Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network.

The embryonic stem (ES) cell transcriptional and chromatin-modifying networks are critical for self-renewal maintenance. However, it remains unclear whether these networks functionally interact and, if so, what factors mediate such interactions. Here, we show that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a regulator of ES cell self-renewal. We demonstrate that Wdr5, an "effector" of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. The Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators, and furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for the maintenance of ES cell self-renewal and somatic cell reprogramming.

ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globin expression.

The histone variant macroH2A generally associates with transcriptionally inert chromatin; however, the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase ATRX (α-thalassemia/MR, X-linked) as a novel macroH2A-interacting protein. Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator of macroH2A's chromatin association. In human erythroleukemic cells deficient for ATRX, macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of α-globin expression. Collectively, our results implicate deregulation of macroH2A's distribution as a contributing factor to the α-thalassemia phenotype of ATRX syndrome.

The histone variant macroH2A suppresses melanoma progression through regulation of CDK8.

Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

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.

RSC functions as an early double-strand-break sensor in the cell's response to DNA damage.

The detection of a DNA double-strand break (DSB) is necessary to initiate DSB repair. Several proteins, including the MRX/N complex, Tel1/ATM (ataxia telangiectasia mutated), and Mec1/ATR (ATM and Rad3 related), have been proposed as sensors of DNA damage, yet how they recognize the breaks is poorly understood. DSBs occur in the context of chromatin, implicating factors capable of altering local and/or global chromatin structure in the cellular response to DNA damage, including DSB sensing. Emerging evidence indicates that ATP-dependent chromatin-remodeling complexes function in DNA repair. Here we describe an important and novel early role for the RSC ATP-dependent chromatin remodeler linked to DSB sensing in the cell's DNA-damage response. RSC is required for full levels of H2A phosphorylation because it facilitates the recruitment of Tel1/ATM and Mec1/ATR to the break site. Consistent with these results, we also show that Rsc2 is needed for efficient activation of the Rad53-dependent checkpoint, as well as for Cohesin's association with the break site. Finally, Rsc2 is needed for the DNA-damage-induced changes in nucleosome structure surrounding the DSB site. Together, these new findings functionally link RSC to DSB sensing, highlighting the importance of ATP-dependent chromatin-remodeling factors in the cell's early response to DNA damage.

Smarcc1/Baf155 couples self-renewal gene repression with changes in chromatin structure in mouse embryonic stem cells.

Little is known about the molecular mechanism(s) governing differentiation decisions in embryonic stem cells (ESCs). To identify factors critical for ESC lineage formation, we carried out a functional genetic screen for factors affecting Nanog promoter activity during mESC differentiation. We report that members of the PBAF chromatin remodeling complex, including Smarca4/Brg1, Smarcb1/Baf47, Smarcc1/Baf155, and Smarce1/Baf57, are required for the repression of Nanog and other self-renewal gene expression upon mouse ESC (mESC) differentiation. Knockdown of Smarcc1 or Smarce1 suppressed loss of Nanog expression in multiple forms of differentiation. This effect occurred in the absence of self-renewal factors normally required for Nanog expression (e.g., Oct4), possibly indicating that changes in chromatin structure, rather than loss of self-renewal gene transcription per se, trigger differentiation. Consistent with this notion, mechanistic studies demonstrated that expression of Smarcc1 is necessary for heterochromatin formation and chromatin compaction during differentiation. Collectively, our data reveal that Smarcc1 plays important roles in facilitating mESCs differentiation by coupling gene repression with global and local changes in chromatin structure.

Deciphering UV-induced DNA Damage Responses to Prevent and Treat Skin Cancer.

Ultraviolet (UV) radiation is among the most prevalent environmental factors that influence human health and disease. Even 1 h of UV irradiation extensively damages the genome. To cope with resulting deleterious DNA lesions, cells activate a multitude of DNA damage response pathways, including DNA repair. Strikingly, UV-induced DNA damage formation and repair are affected by chromatin state. When cells enter S phase with these lesions, a distinct mutation signature is created via error-prone translesion synthesis. Chronic UV exposure leads to high mutation burden in skin and consequently the development of skin cancer, the most common cancer in the United States. Intriguingly, UV-induced oxidative stress has opposing effects on carcinogenesis. Elucidating the molecular mechanisms of UV-induced DNA damage responses will be useful for preventing and treating skin cancer with greater precision. Excitingly, recent studies have uncovered substantial depth of novel findings regarding the molecular and cellular consequences of UV irradiation. In this review, we will discuss updated mechanisms of UV-induced DNA damage responses including the ATR pathway, which maintains genome integrity following UV irradiation. We will also present current strategies for preventing and treating nonmelanoma skin cancer, including ATR pathway inhibition for prevention and photodynamic therapy for treatment.

Microenvironment-derived factors driving metastatic plasticity in melanoma.

Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.

A Quantitative System for Studying Metastasis Using Transparent Zebrafish.

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

ATRX: the case of a peculiar chromatin remodeler.

The SWI/SNF-like chromatin remodeler ATRX has recently garnered renewed attention. ATRX mutations were first identified in patients bearing the syndrome after which it is named, alpha thalassemia/mental retardation, X-linked. While ATRX has long been implicated in transcriptional regulation through multiple mechanisms, recent studies have identified a role for ATRX in the regulation of histone variant deposition. In addition, current reports describe ATRX to be mutated at high percentages in multiple tumor types, suggestive of a potential 'driver' role in cancer. Here we discuss the numerous and seemingly diverse roles for ATRX in transcriptional regulation and histone deposition and suggest that ATRX's effects are mediated by its regulation of histones within the chromatin template.

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. Using fibroblasts derived from macroH2A double knockout (dKO) mice, here 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 (wt) 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 dKO-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.

Regulation of R7 and R8 differentiation by the spalt genes.

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.