Heterochromatic Gene Silencing Research Articles

Heterochromatic gene silencing controls CD4+ T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

Protective immune responses require close interactions between conventional (Tconv) and regulatory T cells (Treg). The extracellular mediators and signaling events that regulate the crosstalk between these CD4+ T cell subsets have been extensively characterized. However, how Tconv translate Treg-dependent suppressive signals at the chromatin level remains largely unknown. Here we show, using a murine bone marrow allograft model in which graft rejection is coordinated by CD4+ T cells and can be inhibited by Treg, that Treg-mediated T cell suppression involves Heterochromatin Protein 1 α (HP1α)-dependent gene silencing. Unexpectedly, our screen also reveals that T cells deficient for HP1γ or the methyltransferase SUV39H1 are better repressed by Treg than their wild-type counterparts. Mechanistically, our transcriptional and epigenetic profiling identifies HP1γ as a negative regulator of a gene network functionally associated with T-cell exhaustion, including those encoding the inhibitory receptors PD-1 and LAG-3. In conclusion, we identify HP1 variants as rheostats that finely tune the balance between tolerance and immunity. While HP1α converts immunosuppressive signals into heterochromatin-dependent gene silencing mechanisms, HP1γ adjusts Tconv sensitivity to inhibitory environmental signals.

Heterochromatic silencing of immune-related genes in glia is required for BBB integrity and normal lifespan in drosophila.

Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. Here, we report that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, we conducted a series of lifespan rescue experiments and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, we showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, our findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan.

A dual, catalytic role for the fission yeast Ccr4-Not complex in gene silencing and heterochromatin spreading.

Heterochromatic gene silencing relies on combinatorial control by specific histone modifications, the occurrence of transcription, and/or RNA degradation. Once nucleated, heterochromatin propagates within defined chromosomal regions and is maintained throughout cell divisions to warrant proper genome expression and integrity. In the fission yeast Schizosaccharomyces pombe, the Ccr4-Not complex partakes in gene silencing, but its relative contribution to distinct heterochromatin domains and its role in nucleation versus spreading have remained elusive. Here, we unveil major functions for Ccr4-Not in silencing and heterochromatin spreading at the mating type locus and subtelomeres. Mutations of the catalytic subunits Caf1 or Mot2, involved in RNA deadenylation and protein ubiquitinylation, respectively, result in impaired propagation of H3K9me3 and massive accumulation of nucleation-distal heterochromatic transcripts. Both silencing and spreading defects are suppressed upon disruption of the heterochromatin antagonizing factor Epe1. Overall, our results position the Ccr4-Not complex as a critical, dual regulator of heterochromatic gene silencing and spreading.

Local chromatin context regulates the genetic requirements of the heterochromatin spreading reaction.

Heterochromatin spreading, the expansion of repressive chromatin structure from sequence-specific nucleation sites, is critical for stable gene silencing. Spreading re-establishes gene-poor constitutive heterochromatin across cell cycles but can also invade gene-rich euchromatin de novo to steer cell fate decisions. How chromatin context (i.e. euchromatic, heterochromatic) or different nucleation pathways influence heterochromatin spreading remains poorly understood. Previously, we developed a single-cell sensor in fission yeast that can separately record heterochromatic gene silencing at nucleation sequences and distal sites. Here we couple our quantitative assay to a genetic screen to identify genes encoding nuclear factors linked to the regulation of heterochromatin nucleation and the distal spreading of gene silencing. We find that mechanisms underlying gene silencing distal to a nucleation site differ by chromatin context. For example, Clr6 histone deacetylase complexes containing the Fkh2 transcription factor are specifically required for heterochromatin spreading at constitutive sites. Fkh2 recruits Clr6 to nucleation-distal chromatin sites in such contexts. In addition, we find that a number of chromatin remodeling complexes antagonize nucleation-distal gene silencing. Our results separate the regulation of heterochromatic gene silencing at nucleation versus distal sites and show that it is controlled by context-dependent mechanisms. The results of our genetic analysis constitute a broad community resource that will support further analysis of the mechanisms underlying the spread of epigenetic silencing along chromatin.

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.

