RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

The role of chromatin remodeler SMARCA4/BRG1 in brain cancers: a potential therapeutic target.

The chromatin remodeler SMARCA4/BRG1 is a key epigenetic regulator with diverse roles in coordinating the molecular programs that underlie brain tumour development. BRG1 function in brain cancer is largely specific to the tumour type and varies further between tumour subtypes, highlighting its complexity. Altered SMARCA4 expression has been linked to medulloblastoma, low-grade gliomas such as oligodendroglioma, high-grade gliomas such as glioblastoma and atypical/teratoid rhabdoid tumours. SMARCA4 mutations in brain cancer predominantly occur in the crucial catalytic ATPase domain, which is associated with tumour suppressor activity. However, SMARCA4 is opposingly seen to promote tumourigenesis in the absence of mutation and through overexpression in other brain tumours. This review explores the multifaceted interaction between SMARCA4 and various brain cancer types, highlighting its roles in tumour pathogenesis, the pathways it regulates, and the advances that have been made in understanding the functional relevance of mutations. We discuss developments made in targeting SMARCA4 and the potential to translate these to adjuvant therapies able to enhance current methods of brain cancer treatment.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

BRG1 knockdown inhibits proliferation through multiple cellular pathways in prostate cancer.

BACKGROUND: BRG1 (encoded by SMARCA4) is a catalytic component of the SWI/SNF chromatin remodelling complex, with key roles in modulating DNA accessibility. Dysregulation of BRG1 is observed, but functionally uncharacterised, in a wide range of malignancies. We have probed the functions of BRG1 on a background of prostate cancer to investigate how BRG1 controls gene expression programmes and cancer cell behaviour. RESULTS: Our investigation of SMARCA4 revealed that BRG1 is over-expressed in the majority of the 486 tumours from The Cancer Genome Atlas prostate cohort, as well as in a complementary panel of 21 prostate cell lines. Next, we utilised a temporal model of BRG1 depletion to investigate the molecular effects on global transcription programmes. Depleting BRG1 had no impact on alternative splicing and conferred only modest effect on global expression. However, of the transcriptional changes that occurred, most manifested as down-regulated expression. Deeper examination found the common thread linking down-regulated genes was involvement in proliferation, including several known to increase prostate cancer proliferation (KLK2, PCAT1 and VAV3). Interestingly, the promoters of genes driving proliferation were bound by BRG1 as well as the transcription factors, AR and FOXA1. We also noted that BRG1 depletion repressed genes involved in cell cycle progression and DNA replication, but intriguingly, these pathways operated independently of AR and FOXA1. In agreement with transcriptional changes, depleting BRG1 conferred G1 arrest. CONCLUSIONS: Our data have revealed that BRG1 promotes cell cycle progression and DNA replication, consistent with the increased cell proliferation associated with oncogenesis.

DNA methylation changes following DNA damage in prostate cancer cells.

Many cancer therapies operate by inducing double-strand breaks (DSBs) in cancer cells, however treatment-resistant cells rapidly initiate mechanisms to repair damage enabling survival. While the DNA repair mechanisms responsible for cancer cell survival following DNA damaging treatments are becoming better understood, less is known about the role of the epigenome in this process. Using prostate cancer cell lines with differing sensitivities to radiation treatment, we analysed the DNA methylation profiles prior to and following a single dose of radiotherapy (RT) using the Illumina Infinium HumanMethylation450 BeadChip platform. DSB formation and repair, in the absence and presence of the DNA hypomethylating agent, 5-azacytidine (5-AzaC), were also investigated using γH2A.X immunofluorescence staining. Here we demonstrate that DNA methylation is generally stable following a single dose of RT; however, a small number of CpG sites are stably altered up to 14 d following exposure. While the radioresistant and radiosensitive cells displayed distinct basal DNA methylation profiles, their susceptibility to DNA damage appeared similar demonstrating that basal DNA methylation has a limited influence on DSB induction at the regions examined. Recovery from DSB induction was also similar between these cells. Treatment with 5-AzaC did not sensitize resistant cells to DNA damage, but rather delayed recruitment of phosphorylated BRCA1 (S1423) and repair of DSBs. These results highlight that stable epigenetic changes are possible following a single dose of RT and may have significant clinical implications for cancer treatment involving recurrent or fractionated dosing regimens.

Age, but Not Amyloidosis, Induced Changes in Global Levels of Histone Modifications in Susceptible and Disease-Resistant Neurons in Alzheimer's Disease Model Mice.

There is increasing interest in the role of epigenetic alterations in Alzheimer's disease (AD). The epigenome of every cell type is distinct, yet data regarding epigenetic change in specific cell types in aging and AD is limited. We investigated histone tail modifications in neuronal subtypes in wild-type and APP/PS1 mice at 3 (pre-pathology), 6 (pathology-onset) and 12 (pathology-rich) months of age. In neurofilament (NF)-positive pyramidal neurons (vulnerable to AD pathology), and in calretinin-labeled interneurons (resistant to AD pathology) there were no global alterations in histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 27 acetylation (H3K27ac) or histone 3 lysine 27 trimethylation (H3K27me3) in APP/PS1 compared to wild-type mice at any age. Interestingly, age-related changes in the presence of H3K27ac and H3K27me3 were detected in NF-labeled pyramidal neurons and calretinin-positive interneurons, respectively. These data suggest that the global levels of histone modifications change with age, whilst amyloid plaque deposition and its sequelae do not result in global alterations of H3K4me3, H3K27ac and H3K27me3 in NF-positive pyramidal neurons or calretinin-labeled interneurons.

Integrated epigenomic analysis stratifies chromatin remodellers into distinct functional groups.

BACKGROUND: ATP-dependent chromatin remodelling complexes are responsible for establishing and maintaining the positions of nucleosomes. Chromatin remodellers are targeted to chromatin by transcription factors and non-coding RNA to remodel the chromatin into functional states. However, the influence of chromatin remodelling on shaping the functional epigenome is not well understood. Moreover, chromatin remodellers have not been extensively explored as a collective group across two-dimensional and three-dimensional epigenomic layers. RESULTS: Here, we have integrated the genome-wide binding profiles of eight chromatin remodellers together with DNA methylation, nucleosome positioning, histone modification and Hi-C chromosomal contacts to reveal that chromatin remodellers can be stratified into two functional groups. Group 1 (BRG1, SNF2H, CHD3 and CHD4) has a clear preference for binding at 'actively marked' chromatin and Group 2 (BRM, INO80, SNF2L and CHD1) for 'repressively marked' chromatin. We find that histone modifications and chromatin architectural features, but not DNA methylation, stratify the remodellers into these functional groups. CONCLUSIONS: Our findings suggest that chromatin remodelling events are synchronous and that chromatin remodellers themselves should be considered simultaneously and not as individual entities in isolation or necessarily by structural similarity, as they are traditionally classified. Their coordinated function should be considered by preference for chromatin features in order to gain a more accurate and comprehensive picture of chromatin regulation.

Distinct mechanisms of regulation of the ITGA6 and ITGB4 genes by RUNX1 in myeloid cells.

Integrins are transmembrane adhesion receptors that play an important role in hematopoiesis by facilitating interactions between hematopoietic cells and extracellular matrix components of the bone marrow and hematopoietic tissues. These interactions are important in regulating the function, proliferation, and differentiation of hematopoietic cells, as well as their homing and mobilization in the bone marrow. Not surprisingly altered expression and function of integrins plays a key role in the development and progression of cancer including leukemias. However, the regulation of integrin gene expression is not well characterized and the mechanisms by which integrin genes are disrupted in cancer remain unclear. Here we demonstrate for the first time that a key regulator of hematopoiesis, RUNX1, binds to and regulates the promoters of both the ITGA6 and ITGB4 genes in myeloid cells. The ITGA6 and ITGB4 integrin genes form the α6ß4 integrin receptor. However, our data indicate that RUNX1 functions differently at these two promoters. RUNX1 regulates ITGA6 through a consensus RUNX1 binding motif in its promoter. In contrast, although the ITGB4 promoter is also activated by RUNX1, it does so in the absence of a recognized consensus RUNX1 binding motif. Furthermore, our data suggest that regulation of ITGB4 may involve interactions between the promoter and upstream regulatory elements.

