs through a G1 to S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, which are highly expressed and acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic cells. We further identified the BET (bromodomain and extraterminal domain) family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and our data suggest that H2A.Z.2 exerts its oncogenic function by maintaining the global levels of BRD2 and histone H4 acetylation. Furthermore, H2A.Z.2 depletion sensitizes melanoma cells to targeted therapies and chemotherapy. Collectively, our findings implicate H2A.Z.2 as a driver of melanoma pathogenesis. Owing to the fact that histone modification is a reversible process, H2A.Z.2 and BRD2 hold translational potential for novel therapeutic strategies. Citation Format: Chiara Vardabasso, Alexandre Gaspar-Maia, Sebastian Punzeler, David Valle-Garcia, Dan Hasson, Tobias Straub, Eva C. Keilhauer, Thomas Strub, Taniya Panda, Miguel F. Segura, Chi-Yeh Chung, Amit K. Verma, Matthias Mann, Eva Hernando, Sandra B. Hake, Emily Bernstein. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A12.