Genetic screen for suppressors of increased silencing in rpd3 mutants in Saccharomyces cerevisiae identifies a potential role for H3K4 methylation.

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

Epigenetic memory independent of symmetric histone inheritance.

Heterochromatic gene silencing is an important form of gene regulation that usually requires specific histone modifications. A popular model posits that inheritance of modified histones, especially in the form of H3-H4 tetramers, underlies inheritance of heterochromatin. Because H3-H4 tetramers are randomly distributed between daughter chromatids during DNA replication, rare occurrences of asymmetric tetramer inheritance within a heterochromatic domain would have the potential to destabilize heterochromatin. This model makes a prediction that shorter heterochromatic domains would experience unbalanced tetramer inheritance more frequently, and thereby be less stable. In contrast to this prediction, we found that shortening a heterochromatic domain in Saccharomyces had no impact on the strength of silencing nor its heritability. Additionally, we found that replisome mutations that disrupt inheritance of H3-H4 tetramers had only minor effects on heterochromatin stability. These findings suggest that histones carry little or no memory of the heterochromatin state through DNA replication.

Ovaries absent links dLsd1 to HP1a for local H3K4 demethylation required for heterochromatic gene silencing.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and regulation of gene expression1–4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help limit inappropriate H3K9me spreading12–15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me leading to aberrant gene silencing and epigenetic instability is not fully understood. Here we reveal an autoinhibited conformation in the conserved fission yeast S. pombe H3K9 methyltransferase Clr4/Suv39h that plays a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits its catalytic activity by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances Clr4 H3K9 methylation activity. Mutations predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains, and growth inhibition, demonstrating the importance of Clr4 intrinsic inhibition and auto-activation in regulating H3K9me deposition and preventing epigenetic instability. Conservation of the Clr4 autoinhibitory loop in other H3K9 methyltransferases, and automethylation of a corresponding lysine in the human SUV39H2 homolog16, suggest that the mechanism described here is broadly conserved.

Selective termination of lncRNA transcription promotes heterochromatin silencing and cell differentiation.

Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes.

Epigenetic regulation of tissue factor inducibility in endothelial cell senescence

Cellular senescence, a programmed state induced by multiple deleterious triggers, is characterised by permanent cell-cycle exit and altered gene expression and cell morphology. In humans it is considered a tumor suppressor mechanism, mediating removal of damaged or mutated cells from the cell-cycle pool, and may also contribute to the ageing process. In this study, we show that senescent human umbilical vein endothelial cells lose their ability to induce tissue factor (TF), a transmembrane protein with important roles in hemostasis and cancer progression, in response to thrombin or – independently of cell-surface receptors – phorbol-12-myristate-13-acetate. This phenomenon could not be explained by senescence-related alterations in the downstream signal transduction cascade or by accelerated TF mRNA degradation. Rather, using chromatin immuno-precipitation we could show that loss of TF gene inducibility during senescence occurs following chromatin remodelling of the TF promoter resulting from hypo-acetylation of histone H3. These findings were reversible after transduction of presenescent cultures with telomerase reverse transcriptase, enabling late-passage cultures to escape senescence. These results extend the involvement of heterochromatic gene silencing in senescence beyond cell cycle-related genes and suggest a novel anti-cancer mechanism of senescence through inhibition of TF inducibility.

HP1 knockdown is associated with abnormal condensation of almost all chromatin types in a grasshopper (Eyprepocnemis plorans)

Heterochromatin protein 1 (HP1) is a highly conserved family of eukaryotic proteins required for heterochromatic gene silencing and euchromatic gene transcription regulation. In addition, HP1 is involved in chromatin organization and protection of chromosome integrity during cell division. Here, we present a cytological and molecular analysis of the effects of HP1 knockdown in Eyprepocnemis plorans, a grasshopper species polymorphic for supernumerary heterochromatic chromosomes. Our results revealed contrasting effects of HP1 knockdown on gene activity. While the Bub1 gene decreased in expression level in HP1 knockdown animals, NOR activity, rRNA and, contrarily to previous reports in Drosophila, Hsp70 gene expression remained unchanged. Furthermore, HP1 knockdown resulted in abnormal chromatin condensation, chromosomal bridges, higher frequency of macrospermatids, loss of muscle mass and hemolymph amount as well as a low number of dividing cells and survival reduction. All these phenotypes are very likely due to the chromatin condensation disruption observed for almost all kinds of chromatin.