Three-dimensional disorganization of the cancer genome occurs coincident with long-range genetic and epigenetic alterations.

A three-dimensional chromatin state underpins the structural and functional basis of the genome by bringing regulatory elements and genes into close spatial proximity to ensure proper, cell-type-specific gene expression profiles. Here, we performed Hi-C chromosome conformation capture sequencing to investigate how three-dimensional chromatin organization is disrupted in the context of copy-number variation, long-range epigenetic remodeling, and atypical gene expression programs in prostate cancer. We find that cancer cells retain the ability to segment their genomes into megabase-sized topologically associated domains (TADs); however, these domains are generally smaller due to establishment of additional domain boundaries. Interestingly, a large proportion of the new cancer-specific domain boundaries occur at regions that display copy-number variation. Notably, a common deletion on 17p13.1 in prostate cancer spanning the TP53 tumor suppressor locus results in bifurcation of a single TAD into two distinct smaller TADs. Change in domain structure is also accompanied by novel cancer-specific chromatin interactions within the TADs that are enriched at regulatory elements such as enhancers, promoters, and insulators, and associated with alterations in gene expression. We also show that differential chromatin interactions across regulatory regions occur within long-range epigenetically activated or silenced regions of concordant gene activation or repression in prostate cancer. Finally, we present a novel visualization tool that enables integrated exploration of Hi-C interaction data, the transcriptome, and epigenome. This study provides new insights into the relationship between long-range epigenetic and genomic dysregulation and changes in higher-order chromatin interactions in cancer.

Alterations in Three-Dimensional Organization of the Cancer Genome and Epigenome.

The structural and functional basis of the genome is provided by the three-dimensional (3D) chromatin state. To enable accurate gene regulation, enhancer elements and promoter regions are brought into close spatial proximity to ensure proper, cell type-specific gene expression. In cancer, genetic and epigenetic processes can deregulate the transcriptional program. To investigate whether the 3D chromatin state is also disrupted in cancer we performed Hi-C chromosome conformation sequencing in normal and prostate cancer cells and compared the chromatin interaction maps with changes to the genome and epigenome. Notably, we find that additional topologically associated domain (TAD) boundaries are formed in cancer cells resulting in smaller TADs and altered gene expression profiles. The new TAD boundaries are commonly associated with copy-number changes observed in the cancer genome. We also identified new cancer-specific chromatin loops within TADs that are enriched for enhancers and promoters. Finally, we find that many of the long-range epigenetically silenced (LRES) and long-range epigenetically active (LREA) regions in cancer are characterized by differential chromatin interactions. Together our data provide a new insight into charting alterations in higher-order structure and the relationship with genetic, epigenetic, and transcriptional changes across the cancer genome.

The Leukemia Inhibitory Factor Receptor Gene Is a Direct Target of RUNX1.

