The DREAM complex promotes gene body H2A.Z for target repression.

The DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex controls cellular quiescence by repressing cell cycle genes, but its mechanism of action is poorly understood. Here we show that Caenorhabditis elegans DREAM targets have an unusual pattern of high gene body HTZ-1/H2A.Z. In mutants of lin-35, the sole p130/Rb-like gene in C. elegans, DREAM targets have reduced gene body HTZ-1/H2A.Z and increased expression. Consistent with a repressive role for gene body H2A.Z, many DREAM targets are up-regulated in htz-1/H2A.Z mutants. Our results indicate that the DREAM complex facilitates high gene body HTZ-1/H2A.Z, which plays a role in target gene repression.We are grateful to D. Fay for providing the 5× outcrossed lin-35 strain, and Robert Horvitz for antibodies. I.L., M.A.C., P.S., A.A., and J.A. were supported by Wellcome Trust Senior Research Fellowships to J.A. (054523 and 101863). J.A. also acknowledges support by core funding from the Wellcome Trust and Cancer Research UK. J.M.G. and S.S. were supported by National Institutes of Health (NIH) R01 grant GM34059. Part of this work was supported by NIH National Human Genome Research Institute (NHGRI) grant U01 HG004270 to the modENCODE consortium headed by J.D. Lieb.This is the final version of the article. It first appeared from CSH Press via http://dx.doi.org/10.1101/gad.255810.11

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Loss of the Caenorhabditis elegans pocket protein LIN-35 reveals MuvB's innate function as the repressor of DREAM target genes.

The DREAM (Dp/Retinoblastoma(Rb)-like/E2F/MuvB) transcriptional repressor complex acts as a gatekeeper of the mammalian cell cycle by establishing and maintaining cellular quiescence. How DREAM's three functional components, the E2F-DP heterodimer, the Rb-like pocket protein, and the MuvB subcomplex, form and function at target gene promoters remains unknown. The current model invokes that the pocket protein links E2F-DP and MuvB and is essential for gene repression. We tested this model by assessing how the conserved yet less redundant DREAM system in Caenorhabditis elegans is affected by absence of the sole C. elegans pocket protein LIN-35. Using a LIN-35 protein null mutant, we analyzed the assembly of E2F-DP and MuvB at promoters that are bound by DREAM and the level of expression of those "DREAM target genes" in embryos. We report that LIN-35 indeed mediates the association of E2F-DP and MuvB, a function that stabilizes DREAM subunit occupancy at target genes. In the absence of LIN-35, the occupancy of E2F-DP and MuvB at most DREAM target genes decreases dramatically and many of those genes become upregulated. The retention of E2F-DP and MuvB at some target gene promoters in lin-35 null embryos allowed us to test their contribution to DREAM target gene repression. Depletion of MuvB, but not E2F-DP, in the sensitized lin-35 null background caused further upregulation of DREAM target genes. We conclude that the pocket protein functions primarily to support MuvB-mediated repression of DREAM targets and that transcriptional repression is the innate function of the evolutionarily conserved MuvB complex. Our findings provide important insights into how mammalian DREAM assembly and disassembly may regulate gene expression and the cell cycle

Defining heterochromatin in C. elegans through genome-wide analysis of the heterochromatin protein 1 homolog HPL-2.

Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In most organisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavily studied model C. elegans, which possesses holocentric chromosomes with dispersed centromeres, we analyzed the genome-wide distribution of the heterochromatin protein 1 (HP1) ortholog HPL-2 and compared its distribution to other features commonly associated with heterochromatin. HPL-2 binding highly correlates with histone H3 mono- and dimethylated at lysine 9 (H3K9me1 and H3K9me2) and forms broad domains on autosomal arms. Although HPL-2, like other HP1 orthologs, binds H3K9me peptides in vitro, the distribution of HPL-2 in vivo appears relatively normal in mutant embryos that lack H3K9me, demonstrating that the chromosomal distribution of HPL-2 can be achieved in an H3K9me-independent manner. Consistent with HPL-2 serving roles independent of H3K9me, hpl-2 mutant worms display more severe defects than mutant worms lacking H3K9me. HPL-2 binding is enriched for repetitive sequences, and on chromosome arms is anticorrelated with centromeres. At the genic level, HPL-2 preferentially associates with well-expressed genes, and loss of HPL-2 results in up-regulation of some binding targets and down-regulation of others. Our work defines heterochromatin in an important model organism and uncovers both shared and distinctive properties of heterochromatin relative to other systems

Defining heterochromatin in C. elegans through genome-wide analysis of the heterochromatin protein 1 homolog HPL-2.

Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In most organisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavily studied model C. elegans, which possesses holocentric chromosomes with dispersed centromeres, we analyzed the genome-wide distribution of the heterochromatin protein 1 (HP1) ortholog HPL-2 and compared its distribution to other features commonly associated with heterochromatin. HPL-2 binding highly correlates with histone H3 mono- and dimethylated at lysine 9 (H3K9me1 and H3K9me2) and forms broad domains on autosomal arms. Although HPL-2, like other HP1 orthologs, binds H3K9me peptides in vitro, the distribution of HPL-2 in vivo appears relatively normal in mutant embryos that lack H3K9me, demonstrating that the chromosomal distribution of HPL-2 can be achieved in an H3K9me-independent manner. Consistent with HPL-2 serving roles independent of H3K9me, hpl-2 mutant worms display more severe defects than mutant worms lacking H3K9me. HPL-2 binding is enriched for repetitive sequences, and on chromosome arms is anticorrelated with centromeres. At the genic level, HPL-2 preferentially associates with well-expressed genes, and loss of HPL-2 results in up-regulation of some binding targets and down-regulation of others. Our work defines heterochromatin in an important model organism and uncovers both shared and distinctive properties of heterochromatin relative to other systems

Loss of the <i>Caenorhabditis elegans</i> pocket protein LIN-35 reveals MuvB's innate function as the repressor of DREAM target genes

<div><p>The DREAM (Dp/Retinoblastoma(Rb)-like/E2F/MuvB) transcriptional repressor complex acts as a gatekeeper of the mammalian cell cycle by establishing and maintaining cellular quiescence. How DREAM’s three functional components, the E2F-DP heterodimer, the Rb-like pocket protein, and the MuvB subcomplex, form and function at target gene promoters remains unknown. The current model invokes that the pocket protein links E2F-DP and MuvB and is essential for gene repression. We tested this model by assessing how the conserved yet less redundant DREAM system in <i>Caenorhabditis elegans</i> is affected by absence of the sole <i>C</i>. <i>elegans</i> pocket protein LIN-35. Using a LIN-35 protein null mutant, we analyzed the assembly of E2F-DP and MuvB at promoters that are bound by DREAM and the level of expression of those "DREAM target genes" in embryos. We report that LIN-35 indeed mediates the association of E2F-DP and MuvB, a function that stabilizes DREAM subunit occupancy at target genes. In the absence of LIN-35, the occupancy of E2F-DP and MuvB at most DREAM target genes decreases dramatically and many of those genes become upregulated. The retention of E2F-DP and MuvB at some target gene promoters in <i>lin-35</i> null embryos allowed us to test their contribution to DREAM target gene repression. Depletion of MuvB, but not E2F-DP, in the sensitized <i>lin-35</i> null background caused further upregulation of DREAM target genes. We conclude that the pocket protein functions primarily to support MuvB-mediated repression of DREAM targets and that transcriptional repression is the innate function of the evolutionarily conserved MuvB complex. Our findings provide important insights into how mammalian DREAM assembly and disassembly may regulate gene expression and the cell cycle.</p></div

Effects of depletion of additional DRM subunits in <i>lin-35</i> null mutants.

<p>(A,B) Genomic profiles of each DRM component in wild-type (WT) and <i>lin-35</i> late embryos near (A) <i>set-21</i> and <i>cdk-1</i> and (B) <i>air-1 and rad-51</i>. E2F-DP subunits (blues), LIN-35 (purple), and MuvB subunits (greens) were overlaid on the same track. Normalized ChIP-seq enrichment values for each data track are indicated on the y-axis, and the chromosomal coordinates of the areas shown are indicated below the x-axis. Gene transcriptional start sites are indicated by black boxes, and coding strand directions are indicated by white arrowheads. Other genes in the region are similarly indicated in grey. Each DRM peak location is indicated by a black rectangle with the peak center highlighted in red. (C,D) RT-qPCR analysis of (C) <i>set-21</i>, <i>cdk-1</i>, <i>F01G4</i>.<i>4</i>, and <i>csc-1</i> and (D) <i>air-1</i>, <i>rad-51</i>, <i>kbp-3</i>, and <i>mes-4</i> following <i>efl-1</i> (blue), <i>lin-9</i> (light green), or <i>lin-54</i> (dark green) RNAi in <i>lin-35</i> null late embryos compared to empty-vector RNAi (Ctrl, purple). Expression values from 2 independent experiments each consisting of 4 biological replicates for each RNAi condition were averaged and are presented as the relative quantity (Rq) compared to <i>act-2</i>. Error bars indicate standard error of the mean, and significance was determined by a student’s T test comparing DRM subunit RNAi to Control RNAi (Ctrl) (* p-value < 0.05, ** p-value < 0.01). Additional genes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007088#pgen.1007088.s015" target="_blank">S5 Table</a>.</p