RNA editing regulates transposon-mediated heterochromatic gene silencing

Heterochromatin formation drives epigenetic mechanisms associated with silenced gene expression. Repressive heterochromatin is established through the RNA interference pathway, triggered by double-stranded RNAs (dsRNAs) that can be modified via RNA editing. However, the biological consequences of such modifications remain enigmatic. Here we show that RNA editing regulates heterochromatic gene silencing in Drosophila. We utilize the binding activity of an RNA-editing enzyme to visualize the in vivo production of a long dsRNA trigger mediated by Hoppel transposable elements. Using homologous recombination, we delete this trigger, dramatically altering heterochromatic gene silencing and chromatin architecture. Furthermore, we show that the trigger RNA is edited and that dADAR serves as a key regulator of chromatin state. Additionally, dADAR auto-editing generates a natural suppressor of gene silencing. Lastly, systemic differences in RNA editing activity generates interindividual variation in silencing state within a population. Our data reveal a global role for RNA editing in regulating gene expression.

Heterochromatic Gene Silencing by Activator Interference and a Transcription Elongation Barrier

Heterochromatin silences transcription, contributing to development, differentiation, and genome stability in eukaryotic organisms. Budding yeast heterochromatic silencing is strictly dependent on the silent information regulator (SIR) complex composed of the Sir2 histone deacetylase and the chromatin-interacting proteins Sir3 and Sir4. We use reconstituted SIR heterochromatin to characterize the steps in transcription that are disrupted to achieve silencing. Transcriptional activator binding is permitted before and after heterochromatin assembly. A comprehensive proteomic approach identified heterochromatin-mediated disruption of activator interactions with coactivator complexes. We also find that if RNA polymerase II (Pol II) is allowed to initiate transcription, the SIR complex blocks elongation on chromatin while maintaining Pol II in a halted conformation. This Pol II elongation barrier functions for even one nucleosome, is more effective when assembled with multiple nucleosomes, and is sensitive to a histone mutation that is known to disrupt silencing. This dual mechanism of silencing suggests a conserved principle of heterochromatin in assembling a specific structure that targets multiple steps to achieve repression.

The effect of JIL-1 on position-effect variegation is proportional to the total amount of heterochromatin in the genome

In this study we have taken advantage of recent whole genome sequencing studies that have determined the DNA content in the heterochromatic regions of each Drosophila chromosome to directly correlate the effect on position-effect variegation of a pericentric insertion reporter line, 118E-10 with the total amount of heterochromatic DNA. Heterochromatic DNA levels were manipulated by adding or subtracting a Y chromosome as well as by the difference in the amount of pericentric heterochromatin between the X and Y chromosome. The results showed a direct, linear relationship between the amount of heterochromatic DNA in the genome and the expression of the w marker gene in the 118E-10 pericentric reporter line and that increasing amounts of heterochromatic DNA resulted in increasing amounts of pigment/gene activity. In Drosophila heterochromatic spreading and gene silencing is counteracted by H3S10 phosphorylation by the JIL-1 kinase, and we further demonstrate that the haplo-enhancer effect of JIL-1 is proportional to the amount of total heterochomatin, suggesting that JIL-1's activity is dynamically modulated to achieve a more or less constant balance depending on the levels of heterochromatic factors present.

