ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex.

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.This work was supported by the Wellcome Trust through a Principal Research Fellowship to P.J.L. (101835/Z/13/Z) and a Ph.D. studentship to I.A.T. The CIMR is in receipt of a Wellcome Trust strategic award.This is the final version of the article. It first appeared from Elsevier (Cell Press) via https://doi.org/10.1016/j.celrep.2016.09.05

ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.This work was supported by the Wellcome Trust through a Principal Research Fellowship to P.J.L. (101835/Z/13/Z) and a Ph.D. studentship to I.A.T. The CIMR is in receipt of a Wellcome Trust strategic award.This is the final version of the article. It first appeared from Elsevier (Cell Press) via https://doi.org/10.1016/j.celrep.2016.09.05

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

GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.This work was supported by a Wellcome Trust Principal Research Fellowship to P.J.L. (084957/Z/08/Z) and studentship to I.A.T., an MRC Centenary Award to R.T.T., and the Cambridge Biomedical Research Centre (UK). The CIMR is in receipt of a Wellcome Trust Strategic Award.This is the author accepted manuscript. The final version is available from AAAS via http://dx.doi.org/10.1126/science.aaa722

Neuropathic MORC2 mutations perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the human silencing hub (HUSH). Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies

Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens.

The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing 'gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.This work was supported by the Wellcome Trust, through a Principal Research Fellowship to P.J.L. and PhD studentships to S.A.M. and I.A.T., the NIHR Cambridge BRC and the Lundbeck Foundation (L.C.C. and L.E.). The CIMR is in receipt of a Wellcome Trust strategic award.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms11786

Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2

Dominant mutations in the $\textit{MORC2}$ gene have recently been shown to cause axonal Charcot–Marie–Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR–Cas9-mediated forward genetic screen, we identified $\textit{MORC2}$ as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of $\textit{MORC2}$ mutations in CMT disease.This work was supported by the Wellcome Trust, through a Principal Research Fellowship to P.J.L. (101835/Z/13/Z), a Senior Research Fellowship to Y.M. (101908/Z/13/Z), a Sir Henry Wellcome Postdoctoral Fellowship to R.T.T. (201387/Z/16/Z) and a PhD studentship to I.A.T., and by the BBSRC, through a Future Leader Fellowship to C.H.D. I.A.T. is supported as a Damon Runyon Fellow by the Damon Runyon Cancer Research Foundation (DRG-2277-16). The CIMR is in receipt of a Wellcome Trust strategic award

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

Abstract: The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation