SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.

The Human Silencing Hub (HuSH) complex is composed of TASOR, MPP8, and PPHLN1 subunits and serves as a conserved protein complex responsible for silencing transposable elements in vertebrate animals. Despite its importance, the regulatory mechanisms and recruitment dynamics governing this complex remain poorly understood. In this study, we have identified a second HuSH complex, termed HuSH2, centered around TASOR2, a paralog of the core TASOR protein in HuSH. Our findings indicate that every subunit in both HuSH and HuSH2 has an important role in achieving precise genomic localization to distinct, non-overlapping genomic loci. We utilized in silico protein structure prediction to simulate the interactions between MPP8 and both TASOR paralogs. Drawing on the insights gained from these predictions, we implemented amino acid substitutions that interfered with the binding of MPP8 to each HuSH complex. Leveraging these MPP8 transgenes and other constructs, we identified an important role played by the relative quantities of HuSH complexes in controlling the activity of LINE-1 elements. Furthermore, our results suggest that dynamic changes in TASOR and TASOR2 expression enable cells to finely tune the extent of HuSH-mediated silencing. Our study provides insights into the intricate interplay between HuSH complexes, illuminating their important role in the regulation of retrotransposon silencing.

The human silencing hub (HUSH) complex is a transcription-dependent, epigenetic repressor complex that provides a genome-wide immunosurveillance system for the recognition and silencing of newly-integrated retroelements. The core HUSH complex of TASOR, MPP8 and Periphilin, represses these retroelements through SETDB1-mediated H3K9me3 deposition and MORC2-dependent chromatin compaction. HUSH-dependent silencing is RNA-mediated, yet no HUSH component contains a recognised RNA-binding domain. Here we used an unbiased approach to identify which HUSH component was able to bind RNA and determine whether RNA-binding was essential for HUSH function. We identify Periphilin as the major RNA-binding component of the HUSH complex and show that Periphilin's N-terminal domain is essential for both RNA binding and HUSH function. Periphilin binding to RNA was independent of its interaction with TASOR or MPP8, as its N-terminal domain was sufficient for RNA targeting. The artificial tethering of Periphilin to a HUSH-insensitive, nascent transcript, enabled the HUSH-dependent silencing of the transcript. This tethering of Periphilin allowed the RNA-binding region of Periphilin to be removed such that only its C-terminal domain was required for oligomerisation and interaction with TASOR. We therefore show that Periphilin is the predominant RNA-binding protein of the HUSH complex and this RNA-binding is essential for HUSH activity.

Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of an SVA retrotransposon reporter. By using naïve versus primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. Although the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages <5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s, and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon noncoding DNA.

The Human Silencing Hub (HuSH) complex silences retrotransposable elements in vertebrates. Here, we identify a second HuSH complex, designated HuSH2, which is centered around TASOR2, a paralog of the core TASOR protein in HuSH. Our findings reveal that HuSH and HuSH2 localize to distinct and non-overlapping genomic loci. Specifically, HuSH localizes to and represses LINE-1 retrotransposons, whereas HuSH2 targets and represses KRAB-ZNFs and interferon signaling and response genes. We use in silico protein structure predictions to simulate MPP8 interactions with TASOR paralogs, guiding amino acid substitutions that disrupted binding to HuSH complexes. These MPP8 transgenes and other constructs reveal the importance of HuSH complex quantities in regulating LINE-1 activity. Furthermore, our results suggest that dynamic changes in TASOR and TASOR2 expression enable cells to finely tune HuSH-mediated silencing. This study offers insights into the interplay of HuSH complexes, highlighting their vital role in retrotransposon regulation.

The human silencing hub (HUSH) complex is an epigenetic repressor complex whose role has emerged as an important guardian of genome integrity. It protects the genome from exogenous DNA invasion and regulates endogenous retroelements by recruiting histone methyltransferases catalyzing histone 3 lysine 9 trimethylation (H3K9me3) and additional proteins involved in chromatin compaction. In particular, its regulation of transcriptionally active LINE1 retroelements, by binding to and neutralizing LINE1 transcripts, has been well characterized. HUSH is required for mouse embryogenesis and is associated with disease, in particular cancer. Here we provide insights into the structural and biochemical features of the HUSH complex. Furthermore, we discuss the molecular mechanisms by which the HUSH complex is recruited to specific genomic regions and how it silences transcription. Finally, we discuss the role of HUSH complex members in mammalian development, antiretroviral immunity, and diseases such as cancer.

