TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.

RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA.

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.

When Down Is Up: Heterochromatin, Nuclear Organization and X Upregulation

Organisms with highly differentiated sex chromosomes face an imbalance in X-linked gene dosage. Male Drosophila solve this problem by increasing expression from virtually every gene on their single X chromosome, a process known as dosage compensation. This involves a ribonucleoprotein complex that is recruited to active, X-linked genes to remodel chromatin and increase expression. Interestingly, the male X chromosome is also enriched for several proteins associated with heterochromatin. Furthermore, the polytenized male X is selectively disrupted by the loss of factors involved in repression, silencing, heterochromatin formation or chromatin remodeling. Mutations in many of these factors preferentially reduce male survival or enhance the lethality of mutations that prevent normal recognition of the X chromosome. The involvement of primarily repressive factors in a process that elevates expression has long been puzzling. Interestingly, recent work suggests that the siRNA pathway, often associated with heterochromatin formation and repression, also helps the dosage compensation machinery identify the X chromosome. In light of this finding, we revisit the evidence that links nuclear organization and heterochromatin to regulation of the male X chromosome.

HP1a-mediated heterochromatin formation inhibits high dietary sugar-induced tumor progression

High dietary sugar (HDS) is a modern dietary concern that involves excessive consumption of carbohydrates and added sugars, and increases the risk of metabolic disorders and associated cancers. However, epigenetic mechanisms by which HDS induces tumor progression remain unclear. Here, we investigate the role of heterochromatin, an important yet poorly understood part of the epigenome, in HDS-induced tumor progression of Drosophila Ras/Src and Ras/scrib tumor systems. We found that increased heterochromatin formation with overexpression of heterochromatin protein 1a (HP1a), specifically in tumor cells, not only decreases HDS-induced tumor growth/burden but also drastically improves survival of Drosophila with HDS and Ras/Src or Ras/scrib tumors. Moreover, HDS reduces heterochromatin levels in tumor cells. Mechanistically, we demonstrated that increased heterochromatin formation decreases wingless (wg) and Hippo (Hpo) signaling, thereby promoting apoptosis, via inhibition of Yorkie (Yki) nuclear accumulation and upregulation of apoptotic genes, and reduces DNA damage in tumor cells under HDS. Taken together, our work identified a novel epigenetic mechanism by which HP1a-mediated heterochromatin formation suppresses HDS-induced tumor progression likely by decreasing wingless and Hippo signaling, increasing apoptosis, and maintaining genome stability. Our model explains that the molecular, cellular, and organismal aspects of HDS-aggravated tumor progression are dependent on heterochromatin formation, and highlights heterochromatin as a therapeutic target for cancers associated with HDS-induced metabolic disorders.

Multiple distinct domains of human XIST are required to coordinate gene silencing and subsequent heterochromatin formation

Background Mammalian dosage compensation is achieved by the inactivation of one X chromosome in XX individuals. In eutheria this process is initiated early in development by the long non-coding RNA XIST. Studies of the initiation of silencing by XIST have focussed on mouse models, so the domains of XIST required to induce silencing in humans, and their relationship with domains required to establish heterochromatin remain to be determined. Methods We have previously established an inducible XIST cDNA in somatic cells and shown it can induce silencing and recruit heterochromatic features. We now assess a series of deletions across the construct for the ability to induce silencing and integrate these results with time-course and chromatin remodelling inhibitor treatments to follow the steps of XIST-induced silencing and heterochromatinization. Discussion We find that in addition to the previously reported necessity of the 5’ A repeat region for XIST-induced silencing, the F repeat region and a non-repetitive region at the 3’ end of the RNA are also required to silence genes. Silencing of genes up to 17Mb from the XIST integration occurs within two days, while formation of a Cot-1 depleted domain is slower, and more dependent on repeat F. The role of Repeat F in both the silencing of actively transcribed genes, the spread of H3K27me3 and the formation of a transcriptionally inert domain suggests a role in a pathway crucial for the spread of XIST across the chromatin to target distal regions of inactivation. Histone deacetylation requires only the A repeat region, with HDAC3 inhibition showing limited effect on silencing, but an impact on H3K27me3 recruitment, and as a result the recruitment of MacroH2A. Global HDAC inhibition impacted silencing in both a distance and dose-dependent fashion. The E repeat region was required for CIZ1 and H4K20me1 recruitment as well as H3K27me3; however, these appeared to act relatively independently. The H3K27me3 mark established by PRC2 integrated silencing and many of the heterochromatic features, while the PRC1 mark ubH2A appeared to be downstream of silencing in these human somatic cells.

An Inducible System for Silencing Establishment Reveals a Stepwise Mechanism in Which Anchoring at the Nuclear Periphery Precedes Heterochromatin Formation

In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

To export, or not to export: how nuclear export factor variants resolve Piwi's dilemma

Piwi-interacting RNAs (piRNAs) defend animal gonads by guiding PIWI-clade Argonaute proteins to silence transposons. The nuclear Piwi/piRNA complexes confer transcriptional repression of transposons, which is accompanied with heterochromatin formation at target loci. On the other hand, piRNA clusters, genomic loci that transcribe piRNA precursors composed of transposon fragments, are often recognized by piRNAs to define their heterochromatic identity. Therefore, Piwi/piRNA complexes must resolve this conundrum of silencing transposons while allowing the expression of piRNA precursors, at least in Drosophila germlines. This review is focused on recent advances how the piRNA pathway deals with this genetic conflict.

Polycomb condensates can promote epigenetic marks but are not required for sustained chromatin compaction

Organization of the genome into transcriptionally active euchromatin and silenced heterochromatin is essential for eukaryotic cell function. Phase-separation has been implicated in heterochromatin formation, but it is unclear how phase-separated condensates can contribute to stable repression, particularly for heritable epigenetic changes. Polycomb complex PRC1 is key for heterochromatin formation, but the multitude of Polycomb proteins has hindered our understanding of their collective contribution to chromatin repression. Here, we show that PRC1 forms multicomponent condensates through hetero-oligomerization. They preferentially seed at H3K27me3 marks, and subsequently write H2AK119Ub marks. We show that inducing Polycomb phase-separation can cause chromatin compaction, but polycomb condensates are dispensable for maintenance of the compacted state. Our data and simulations are consistent with a model in which the time integral of Polycomb phase-separation is progressively recorded in repressive histone marks, which subsequently drive compaction. These findings link the equilibrium thermodynamics of phase-separation with the fundamentally non-equilibrium concept of epigenetic memory.

The Paramount Role of Drosophila melanogaster in the Study of Epigenetics: From Simple Phenotypes to Molecular Dissection and Higher-Order Genome Organization

Drosophila melanogaster has played a paramount role in epigenetics, the study of changes in gene function inherited through mitosis or meiosis that are not due to changes in the DNA sequence. By analyzing simple phenotypes, such as the bristle position or cuticle pigmentation, as read-outs of regulatory processes, the identification of mutated genes led to the discovery of major chromatin regulators. These are often conserved in distantly related organisms such as vertebrates or even plants. Many of them deposit, recognize, or erase post-translational modifications on histones (histone marks). Others are members of chromatin remodeling complexes that move, eject, or exchange nucleosomes. We review the role of D. melanogaster research in three epigenetic fields: Heterochromatin formation and maintenance, the repression of transposable elements by piRNAs, and the regulation of gene expression by the antagonistic Polycomb and Trithorax complexes. We then describe how genetic tools available in D. melanogaster allowed to examine the role of histone marks and show that some histone marks are dispensable for gene regulation, whereas others play essential roles. Next, we describe how D. melanogaster has been particularly important in defining chromatin types, higher-order chromatin structures, and their dynamic changes during development. Lastly, we discuss the role of epigenetics in a changing environment.