Activation of cytokine signaling via the leukemia inhibitory factor receptor (LIFR) plays an integral role in hematopoiesis, osteogenesis, and placental development, along with mediating neurotrophic mechanisms. However, the regulatory control of the LIFR gene has remained largely unexplored. Here, we characterize the LIFR gene as a novel target of the RUNX1 transcription factor. The RUNX1 transcription factor is an essential regulator of hematopoiesis and is a frequent target of point mutations and chromosomal alterations in leukemia. RUNX1 regulates hematopoiesis through its control of genes important for hematopoietic cell growth, proliferation, and differentiation, including a number of cytokines and cytokine receptors. LIFR is regulated by two alternate promoters: a placental-specific and a ubiquitously active general promoter. We show that both of these promoters are regulated by RUNX1. However, in myeloid cells LIFR expression is driven solely by the general LIFR promoter with our data indicating that the placental promoter is epigenetically silenced in these cells. While RUNX1 activates the LIFR general promoter, the oncogenic RUNX1-ETO fusion protein generated by the t(8;21) translocation commonly associated with acute myeloid leukemia represses promoter activity. The data presented here establish LIFR as a transcriptional target of RUNX1 and suggest that disruption of RUNX1 activity in myeloid cells may result in altered LIFR signaling in these cells.

Interplay between Transcription Factors and the Epigenome: Insight from the Role of RUNX1 in Leukemia.

The genome has the ability to respond in a precise and co-ordinated manner to cellular signals. It achieves this through the concerted actions of transcription factors and the chromatin platform, which are targets of the signaling pathways. Our understanding of the molecular mechanisms through which transcription factors and the chromatin landscape each control gene activity has expanded dramatically over recent years, and attention has now turned to understanding the complex, multifaceted interplay between these regulatory layers in normal and disease states. It has become apparent that transcription factors as well as the components and modifiers of the epigenetic machinery are frequent targets of genomic alterations in cancer cells. Through the study of these factors, we can gain unique insight into the dynamic interplay between transcription factors and the epigenome, and how their dysregulation leads to aberrant gene expression programs in cancer. Here, we will highlight how these factors normally co-operate to establish and maintain the transcriptional and epigenetic landscape of cells, and how this is reprogramed in cancer, focusing on the RUNX1 transcription factor and oncogenic derivative RUNX1-ETO in leukemia as paradigms of transcriptional and epigenetic reprograming.

Genome-wide nucleosome occupancy and DNA methylation profiling of four human cell lines.

DNA methylation and nucleosome positioning are two key mechanisms that contribute to the epigenetic control of gene expression. During carcinogenesis, the expression of many genes is altered alongside extensive changes in the epigenome, with repressed genes often being associated with local DNA hypermethylation and gain of nucleosomes at their promoters. However the spectrum of alterations that occur at distal regulatory regions has not been extensively studied. To address this we used Nucleosome Occupancy and Methylation sequencing (NOMe-seq) to compare the genome-wide DNA methylation and nucleosome occupancy profiles between normal and cancer cell line models of the breast and prostate. Here we describe the bioinformatic pipeline and methods that we developed for the processing and analysis of the NOMe-seq data published by (Taberlay et al., 2014 [1]) and deposited in the Gene Expression Omnibus with accession GSE57498.

Chromatin remodeler mutations in human cancers: epigenetic implications.

Chromatin remodeler complexes exhibit the ability to alter nucleosome composition and positions, with seemingly divergent roles in the regulation of chromatin architecture and gene expression. The outcome is directed by subunit variation and interactions with accessory factors. Recent studies have revealed that subunits of chromatin remodelers display an unexpectedly high mutation rate and/or are inactivated in a number of cancers. Consequently, a repertoire of epigenetic processes are likely to be affected, including interactions with histone modifying factors, as well as the ability to precisely modulate nucleosome positions, DNA methylation patterns and potentially, higher-order genome structure. However, the true significance of chromatin remodeler genetic aberrations in promoting a cascade of epigenetic changes, particularly during initiation and progression of cancer, remains largely unknown.

Reconfiguration of nucleosome-depleted regions at distal regulatory elements accompanies DNA methylation of enhancers and insulators in cancer.