The Human Silencing Hub (HUSH) complex is necessary for epigenetic repression of LINE-1 elements. We show that HUSH-depletion in human cell lines and primary fibroblasts leads to induction of interferon-stimulated genes (ISGs) through JAK/STAT signaling. This effect is mainly attributed to MDA5 and RIG-I sensing of double-stranded RNAs (dsRNAs). This coincides with upregulation of primate-conserved LINE-1s, as well as increased expression of full-length hominid-specific LINE-1s that produce bidirectional RNAs, which may form dsRNA. Notably, LTRs nearby ISGs are derepressed likely rendering these genes more responsive to interferon. LINE-1 shRNAs can abrogate the HUSH-dependent response, while overexpression of an engineered LINE-1 construct activates interferon signaling. Finally, we show that the HUSH component, MPP8 is frequently downregulated in diverse cancers and that its depletion leads to DNA damage. These results suggest that LINE-1s may drive physiological or autoinflammatory responses through dsRNA sensing and gene-regulatory roles and are controlled by the HUSH complex.

Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, though, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2–4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5–8. A screen for factors that mimic these effects identified the Human Silencing Hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN), and MORC29–13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.

Much of what we understand about heterochromatin formation in mammals has been extrapolated from forward genetic screens for modifiers of position-effect variegation (PEV) in the fruit fly Drosophila melanogaster. The recent identification of the HUSH (Human Silencing Hub) complex suggests that more recent evolutionary developments contribute to the mechanisms underlying PEV in human cells. Although HUSH-mediated repression also involves heterochromatin spreading through the reading and writing of the repressive H3K9me3 histone modification, clear orthologues of HUSH subunits are not found in Drosophila but are conserved in vertebrates. Here we compare the insights into the mechanisms of PEV derived from genetic screens in the fly, the mouse and in human cells, review what is currently known about the HUSH complex and discuss the implications of HUSH-mediated silencing for viral latency. Future studies will provide mechanistic insight into HUSH complex function and reveal the relationship between HUSH and other epigenetic silencing complexes.

The HUSH (human silencing hub) complex contains the H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 as well as Microrchidia CW-type zinc finger protein 2 (MORC2). Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive. Here, we show that genetic inactivation of Mphosph8 or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, in both mouse brains and human cerebral organoids, MPP8 and MORC2 suppress the repetitive-like protocadherin gene cluster in an H3K9me3-dependent manner. Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

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.

The interpretation of histone post-translational modifications (PTMs), specifically lysine methylation, by specific classes of "reader" proteins marks an important aspect of epigenetic control of gene expression. Methyl-lysine (Kme) readers often regulate gene expression patterns through the recognition of a specific Kme PTM while participating in or recruiting large protein complexes that contain enzymatic or chromatin remodeling activity. Understanding the composition of these Kme-reader-containing protein complexes can serve to further our understanding of the biological roles of Kme readers, while small molecule chemical tools can be valuable reagents in interrogating novel protein-protein interactions. Here, we describe our efforts to target the chromodomain of M-phase phosphoprotein 8 (MPP8), a member of the human silencing hub (HUSH) complex and a histone 3 lysine 9 trimethyl (H3K9me3) reader that is vital for heterochromatin formation and has specific roles in cancer metastasis. Utilizing a one-bead, one-compound (OBOC) combinatorial screening approach, we identified UNC5246, a peptidomimetic ligand capable of interacting with the MPP8 chromodomain in the context of the HUSH complex. Additionally, a biotinylated derivative of UNC5246 facilitated chemoproteomics studies which revealed hepatoma-derived growth factor-related protein 2 (HRP2) as a novel protein associated with MPP8. HRP2 was further shown to colocalize with MPP8 at the E-cadherin gene locus, suggesting a possible role in cancer cell plasticity.

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.

The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.

The human silencing hub (HUSH) maintains the genome integrity by silencing invasive genetic elements. However, despite essential roles of the HUSH complex during early embryonic development, the function of the complex in the maintenance of pluripotency is mostly unknown. Here, using CRISPR-based genome editing, we demonstrate that in mouse embryonic stem cells (mESCs), deficiency of HUSH core members MPP8 or TASOR, but not PPHLN1, triggers alteration of colony morphology, and mild defect in exit from pluripotency. Importantly, Mpp8/Tasor double mutants are locked in pluripotent state even upon differentiation stimuli. We further demonstrate that these phenotypes are accompanied by decreased expression of adhesion-related genes. Strikingly, the abnormalities observed in Mpp8/Tasor-null mESCs were largely rescued by ectopic coexpression of cell-adhesion associated genes keratins 18 and 19. Thus, our study provides new insights into the mechanisms by which the HUSH complex coordinates the exit from pluripotency through regulation of the cell adhesion machinery.