Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis

Role of the Epigenetic Regulator HP1γ in the Control of Embryonic Stem Cell Properties

The unique properties of embryonic stem cells (ESC) rely on long-lasting self-renewal and their ability to switch in all adult cell type programs. Recent advances have shown that regulations at the chromatin level sustain both ESC properties along with transcription factors. We have focused our interest on the epigenetic modulator HP1γ (Heterochromatin Protein 1, isoform γ) that binds histones H3 methylated at lysine 9 (meH3K9) and is highly plastic in its distribution and association with the transcriptional regulation of specific genes during cell fate transitions. These characteristics of HP1γ make it a good candidate to sustain the ESC flexibility required for rapid program changes during differentiation. Using RNA interference, we describe the functional role of HP1γ in mouse ESC. The analysis of HP1γ deprived cells in proliferative and in various differentiating conditions was performed combining functional assays with molecular approaches (RT-qPCR, microarray). We show that HP1γ deprivation slows down the cell cycle of ESC and decreases their resistance to differentiating conditions, rendering the cells poised to differentiate. In addition, HP1γ depletion hampers the differentiation to the endoderm as compared with the differentiation to the neurectoderm or the mesoderm. Altogether, our results reveal the role of HP1γ in ESC self-renewal and in the balance between the pluripotent and the differentiation programs

Depletion of somatic mutations in splicing-associated sequences in cancer genomes

Abstract Background An important goal of cancer genomics is to identify systematically cancer-causing mutations. A common approach is to identify sites with high ratios of non-synonymous to synonymous mutations; however, if synonymous mutations are under purifying selection, this methodology leads to identification of false-positive mutations. Here, using synonymous somatic mutations (SSMs) identified in over 4000 tumours across 15 different cancer types, we sought to test this assumption by focusing on coding regions required for splicing. Results Exon flanks, which are enriched for sequences required for splicing fidelity, have ~ 17% lower SSM density compared to exonic cores, even after excluding canonical splice sites. While it is impossible to eliminate a mutation bias of unknown cause, multiple lines of evidence support a purifying selection model above a mutational bias explanation. The flank/core difference is not explained by skewed nucleotide content, replication timing, nucleosome occupancy or deficiency in mismatch repair. The depletion is not seen in tumour suppressors, consistent with their role in positive tumour selection, but is otherwise observed in cancer-associated and non-cancer genes, both essential and non-essential. Consistent with a role in splicing modulation, exonic splice enhancers have a lower SSM density before and after controlling for nucleotide composition; moreover, flanks at the 5’ end of the exons have significantly lower SSM density than at the 3’ end. Conclusions These results suggest that the observable mutational spectrum of cancer genomes is not simply a product of various mutational processes and positive selection, but might also be shaped by negative selection

Oligosaccharyltransferase Inhibition Induces Senescence in RTK-Driven Tumor Cells

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high throughput screen and lead compound optimization campaign that delivered a cell permeable inhibitor (NGI-1). NGI-1 targets the oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small cell lung cancer cells NGI-1 blocks cell surface localization and signaling of the EGFR glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or FGFR) for survival. In these cell lines OST inhibition causes cell cycle arrest accompanied by induction of p21, autofluorescence, and changes in cell morphology; all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor tyrosine kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells

Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner

SETDB1 Is Involved in Postembryonic DNA Methylation and Gene Silencing in Drosophila

DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3–9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian “Heterochromatin Protein 1”, to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development

MLL-fusion-driven leukemia requires SETD2 to safeguard genomic integrity

MLL-fusions represent a large group of leukemia drivers, whose diversity originates from the vast molecular heterogeneity of C-terminal fusion partners of MLL. While studies of selected MLL-fusions have revealed critical molecular pathways, unifying mechanisms across all MLL-fusions remain poorly understood. We present the first comprehensive survey of protein-protein interactions of seven distantly related MLL-fusion proteins. Functional investigation of 128 conserved MLL-fusion-interactors identifies a specific role for the lysine methyltransferase SETD2 in MLL-leukemia. SETD2 loss causes growth arrest and differentiation of AML cells, and leads to increased DNA damage. In addition to its role in H3K36 tri-methylation, SETD2 is required to maintain high H3K79 di-methylation and MLL-AF9-binding to critical target genes, such as Hoxa9. SETD2 loss synergizes with pharmacologic inhibition of the H3K79 methyltransferase DOT1L to induce DNA damage, growth arrest, differentiation, and apoptosis. These results uncover a dependency for SETD2 during MLL-leukemogenesis, revealing a novel actionable vulnerability in this disease

Screening for Factors Involved in X Chromosome Inactivation Using Haploid ESCs

ISSN:1064-3745ISSN:1940-602