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Title: Embryonic Stem Cell (ES)-Specific Enhancers Specify the Expression Potential of ES Genes in Cancer}
Article:
Abstract:
Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers( ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer.
Introduction:
Disruption of normal cellular identity is a hallmark of cancer cells[1]. Various cancer types display gene expression profiles that show similarities to the expression program of embryonic stem cells( ES), but are absent from the differentiated tissue from which the cancer evolves. These ES and cancer-specific expression signatures comprise genes of the ES core module, including NANOG, OCT4, SOX2 and KLF4, the polycomb repressive complex 2( PRC2) module including EZH2, EED, and SUZ12, the MYC module and other genes[2–6]( reviewed in[7]). Many of these genes participate in inter- and intra-cellular signaling pathways. ES-like gene expression profiles may result from re-wiring of stem cell regulatory circuits in cancer. In line with this theory, DNase I hypersensitive sites profiling revealed extensive reprograming of ES-specific regulatory elements in cancer[8]. Moreover, ES-like chromatin patterns mediated by the PRC2, which predispose genes to DNA hypermethylation and permanent silencing in cancer, have been reported in loci proximal to transcription start sites( TSSs)[9, 10]. However, the regulatory mechanisms underlying the establishment of ES-related expression profiles in cancer are not well understood. Transcriptional enhancers control the expression of genes independently of their orientation, position or distance relative to TSS[11, 12]. Their genomic locations, covering more than 10% of the human genome, are readily detected on the basis of characteristic chromatin marks, primarily the monomethylation of histone 3 lysine-4( H3K4me1)[13, 14]. Many enhancers are specific to certain tissues or cell types, including a particularly large group of enhancers that are exclusive to embryonic and induced pluripotent stem cells[15–17]. In contrast to gene promoters, which tend to maintain their core chromatin structure across expressing and non-expressing cells, with some minor activity-related modifications, enhancer chromatin is not constant across cell types. Rather, enhancer sites may display active, inactive, or entirely naïve non-regulatory chromatin states across cells. The generation of the enhancer chromatin landscape is a complex multilayered process that involves the recruitment of chromatin modulators and pioneer transcription factors to certain genomic sites upon specification of particular cell lineages. Once established, enhancers can be found in active or inactive states, characterized by H3K27ac and H3K27me3, respectively, and by other chromatin features. However, in other cell lineages the very same sites do not display regulatory chromatin structures, and are unable to support transcription[18–21]. Interestingly, epigenetic reprograming of enhancer elements was previously shown to be prevalent in multiple cancer types, and evidence showing their role in cancer development has been reported[22–24]. However, the rules that govern the activation or silencing of these regulatory elements in cancer are still an enigma. Here we hypothesize that across the cancer genome, sites that once had developed an enhancer chromatin state throughout the history of the cancer cells would be more prone to be activated and to stimulate gene transcription in cancer than sites that had never been enhancers. Thus, the repertoire of enhancers established during the developmental history of cancer cells may define their expression potential. We and others have previously shown that the DNA methylation level is closely linked to enhancer activity states[25–28] and to expression levels of associated cancer genes[22, 25]. We now utilized DNA methylation data to investigate the fate of ES enhancers during normal hematopoietic development and in cancer. Surprisingly, we discovered that enhancers present in ES cells and absent from differentiated hematopoietic cells are especially prone to DNA methylation alterations in cancer. Moreover, these changes in methylation are in accordance with the expression levels of adjacent genes, and thus may provide a basis for the reactivation of ES genes in cancer.