It is well established that cancer-associated epigenetic repression occurs concomitant with CpG island hypermethylation and loss of nucleosomes at promoters, but the role of nucleosome occupancy and epigenetic reprogramming at distal regulatory elements in cancer is still poorly understood. Here, we evaluate the scope of global epigenetic alterations at enhancers and insulator elements in prostate and breast cancer cells using simultaneous genome-wide mapping of DNA methylation and nucleosome occupancy (NOMe-seq). We find that the genomic location of nucleosome-depleted regions (NDRs) is mostly cell type specific and preferentially found at enhancers in normal cells. In cancer cells, however, we observe a global reconfiguration of NDRs at distal regulatory elements coupled with a substantial reorganization of the cancer methylome. Aberrant acquisition of nucleosomes at enhancer-associated NDRs is associated with hypermethylation and epigenetic silencing marks, and conversely, loss of nucleosomes with demethylation and epigenetic activation. Remarkably, we show that nucleosomes remain strongly organized and phased at many facultative distal regulatory elements, even in the absence of a NDR as an anchor. Finally, we find that key transcription factor (TF) binding sites also show extensive peripheral nucleosome phasing, suggesting the potential for TFs to organize NDRs genome-wide and contribute to deregulation of cancer epigenomes. Together, our findings suggest that "decommissioning" of NDRs and TFs at distal regulatory elements in cancer cells is accompanied by DNA hypermethylation susceptibility of enhancers and insulator elements, which in turn may contribute to an altered genome-wide architecture and epigenetic deregulation in malignancy.

Mining cancer methylomes: prospects and challenges.

There are over 28 million CpG sites in the human genome. Assessing the methylation status of each of these sites will be required to understand fully the role of DNA methylation in health and disease. Genome-wide analysis, using arrays and high-throughput sequencing, has enabled assessment of large fractions of the methylome, but each protocol comes with unique advantages and disadvantages. Notably, except for whole-genome bisulfite sequencing, most commonly used genome-wide methods detect <5% of all CpG sites. Here, we discuss approaches for methylome studies and compare genome coverage of promoters, genes, and intergenic regions, and capacity to quantitate individual CpG methylation states. Finally, we examine the extent of published cancer methylomes that have been generated using genome-wide approaches.

DNA methylation screening identifies driver epigenetic events of cancer cell survival.

Cancer cells typically exhibit aberrant DNA methylation patterns that can drive malignant transformation. Whether cancer cells are dependent on these abnormal epigenetic modifications remains elusive. We used experimental and bioinformatic approaches to unveil genomic regions that require DNA methylation for survival of cancer cells. First, we surveyed the residual DNA methylation profiles in cancer cells with highly impaired DNA methyltransferases. Then, we clustered these profiles according to their DNA methylation status in primary normal and tumor tissues. Finally, we used gene expression meta-analysis to identify regions that are dependent on DNA methylation-mediated gene silencing. We further showed experimentally that these genes must be silenced by DNA methylation for cancer cell survival, suggesting these are key epigenetic events associated with tumorigenesis.

DNA methylation and cancer.

DNA methylation acts in concert with other epigenetic mechanisms to regulate normal gene expression and facilitate chromatin organization within cells. Aberrant DNA methylation patterns are acquired during carcinogenic transformation; such events are often accompanied by alterations in chromatin structure at gene regulatory regions. The expression pattern of any given gene is achieved by interacting epigenetic mechanisms. First, the insertion of nucleosomes at transcriptional start sites prevents the binding of the transcriptional machinery and additional cofactors that initiate gene expression. Second, nucleosomes anchor all of the DNMT3A and DNMT3B methyltransferase proteins in the cell, which suggests a role for histone octamers in the establishment of DNA methylation patterns. During carcinogenesis, epigenetic switching and 5-methylcytosine reprogramming result in the aberrant hypermethylation of CpG islands, reducing epigenetic plasticity of critical developmental and tumor suppressor genes, rendering them unresponsive to normal stimuli. Here, we will discuss the importance of both established and novel molecular concepts that may underlie the role of DNA methylation in cancer.