Many genes are regulated at the level of a Pol II that is recruited to a nucleosome-free region upstream of the +1 nucleosome. How the Mediator coactivator complex, which functions at multiple steps, ...affects transcription through the promoter proximal region, including this nucleosome, remains largely unaddressed. We have established a fully defined in vitro assay system to delineate mechanisms for Pol II transit across the +1 nucleosome. Our results reveal cooperative functions of multiple cofactors, particularly of Mediator and elongation factor SII, in transcribing into this nucleosome. This is achieved, in part, through an unusual activity of SII that alters the intrinsic catalytic properties of promoter-proximal Pol II and, in concert with the Mediator, leads to enhancement in transcription of nucleosomal DNA. Our data provide additional mechanistic bases for Mediator function after recruitment of Pol II and, potentially, for regulation of genes controlled via nucleosome-mediated promoter-proximal pausing.
► Development of an in vitro system to analyze transcription through a +1 nucleosome ► There is functional interplay between Mediator and chromatin cofactors ► SII induces reiterative oligomeric RNA synthesis by promoter-bound Pol II ► Mediator translates this activity of SII into processive transcription
Individual cell fate decisions can vary according to changes in gene expression in response to environmental, developmental, or metabolic cues. This plasticity is tightly regulated during embryonic ...development and mediated by the exquisitely coordinated activation and repression of groups of genes. Genes that become repressed are immersed in a condensed chromatin environment that renders them refractory to stimulation. This mechanism is responsible for both the loss of cell plasticity during differentiation and the preservation of cell identity. Understanding the molecular events involved in the establishment and maintenance of these restrictive domains will benefit the design of strategies for cellular reprogramming, differentiation, and cancer treatment.
We identify LSD1 (lysine-specific demethylase 1; also known as KDM1A and AOF2) as a key histone modifier that participates in the maintenance of pluripotency through the regulation of bivalent ...domains, a chromatin environment present at the regulatory regions of developmental genes that contains both H3K4 di/trimethylation and H3K27 trimethylation marks. LSD1 occupies the promoters of a subset of developmental genes that contain bivalent domains and are co-occupied by OCT4 and NANOG in human embryonic stem cells, where it controls the levels of H3K4 methylation through its demethylase activity. Thus, LSD1 has a role in maintaining the silencing of several developmental genes in human embryonic stem cells by regulating the critical balance between H3K4 and H3K27 methylation at their regulatory regions.
The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for ...reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.
Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent ...and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.
► hESCs and hiPSCs show more gene copy number variation than somatic cells ► Degree of abnormality differs more between hESC lines than hiPSC lines ► Deletions common in hiPSCs after reprogramming, duplications appear over time ► Recurrent duplications occur at specific genomic loci in pluripotent cells
Nuclear receptors, like other transcriptional activators, switch on gene transcription by recruiting a complex network of coregulatory proteins. Here, we have identified the arginine ...methyltransferase PRMT1 as a coactivator for HNF4, an orphan nuclear receptor that regulates the expression of genes involved in diverse metabolic pathways. Remarkably, PRMT1, whose methylation activity on histone H4 strongly correlates with induction of HNF4 target genes in differentiating enterocytes, regulates HNF4 activity through a bipartite mechanism. First, PRMT1 binds and methylates the HNF4 DNA-binding domain (DBD), thereby enhancing the affinity of HNF4 for its binding site. Second, PRMT1 is recruited to the HNF4 ligand-binding domain (LBD) through a mechanism that involves the p160 family of coactivators and methylates histone H4 at arginine 3. This, together with recruitment of the histone acetyltransferase p300, leads to nucleosomal alterations and subsequent RNA polymerase II preinitiation complex formation.
