Roberto Ferrari

Epigenetic Reprogramming by Adenovirus e1a

Adenovirus e1a induces quiescent human cells to replicate. We found that e1a causes global relocalization of the RB (retinoblastoma) proteins (RB, p130, and p107) and p300/CBP histone acetyltransferases on promoters, the effect of which is to restrict the acetylation of histone 3 lysine-18 (H3K18ac) to a limited set of genes, thereby stimulating cell cycling and inhibiting antiviral responses and cellular differentiation. Soon after expression, e1a binds transiently to promoters of cell cycle and growth genes, causing enrichment of p300/CBP, PCAF (p300/CBP-associated factor), and H3K18ac; depletion of RB proteins; and transcriptional activation. e1a also associates transiently with promoters of antiviral genes, causing enrichment for RB, p130, and H4K16ac; increased nucleosome density; and transcriptional repression. At later times, e1a and p107 bind mainly to promoters of development and differentiation genes, repressing transcription. The temporal order of e1a binding requires its interactions with p300/CBP and RB proteins. Our data uncover a defined epigenetic reprogramming leading to cellular transformation.

Scl Represses Cardiomyogenesis in Prospective Hemogenic Endothelium and Endocardium

Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl(-/-) embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl(-/-) hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Scl(fl/fl)Rosa26Cre-ER(T2) embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl(-/-) endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.

New small nuclear RNA gene-like transcriptional units as sources of regulatory transcripts

By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.

The effects of ruthenium red on mitochondrial function during post-ischaemic reperfusion

Abstract Recently several attempts have been made to reduce reperfusion-induced calcium accumulation in the myocardium and in the mitochondria by using inhibitors of slow channel calcium transport such as verapamil, nifedipine and diltiazem. When these substances were given to the animals before death, or at the onset of ischaemia, they have been shown to prevent calcium accumulation in whole tissue and in isolated mitochondria [7–9, 15], while when added at the time of reperfusion, they failed to prevent myocardial calcium overloading [3, 15]. This supports the view that reperfusion-induced tissue calcium accumulation does not necessarily occur through the slow calcium channels and, at the present, the cause and the way in which this phenomenon can be modified by pharma-cological or other interventions remains unclear. For this reason, in this present study, we used ruthenium red in an attempt to reduce directly the reperfusion-induced mitochondrial calcium accumulation. Ruthenium is a polysaccaride dye which specifically inhibits calcium transport and binding by liver and heart mitochondria [7, 11].

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse

Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5meC levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. DOI: http://dx.doi.org/10.7554/eLife.06205.001

Viral manipulation of the host epigenome for oncogenic transformation.

The cancerous cellular state is associated with multiple epigenetic alterations, but elucidating the precise order of such alterations during tumorigenic progression and their contributions to the transformed phenotype remains a significant challenge in cancer biology. Here we discuss recent findings on how viral oncoproteins exploit specific epigenetic processes to coerce normal cells to replicate when they should remain quiescent — a hallmark of cancer. These findings may highlight roles of epigenetic processes in normal biology and shed light on epigenetic events occurring along the path of non-viral neoplastic transformation.

Adenovirus small E1A employs the lysine acetylases p300/CBP and tumor suppressor Rb to repress select host genes and promote productive virus infection.

Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication.

Polycomb Repressive Complex 1 (PRC1) Disassembles RNA Polymerase II Preinitiation Complexes

Background: PRC1 silences transcription by an unknown mechanism. Results: PRC1 can both block and dissociate PICs in general with the exception of TFIID. Conclusion: PRC1 gene silencing may involve the ability of TFIID to remain bound to gene promoters, leaving them in a poised state. Significance: Understanding how PRC1 regulates transcription deepens our basic understanding of key developmental processes. Despite the important role of Polycomb in genome-wide silencing, little is known of the specific biochemical mechanism by which it inactivates transcription. Here we address how recombinant Polycomb repressive complex 1 (PRC1) inhibits activated RNA polymerase II preinitiation complex (PIC) assembly using immobilized H3K27-methylated chromatin templates in vitro. Recombinant PRC1 inhibited transcription, but had little effect on binding of the activator as reported previously. In contrast, Mediator and the general transcription factors were blocked during assembly or dissociated from preassembled PICs. Importantly, among the PIC components, Tata Binding Protein (TBP) was the most resistant to eviction by PRC1. Immobilized template experiments using purified PRC1, transcription factor II D (TFIID), and Mediator indicate that PRC1 blocks the recruitment of Mediator, but not TFIID. We conclude that PRC1 functions to block or dissociate PICs by interfering with Mediator, but leaves TBP and perhaps TFIID intact, highlighting a specific mechanism for PRC1 transcriptional silencing. Analysis of published genome-wide datasets from mouse embryonic stem cells revealed that the Ring1b subunit of PRC1 and TBP co-enrich at developmental genes. Further, genes enriched for Ring1b and TBP are expressed at significantly lower levels than those enriched for Mediator, TBP, and Ring1b. Collectively, the data are consistent with a model in which PRC1 and TFIID could co-occupy genes poised for activation during development.

Reorganization of the host epigenome by a viral oncogene

Adenovirus small e1a oncoprotein causes ~70% reduction in cellular levels of histone H3 lysine 18 acetylation (H3K18ac). It is unclear, however, where this dramatic reduction occurs genome-wide. ChIP-sequencing revealed that by 24 h after expression, e1a erases 95% of H3K18ac peaks in normal, contact-inhibited fibroblasts and replaces them with one-third as many at new genomic locations. The H3K18ac peaks at promoters and intergenic regions of genes with fibroblast-related functions are eliminated after infection, and new H3K18ac peaks are established at promoters of highly induced genes that regulate cell cycling and at new putative enhancers. Strikingly, the regions bound by the retinoblastoma family of proteins in contact-inhibited fibroblasts gain new peaks of H3K18ac in the e1a-expressing cells, including 55% of RB1-bound loci. In contrast, over half of H3K9ac peaks are similarly distributed before and after infection, independently of RB1. The strategic redistribution of H3K18ac by e1a highlights the importance of this modification for transcriptional activation and cellular transformation as well as functional differences between the RB-family member proteins.

Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis

The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced, whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore, megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.