Discussion:
Many tumors display mixtures of co-expressed lineage-specific and stem cell genes. This has led to the assumptions that tumors originate in undifferentiated cells embedded in the tissue, from dedifferentiation of tissue cells induced by carcinogenetic signals, or from activation of particular stem cell genes through stochastic regulatory mutations later selected by the developing cancer. Our results support an alternative scenario, in which upon loss of control over the global methylome typical of many cancer types, sites that already carry particular regulatory chromatin structures are prone to specific silencing or reactivating methylation modifications. In turn, these modifications may alter gene expression. Among gene regulatory sites, enhancers are particularly versatile owing to their tissue-specific chromatin structures. Indeed, several studies have shown that reprogramming of enhancer activity during cellular transformation such as acquisition of a drug resistance phenotype and other cancer-specific characteristics[35, 36] plays an essential role in cancer development and progression[22–24]. However, little is known about the mechanisms that underlie epigenetic rewiring. Our results indicate that patterning of enhancer chromatin along the developmental history of cells provides frameworks for gene activity from which cancers may further select their expression repertoire. The first important observation is that enhancers that appear only at the very beginning of the hematopoietic cell lineage commitment are specifically prone to methylation perturbation in blood cancers versus gene promoters and enhancers that are generated in blood stem or differentiated cells( Figs 1 and 2). These results are in line with recently published data showing that ES-specific accessible chromatin elements are target for epigenetic reprograming during cancer development, as identified by DNase I hypersensitive site mapping[8]. Second, we show that the level of DNA methylation characterizes the activity state of enhancers and associated genes( Figs 3, 4, 5 and 9), consistent with previous reports from our and other research groups in other model systems[22, 25],[37, 38]. The mechanism underlying this association is unclear, and may involve active adaptation of the DNA methylation level upon enhancer specifications, the effect of methylation on transcription factor binding, and possible feedbacks from the gene transcription machinery. In addition, genes are frequently controlled by sets of enhancers, which may be affected by various activity factors. Therefore, unmethylated enhancers do not necessarily dictate gene activity and methylated enhancers do not automatically eliminate gene expression. In line with these notions, our findings show that enhancer hypomethylation is not a simple outcome of the gene expression status or an absolute requirement for gene expression( Fig 9). Rather, we suggest that ES-specific enhancers provide a framework for gene activity profiles upon global methylation perturbation in cancer. Indeed, our genomic analyses of open resource data revealed a highly significant association between the methylation state of the predicted ESSE sites and the activity state of the corresponding genes in ES and cancers. To reveal whether these analyses identify actual regulatory sites that affect the expression potential of ES genes in cancer, we further performed a set of validating experiments. As a model gene we chose VEGFA, an important cancer gene that is expressed in many types of cancer, including lymphomas, and in ES cells, but not in differentiated white blood cells. Beyond the significant co-localization of ESSEs with the VEGF-A signaling pathway genes, including VEGFA itself( Fig 4A–4D), and the tendency of these genes to be upregulated in the cancer( Fig 4E and 4F), we also characterized a particular site predicted to be a novel VEGFA enhancer. We show that the predicted site displays the full list of essential enhancer characteristics, such as the ability to drive expression from a minimal promoter in the relevant cell type when placed upstream or downstream to the gene, and the physical interaction with the gene( Fig 5). Moreover, the predicted site does not only interact, but plays a causative role in controlling VEGFA activity in the cancer, as shown by the genome-editing experiments( Fig 6). Finally, we provide experimental evidence that the DNA methylation state of this ESSE site strongly affects its activity level as a transcriptional enhancer in leukemia cells( Fig 7). Cumulatively, these experiments strongly support our suggestion that the predicted sites, initially recognized through genomic correlation analyses, are cis-acting regulators that support the reactivation of ES genes in cancer. Because ES enhancers are present at the origin of all cell types, they are expected to affect gene expression in various types of cancer. Our pan-cancer analyses( Figs 8 and 9) support this assumption. Interestingly, HSC enhancers were not prone to hypomethylation in hematopoietic cancers( Figs 2B, 2C and S4). This may indicate that enhancers of a given cell lineage are relatively immune to the global hypomethylation of cancer cells. This could be due to lineage-specific chromatin structures, which help to maintain an appropriate chromatin activity state in the face of the disturbed epigenome of cancer cells. ESSEs may lack these additional structures found in differentiated cells, and are thus more prone to the global methylation perturbations typical of cancer cells. In conclusion, we reveal a novel pattern of regulatory epigenetic mutations in cancer. The new pattern is significantly more effective in ESSEs than in non ES-specific enhancers and promoters. Whereas the most reported pattern in gene promoters is hypermethylation of already-repressed genes, namely polycomb-repressed genes( with the exception of differentially methylated valleys( DMVs) in CRC)[4], the new pattern involves both hyper- and hypomethylations. This new pattern could participate in silencing particular ES genes in cancer, as well as lead to reactivation of ES genes that are silenced in normal somatic cells. The new pattern appears in both cancer cell lines and fresh tumor samples, and may predict the expression levels of important oncogenes across cancers and patients, as demonstrated for the VEGFA gene. The ability to detect this program with the limited genomic coverage provided by the available methylation methods highlights the potential for the discovery of additional key regulatory circuits in normal and cancer cells, once appropriate enhancer mapping methods have been developed.
We demonstrate that a particular subtype of transcriptional enhancers , which we termed ES-specific enhancers ( ESSEs ) , are especially prone to DNA methylation perturbations in cancer . Moreover , the methylation status of ESSEs , but not that of the enhancers of tissue stem cells , is associated with gene activity in cancer cell lines and in fresh tumor samples . Extending the previous reports on the hypermethylation of particular gene promoters in cancer , our analyses revealed both hyper- and hypo-methylation of regulatory elements in cancer , in association with silencing and enhancing , respectively , of important cancer genes . Thus , the discovered sites are appealing candidates for epigenetic control of cancer expression profiles .