Inhibitors that prevent the binding of bromodomains to acetylated histones hold therapeutic potential. However, the effects of targeting most of the 60 different bromodomains found in the human ...proteome remain unexplored. Here, we investigate the molecular mechanisms responsible for the antiproliferative properties of CREBBP/EP300 bromodomain inhibition in ER-negative breast cancer cell lines. We show using genetic and chemical approaches that CREBBP/EP300 bromodomains are critical to support the proliferation of the triple-negative breast cancer cell line MDA-MB-453. Analysis of the transcriptional pathways affected by CREBBP/EP300 bromodomain inhibitors reveals that the expression of genes associated with super-enhancers is downregulated, which in turn are occupied by very high levels of androgen receptor (AR) in MDA-MB-453 cells. Treatment of MDA-MB-453 with CREBBP/EP300 bromodomain inhibitors downregulates the expression of an AR-dependent signature distinctive of breast cancer tumors that express AR and causes a decrease in H3K27ac levels at AR-binding sites. In accordance, in prostate cancer cell lines that express AR CREBBP/EP300 bromodomain inhibitors downregulate the expression of genes bound by AR and associated with super-enhancers. In summary, we report that triple-negative breast cancer cell lines that express AR are particularly sensitive to CREBBP/EP300 bromodomain inhibitors and consequently these inhibitors hold potential to treat this type of cancer. IMPLICATIONS: AR-dependent cancer cell lines are sensitive to CREBBP/EP300 bromodomain inhibitors.
Transcription-factor-induced reprogramming of somatic cells to pluripotency is a very inefficient process, probably due to the existence of important epigenetic barriers that are imposed during ...differentiation and that contribute to preserving cell identity. In an effort to decipher the molecular nature of these barriers, we followed a genome-wide approach, in which we identified macrohistone variants (macroH2A) as highly expressed in human somatic cells but downregulated after reprogramming to pluripotency, as well as strongly induced during differentiation. Knockdown of macrohistone variants in human keratinocytes increased the efficiency of reprogramming to pluripotency, whereas overexpression had opposite effects. Genome-wide occupancy profiles show that in human keratinocytes, macroH2A.1 preferentially occupies genes that are expressed at low levels and are marked with H3K27me3, including pluripotency-related genes and bivalent developmental regulators. The presence of macroH2A.1 at these genes prevents the regain of H3K4me2 during reprogramming, imposing an additional layer of repression that preserves cell identity.
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► Macrohistone variants are regulated during reprogramming and differentiation ► The levels of macrohistone variants modulate the efficiency of reprogramming ► MacroH2A.1 occupies pluripotency and differentiation genes in keratinocytes ► MacroH2A.1 occupancy prevents the gain of H3K4me2 during reprogramming
The epigenetic mechanisms responsible for maintaining the identity of somatic cells are not fully understood. Here, Izpisua Belmonte and colleagues show that macrohistone variants play an important role in preserving somatic cell identity. Macrohistone variants occupy the regulatory regions of bivalent and pluripotency-related genes in somatic cells, where they prevent the regain of the active chromatin mark H3K4me2 during the process of reprogramming to pluripotency.
The contribution of the non-coding genome to disease and its therapeutic potential have been largely unexplored. Recently, several epigenetic drugs developed for cancer treatment have been described ...to mediate therapeutic effects through the reactivation of the expression of transposable elements in cancer cells. This event activates innate immunity-related pathways and promotes the generation of neoantigens in tumor cells, improving the efficacy of immunotherapeutic treatments. This review focuses on the regulation of transposable elements by epigenetic inhibitors and its implications for immuno-oncology.
Rare diseases affect more than 300 million people worldwide. Diagnosing rare diseases is a major challenge as they have different causes and etiologies. Careful assessment of clinical symptoms often ...leads to the testing of the most common genetic alterations that could explain the disease. Patients with negative results for these tests frequently undergo whole exome or genome sequencing, leading to the identification of the molecular cause of the disease in 50% of patients at best. Therefore, a significant proportion of patients remain undiagnosed after sequencing their genome. Recently, approaches based on functional aspects of the genome, including transcriptomics and epigenomics, are beginning to emerge. Here, we will review these approaches, including studies that have successfully provided diagnoses for complex undiagnosed cases.
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