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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 trans-membrane 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.

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

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

Histone variants are emerging as key regulatory molecules in cancer. We report a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z-interacting protein, levels of which are also elevated in melanoma. We further demonstrate that H2A.Z.2-regulated genes are bound by BRD2 and E2F1 in an H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.

This unit describes a streamlined two-step protocol for the isolation of adult murine cardiomyocytes with subsequent Chromatin ImmunoPrecipitation (ChIP). Isolation and culturing of cardiomyocytes is a delicate process and the protocol presented here optimizes the combination of cardiomyocyte isolation with ChIP. ChIP is an invaluable method for analyzing molecular interactions occurring between a specific protein (or its post-translationally modified form) and a region of genomic DNA. ChIP has become a widely used technique in the last decade since several groundbreaking studies have focused attention on epigenetics and have identified many epigenetic regulatory mechanisms. However, epigenetics within cardiovascular biology is a new area of focus for many investigators, and we have optimized a method for performing ChIP in adult murine cardiomyocytes, as we feel this will be an important aid to both the cardiovascular field and for the development of cell- and tissue-specific ChIP.

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.

Various members of tripartite motif (TRIM) protein family display antiviral properties, targeting retroviruses in particular. The potential activity of TRIM19, better known as promyelocytic leukemia protein (PML) against several viruses has been well documented, yet it's role in HIV-1 infection remains elusive. One of the most important cellular partners of HIV-1, P-TEFb kinase complex, composed of CDK9 and CyclinT1 plays a crucial role in regulation of HIV-1 transcription. We have previously demonstrated that both members of P-TEFb interact with PML protein and localize inside the PML Nuclear Bodies (PML NB) [1,2]. In particular, we found that the acetylated and enzymaticaly inactive form of CDK9 binds PML and can be separated with PML in the insoluble chromatin fraction. Our ChIP analysis revealed that acetylated form of CDK9 localizes to the integrated and transcriptionally silent viral genome, thus indicating that PML might have yet unidentified role in regulation of HIV-1 infection and latency [2]. We indeed identified PML protein bound to the silent viral genome in Jurkat cells harboring single integrations of HIV-1 provirus. The distribution of PML protein along the viral genome could be correlated with bimethylated lysine 9 (K9) on Histone 3 (2MetK9 H3), a mark of facultative heterochromatin. Transcriptional activation of the provirus with TNF-α led to a displacement of PML protein from the genome and to a substantial loss of 2MetK9H3 mark. By three-dimensional immuno fluorescent in situ hybridization (3D Immuno DNA FISH) that we developed we visualized the viral genome in close proximity to PML NB and we characterized this position as adjacent. Within the population of cells tested in the silenced state, 40% of the cells showed proximity at the distance of less that 0.6μm. PML NB were recently described to have a close spatial relationship with the so called KAKA foci, characterized by the presence of KAP-1 (or Trim28) and KRAB-Zinc Finger proteins [3] thus prompting us to analyse the relationship of the silent HIV-1 provirus with these nuclear structures. We detected numerous PML and KAKA foci in both Jurkat cells (J-LAT model of latency) and primary human CD4+T lymphocytes, with HIV-1 residing either in close proximity to PML or to KAKA foci (~40% showing less than 0.6μm distance) in J-LAT latency model. Whether this spatial localization of HIV-1 provirus is correlated to silencing of the virus genome or is a general feature of HIV-1 integration and replication remains still to be determined.