05-623 Sigma-AldrichAnti-RNA polymerase II Antibody, clone CTD4H8

Antiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4(+) T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4(+) T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs.Antiretroviral therapy (ART) reduces HIV-1 replication to very low levels, but the virus persists in latently infected memory CD4(+) T cells, representing a long-lasting source of resurgent virus upon ART interruption. Based on the mode of action of didehydro-cortistatin A (dCA), a Tat-dependent transcription inhibitor, our work highlights an alternative approach to current HIV-1 eradication strategies to decrease the latent reservoir. In our model, dCA blocks the Tat feedback loop initiated after low-level basal reactivation, blocking transcriptional elongation and hence viral production from latently infected cells. Therefore, dCA combined with ART would be aimed at delaying or halting ongoing viral replication, reactivation, and replenishment of the latent viral reservoir. Thus, the latent pool of cells in an infected individual would be stabilized, and death of the long-lived infected memory T cells would result in a continuous decay of this pool over time, possibly culminating in the long-awaited sterilizing cure.

Rag1 and Rag2 gene expression in CD4(+)CD8(+) double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.

Insulin controls transcription to sustain its physiologic effects for the organism to adapt to environmental changes added to genetic predisposition. Nevertheless, insulin-induced transcriptional regulation by epigenetic factors and in defined nuclear territory remains elusive. Here we show that inner nuclear membrane (INM)-integrated caveolin-2 (Cav-2) regulates insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery. INM-targeted pY19-Cav-2 in response to insulin associates specifically with the A-type lamin, disengages the repressed Egr-1 and JunB promoters from lamin A/C through disassembly of H3K9me3, and facilitates assembly of H3K9ac, H3K18ac and H3K27ac by recruitment of GCN5 and p300 and the subsequent enrichment of RNA polymerase II (Pol II) on the promoters at the nuclear periphery. Our findings show that Cav-2 is an epigenetic regulator of histone H3 modifications, and provide novel mechanisms of insulin-response epigenetic activation at the nuclear periphery.

Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3'UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1-ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in this crop.

Macrophages play a pivotal role in tissue fibrogenesis, which underlies the pathogenesis of many end-stage chronic inflammatory diseases. MicroRNAs are key regulators of immune cell functions, but their roles in macrophage's fibrogenesis have not been characterized. Here we show that IL-4 and IL-13 induce miR-142-5p and downregulate miR-130a-3p in macrophages; these changes sustain the profibrogenic effect of macrophages. In vitro, miR-142-5p mimic prolongs STAT6 phosphorylation by targeting its negative regulator, SOCS1. Blocking miR-130a relieves its inhibition of PPARγ, which coordinates STAT6 signalling. In vivo, inhibiting miR-142-5p and increasing miR-130a-3p expression with locked nucleic acid-modified oligonucleotides inhibits CCL4-induced liver fibrosis and bleomycin-induced lung fibrosis in mice. Furthermore, macrophages from the tissue samples of patients with liver cirrhosis and idiopathic pulmonary fibrosis display increased miR-142-5p and decreased miR-130a-3p expression. Therefore, miR-142-5p and miR-130a-3p regulate macrophage profibrogenic gene expression in chronic inflammation.

Mutations in the TP53 tumour suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumour progression remain only partially understood. Here, using data from the Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumours from 10 human cancer types. We show that tumours with TP53 mutations have altered gene expression profiles compared to tumours retaining two wild-type TP53 alleles. Among 113 known p53-up-regulated target genes identified from cell culture assays, 10 were consistently up-regulated in at least eight of 10 cancer types that retain both copies of wild-type TP53. RPS27L, CDKN1A (p21(CIP1)) and ZMAT3 were significantly up-regulated in all 10 cancer types retaining wild-type TP53. Using this p53-based expression analysis as a discovery tool, we used cell-based assays to identify five novel p53 target genes from genes consistently up-regulated in wild-type p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2 and FOXM1 were disproportionately up-regulated in many TP53 mutant cancer types. Finally, greater than 93% of tumours with a TP53 mutation exhibited greatly reduced wild-type p53 messenger expression, due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumour suppressor. The data indicate that tumours with wild-type TP53 retain some aspects of p53-mediated growth inhibitory signalling through activation of p53 target genes and suppression of cell cycle regulatory genes.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.

Retinoic acid (RA) has diverse biological effects. The liver stores vitamin A, generates RA, and expresses receptors for RA. The current study examines the hepatic binding profile of two RA receptor isoforms, RARA (RARα) and RARB (RARβ), in response to RA treatment in mouse livers. Our data uncovered 35,521, and 14,968 genomic bindings for RARA and RARB, respectively. Each expressed unique and common bindings, implying their redundant and specific roles. RARB has higher RA responsiveness than RARB. RA treatment generated 18,821 novel RARB bindings but only 14,798 of RARA bindings, compared with the control group. RAR frequently bound the consensus hormone response element [HRE; (A/G)G(G/T)TCA], which often contained the motifs assigned to SP1, GABPA, and FOXA2, suggesting potential interactions between those transcriptional factors. Functional annotation coupled with principle component analysis revealed that the function of RAR target genes were motif dependent. Taken together, the cistrome of RARA and RARB revealed their extensive biological roles in the mouse liver. RAR target genes are enriched in various biological processes. The hepatic RAR genome-wide binding data can help us understand the global molecular mechanisms underlying RAR and RA-mediated gene and pathway regulation.