Trypanosoma bruceiOrc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching

Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host's immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

A novel RNAi protein, Dsh1, assembles RNAi machinery on chromatin to amplify heterochromatic siRNA

In fission yeast, siRNA is generated from pericentromeric noncoding RNA by the RNAi machinery. siRNA synthesis and heterochromatin formation are interdependent, forming a self-reinforcing loop on chromatin. In this system, siRNA is amplified by the RNA-dependent RNA polymerase complex (RDRC) and the endoribonuclease Dcr1, which synthesizes dsRNA and processes the dsRNA, respectively. The amplification is essential for stable heterochromatin formation. Here, a novel gene, dsh1(+) (defect of the gene silencing at centromeric heterochromatin), is identified as an essential component of RNAi-directed heterochromatin assembly. Loss of dsh1(+) abolishes normal RNAi function and heterochromatic gene silencing at pericentromeres. Dsh1 interacts with Dcr1 and RDRC and couples the reactions of both proteins to the effective production of siRNA in vivo. Dsh1 binds to heterochromatin in the absence of RDRC, while RDRC requires Dsh1 for its chromatin-binding activity, suggesting that Dsh1 recruits RDRC to chromatin. Immunofluorescence analysis shows that Dsh1 forms foci at the nuclear periphery, and some Dsh1 foci colocalize with Dcr1 and RDRC. Dsh1 is required for the colocalization of Dcr1 and RDRC. Moreover, loss of the nuclear periphery localization of Dsh1 abolishes Dsh1 function. Taken together, these results suggest that Dsh1 assembles the RNAi machinery on heterochromatin and forms a perinuclear compartment for amplification of heterochromatic siRNA.

Nucleolar Dominance of the Y Chromosome in Drosophila melanogaster

The rDNA genes are transcribed by RNA polymerase I to make structural RNAs for ribosomes. Hundreds of rDNA genes are typically arranged in an array that spans megabase pairs of DNA. These arrays are the major sites of transcription in growing cells, accounting for as much as 50% of RNA synthesis. The repetitive rDNA arrays are thought to use heterochromatic gene silencing as a mechanism for metabolic regulation, since repeated sequences nucleate heterochromatin formation in eukaryotes. Drosophila melanogaster carries an rDNA array on the X chromosome and on the Y chromosome, and genetic analysis has suggested that both are transcribed. However, using a chromatin-marking assay, we find that the entire X chromosome rDNA array is normally silenced in D. melanogaster males, while the Y chromosome rDNA array is dominant and expressed. This resembles "nucleolar dominance," a phenomenon that occurs in interspecific hybrids where an rDNA array from one parental species is silenced, and that from the other parent is preferentially transcribed. Interspecies nucleolar dominance is thought to result from incompatibilities between species-specific transcription factors and the rDNA promoters in the hybrid, but our results show that nucleolar dominance is a normal feature of rDNA regulation. Nucleolar dominance within D. melanogaster is only partially dependent on known components of heterochromatic gene silencing, implying that a distinctive chromatin regulatory system may act at rDNA genes. Finally, we isolate variant Y chromosomes that allow X chromosome array expression and suggest that the large-scale organization of rDNA arrays contribute to nucleolar dominance. This is the first example of allelic inactivation in D. melanogaster.

Intrinsic Nucleic Acid-Binding Activity of Chp1 Chromodomain Is Required for Heterochromatic Gene Silencing

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.

RNA‐guided DNA rearrangements in ciliates: Is the best genome defence a good offence?

Genomes, like crazy patchwork quilts, are stitched together over evolutionary time from diverse elements, including some unwelcome invaders. To deal with parasitic mobile elements, most eukaryotes employ a genome self-defensive manoeuvre to recognise and silence such elements by homology-dependent interactions with RNA-protein complexes that alter chromatin. Ciliated protozoa employ more 'offensive' tactics by actually unstitching and reassembling their somatic genomes at every sexual generation to eliminate transposons and their remnants, using as patterns the maternal genomes that were rearranged in the previous cycle. Genetic and genomic studies of the distant relatives Paramecium and Tetrahymena have begun to reveal how such events are carried out with remarkable precision. Whole genome, non-coding transcripts from the maternal genome are compared with transcripts from the zygotic genome that are processed through an RNA interference (RNAi)-related process. Sequences found only in the latter are targeted for elimination by the resulting short 'scanRNAs' in many thousand DNA splicing reactions initiated by a domesticated transposase. The involvement of widely conserved mechanisms and protein factors clearly shows the relatedness of these phenomena to RNAi-mediated heterochromatic gene silencing. Such malleability of the genome on a generational time scale also has profound evolutionary implications, possibly including the epigenetic inheritance of acquired adaptive traits.