TASOR expression in naive embryonic stem cells safeguards their developmental potential.

The seamless transition through stages of pluripotency relies on a balance between transcription factor networks and epigenetic mechanisms. Here, we reveal the crucial role of the transgene activation suppressor (TASOR), a component of the human silencing hub (HUSH) complex, in maintaining cell viability during the transition from naive to primed pluripotency. TASOR loss in naive pluripotent stem cells (PSCs) triggers replication stress, disrupts H3K9me3 heterochromatin, and impairs silencing of LINE-1 (L1) transposable elements, with more severe effects in primed PSCs. Notably, the survival of Tasor knockout PSCs during this transition can be restored by inhibiting caspase or deleting the mitochondrial antiviral signaling protein (MAVS). This suggests that unscheduled L1 expression activates an innate immune response, leading to cell death specifically in cells exiting naive pluripotency. Our findings highlight the importance of epigenetic programs established in naive pluripotency for normal development.

Protein UFMylation regulates early events during ribosomal DNA-damage response.

The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ataxia-telangiectasia-mutated (ATM)-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA-damage response in human cells. We further show the only ubiquitin-fold modifier 1 (UFM1)-E3 ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. Moreover, analysis of nuclear and nucleolar UFMylation targets in response to DSB induction further identifies key DNA-repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provide evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.

Evolution of retrocopies in the context of HUSH silencing.

Retrotransposition is one of the main factors responsible for gene duplication and thus genome evolution. However, the sequences that undergo this process are not only an excellent source of biological diversity, but in certain cases also pose a threat to the integrity of the DNA. One of the mechanisms that protects against the incorporation of mobile elements is the HUSH complex, which is responsible for silencing long, intronless, transcriptionally active transposed sequences that are rich in adenine on the sense strand. In this study, broad sets of human and porcine retrocopies were analysed with respect to the above factors, taking into account evolution of these molecules. Analysis of expression pattern, genomic structure, transcript length, and nucleotide substitution frequency showed the strong relationship between the expression level and exon length as well as the protective nature of introns. The results of the studies also showed that there is no direct correlation between the expression level and adenine content. However, protein-coding retrocopies, which have a lower adenine content, have a significantly higher expression level than the adenine-rich non-coding but expressed retrocopies. Therefore, although the mechanism of HUSH silencing may be an important part of the regulation of retrocopy expression, it is one component of a more complex molecular network that remains to be elucidated.

Epigenetic Restriction Factors (eRFs) in Virus Infection.

The ongoing arms race between viruses and their hosts is constantly evolving. One of the ways in which cells defend themselves against invading viruses is by using restriction factors (RFs), which are cell-intrinsic antiviral mechanisms that block viral replication and transcription. Recent research has identified a specific group of RFs that belong to the cellular epigenetic machinery and are able to restrict the gene expression of certain viruses. These RFs can be referred to as epigenetic restriction factors or eRFs. In this review, eRFs have been classified into two categories. The first category includes eRFs that target viral chromatin. So far, the identified eRFs in this category include the PML-NBs, the KRAB/KAP1 complex, IFI16, and the HUSH complex. The second category includes eRFs that target viral RNA or, more specifically, the viral epitranscriptome. These epitranscriptomic eRFs have been further classified into two types: those that edit RNA bases-adenosine deaminase acting on RNA (ADAR) and pseudouridine synthases (PUS), and those that covalently modify viral RNA-the N6-methyladenosine (m6A) writers, readers, and erasers. We delve into the molecular machinery of eRFs, their role in limiting various viruses, and the mechanisms by which viruses have evolved to counteract them. We also examine the crosstalk between different eRFs, including the common effectors that connect them. Finally, we explore the potential for new discoveries in the realm of epigenetic networks that restrict viral gene expression, as well as the future research directions in this area.

The DUF3715 domain has a conserved role in RNA-directed transposon silencing.

RNA-directed transposon silencing operates in the mammalian soma and germline to safeguard genomic integrity. The piRNA pathway and the HUSH complex identify active transposons through recognition of their nascent transcripts, but mechanistic understanding of how these distinct pathways evolved is lacking. TASOR is an essential component of the HUSH complex. TASOR's DUF3715 domain adopts a pseudo-PARP structure and is required for transposon silencing in a manner independent of complex assembly. TEX15, an essential piRNA pathway factor, also contains the DUF3715 domain. Here, we show that TASOR's and TEX15's DUF3715 domain share extensive structural homology. We found that the DUF3715 domain arose in early eukaryotes and that in vertebrates it is restricted to TEX15, TASOR, and TASORB orthologs. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate-specific. The branching of TEX15 and the TASOR-like DUF3715 domain likely occurred in early metazoan evolution. Remarkably, despite this vast evolutionary distance, the DUF3715 domain from divergent TEX15 sequences can functionally substitute the DUF3715 domain of TASOR and mediates transposon silencing. We have thus termed this domain of unknown function as the RNA-directed pseudo-PARP transposon silencing (RDTS) domain. In summary, we show an unexpected functional link between these critical transposon silencing pathways.

Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

The sound of silence: mechanisms and implications of HUSH complex function.

The vertebrate genome is under constant threat of invasion by genetic parasites. Whether the host can immediately recognize and respond to invading elements has been unclear. The discovery of the human silencing hub (HUSH) complex, and the finding that it provides immediate protection from genome invasion by silencing products of reverse transcription, have important implications for mammalian genome evolution. In this review, we summarize recent insights into HUSH function and describe how cellular introns provide a novel means of self-nonself discrimination, allowing HUSH to recognize and transcriptionally repress a broad range of intronless genetic elements. We discuss how HUSH contributes to genome evolution, and highlight studies reporting the critical role of HUSH in development and implicating HUSH in the control of immune signaling and cancer progression.

Inhibition of a Chromatin and Transcription Modulator, SLTM, Increases HIV-1 Reactivation Identified by a CRISPR Inhibition Screen.

Despite effective antiretroviral therapy, HIV-1 persistence in latent reservoirs remains a major obstacle to a cure. We postulate that HIV-1 silencing factors suppress HIV-1 reactivation and that inhibition of these factors will increase HIV-1 reactivation. To identify HIV-1 silencing factors, we conducted a genome-wide CRISPR inhibition (CRISPRi) screen using four CRISPRi-ready, HIV-1-d6-GFP-infected Jurkat T cell clones with distinct integration sites. We sorted cells with increased green fluorescent protein (GFP) expression and captured single guide RNAs (sgRNAs) via targeted deep sequencing. We identified 18 HIV-1 silencing factors that were significantly enriched in HIV-1-d6-GFPhigh cells. Among them, SLTM (scaffold attachment factor B-like transcription modulator) is an epigenetic and transcriptional modulator having both DNA and RNA binding capacities not previously known to affect HIV-1 transcription. Knocking down SLTM by CRISPRi significantly increased HIV-1-d6-GFP expression (by 1.9- to 4.2-fold) in three HIV-1-d6-GFP-Jurkat T cell clones. Furthermore, SLTM knockdown increased the chromatin accessibility of HIV-1 and the gene in which HIV-1 is integrated but not the housekeeping gene POLR2A. To test whether SLTM inhibition can reactivate HIV-1 and further induce cell death of HIV-1-infected cells ex vivo, we established a small interfering RNA (siRNA) knockdown method that reduced SLTM expression in CD4+ T cells from 10 antiretroviral therapy (ART)-treated, virally suppressed, HIV-1-infected individuals ex vivo. Using limiting dilution culture, we found that SLTM knockdown significantly reduced the frequency of HIV-1-infected cells harboring inducible HIV-1 by 62.2% (0.56/106 versus 1.48/106 CD4+ T cells [P = 0.029]). Overall, our study indicates that SLTM inhibition reactivates HIV-1 in vitro and induces cell death of HIV-1-infected cells ex vivo. Our study identified SLTM as a novel therapeutic target. IMPORTANCE HIV-1-infected cells, which can survive drug treatment and immune cell killing, prevent an HIV-1 cure. Immune recognition of infected cells requires HIV-1 protein expression; however, HIV-1 protein expression is limited in infected cells after long-term therapy. The ways in which the HIV-1 provirus is blocked from producing protein are unknown. We identified a new host protein that regulates HIV-1 gene expression. We also provided a new method of studying HIV-1-host factor interactions in cells from infected individuals. These improvements may enable future strategies to reactivate HIV-1 in infected individuals so that infected cells can be killed by immune cells, drug treatment, or the virus itself.

Microrchidia CW-Type Zinc Finger 2, a Chromatin Modifier in a Spectrum of Peripheral Neuropathies.

Microrchidia CW-type zinc finger 2 (MORC2) gene encodes a protein expressed in all tissues and enriched in the brain. MORC2 protein is composed of a catalytic ATPase domain, three coil-coiled domains allowing dimerization or protein complex interaction, a zinc-finger CW domain allowing DNA interaction, and a CHROMO-like (CHRromatin Organization Modifier) domain. Recently, de novo or dominantly inherited heterozygous mutations have been associated with a spectrum of disorders affecting the peripheral nervous system such as the Charcot-Marie-Tooth disease, spinal muscular atrophy-like phenotype disorder, or a neurodevelopmental syndrome associated with developmental delay, impaired growth, dysmorphic facies, and axonal neuropathy (DIGFAN). In this review, we detail the various mutations of MORC2 and their consequences on clinical manifestations. Possible genotype-phenotype correlations as well as intra and inter-family variability are discussed. MORC2 molecular functions such as transcriptional modulation, DNA damage repair, and lipid metabolism are then reviewed. We further discuss the impact of MORC2 mutations on the epigenetic landscape in the neuromuscular system and hypothesize probable pathophysiological mechanisms underlying the phenotypic variability observed.

Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression.

Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.

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.

Position-effect variegation revisited: HUSHing up heterochromatin in human cells.

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.