Biology subjects

Al-Sady, B.

Publications and source records attributed to Al-Sady, B..

11 recordsLinked to original sources

bioRxiv2026

Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

Repressive histone methyltransferases carry a catalytic ("write") domain and a separate domain specialized for recognizing ("reading") the reaction product. This read-write configuration acts as a positive feedback mechanism for epigenetic maintenance and the growth of repressive chromatin domains. Feedback exhibits as catalytic stimulation and is understood to act towards a proximal (trans) nucleosome. Whether this stimulation affects a specific methylation transition and whether it is restricted to trans-stimulation remains opaque. Here, we dissect the positive feedback in the heterodimeric histone 3 lysine 9 (H3K9) mono- and dimethlyase G9a-GLP, which carries two catalytic SET and two product-reading Ankyrin repeat (ANK) domains. We find that reading by both ANK domains is required for H3K9 di-, but not monomethylation on nucleosomes and for tight binding to them. As this read-writing occurs on dilute mononucleosomes, we propose that intranucleosomal feedback occurs for G9a-GLP. Swapping the ANK domains results in loss of dimethylation while maintaining nucleosome binding, indicating catalytic coupling of nucleosome methylation intermediates to reading. Crosslinking mass spectrometry reveals specific G9a surfaces that contact nucleosomal methylation intermediates. Structural approaches reveal how these surfaces position the G9a ANK domain on the methylation-intermediate nucleosome and stabilize G9a-GLP on chromatin during the reaction.

biochemistry↗

bioRxiv2025

Discrete Subdomains Establish Epigenetic Diversity in Subtelomeric Heterochromatin

Subtelomeres are imperfect repeats adjacent to telomeres that are transcriptionally repressed by heterochromatin. Although essential for genome integrity, their repetitive nature has thwarted dissection of local heterochromatin assembly and maintenance mechanisms. By engineering Schizosaccharomyces pombe strains carrying fluorescent reporters at a single subtelomere, we uncovered distinct subdomains. These subdomains have different silencing requirements: Telomere-proximal regions rely on canonical shelterin- or RNAi-dependent nucleation pathways, whereas telomere-distal regions involve nucleosome remodelers, histone chaperones, and boundary-associated factors. Subdomains also exhibit discrete epigenetic states emerging both at homologous loci on different chromosome arms and along the same subtelomeric sequence. We document these epigenetic states using multi-generational live imaging and targeted perturbations. These analyses show that subtelomeric subdomains display position-specific, clonally variable silencing across a spectrum from robust to fragile epigenetic states. Interestingly, structural variants, common in subtelomeric sequences across eukaryotes, dictate local epigenetic stability. These findings reveal that subtelomeres, long recognized for their sequence variability, form a dynamic mosaic of coexisting epigenetic domains. This implies a wide range of gene-repressive regulatory logic at the chromosome ends, from environmental responsiveness to silencing stability.

genomics↗

bioRxiv2024

Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading.

Heterochromatin formation in Schizosaccharomyces pombe requires the spreading of histone 3 (H3) Lysine 9 (K9) methylation (me) from nucleation centers by the H3K9 methylase, Suv39/Clr4, and the reader protein, HP1/Swi6. To accomplish this, Suv39/Clr4 and HP1/Swi6 have to associate with nucleosomes both nonspecifically, binding DNA and octamer surfaces and specifically, via recognition of methylated H3K9 by their respective chromodomains. However, how both proteins avoid competition for the same nucleosomes in this process is unclear. Here, we show that phosphorylation tunes oligomerization and the nucleosome affinity of HP1/Swi6 such that it preferentially partitions onto Suv39/Clr4s trimethyl product rather than its unmethylated substrates. Preferential partitioning enables efficient conversion from di-to trimethylation on nucleosomes in vitro and H3K9me3 spreading in vivo. Together, our data suggests that phosphorylation of HP1/Swi6 creates a regime that increases oligomerization and relieves competition with the "read-write" mechanism of Suv39/Clr4, together promoting for productive heterochromatin spreading.

molecular biology↗

bioRxiv2024

The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate

Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the effect of spatial position on heterochromatin function has not been defined. Here, we remove three nuclear lamins and lamin B receptor (LBR) in mouse embryonic stem cells (mESCs) and show that heterochromatin detaches from the nuclear periphery. Mutant mESCs sustain naive pluripotency and maintain H3K9me2 across the genome but cannot repress H3K9me2-marked genes or transposons. Further, mutant cells fail to differentiate into epiblast-like cells (EpiLCs), a transition that requires the expansion of H3K9me2 across the genome. Mutant EpiLCs can silence naive pluripotency genes and activate epiblast-stage genes. However, H3K9me2 cannot repress markers of alternative fates, including primitive endoderm. We conclude that the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of H3K9me2-marked heterochromatin to shape cell fate decisions.

cell biology↗

bioRxiv2024

A systematic quantitative approach comprehensively defines domain-specific functional pathways linked to Schizosaccharomyces pombe heterochromatin regulation

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres, and the silent mating type locus in Schizosaccharomyces pombe. Using quantitative measures, iterative genetic screening, and domain-specific heterochromatin reporters, we identified 369 mutants with different degrees of reduced or enhanced silencing. As expected, mutations in the core heterochromatin machinery globally decreased silencing. However, most other mutants exhibited distinct qualitative and quantitative profiles that indicate domain-specific functions. For example, decreased mating type silencing was linked to mutations in heterochromatin maintenance genes, while compromised subtelomere silencing was associated with metabolic pathways. Furthermore, similar phenotypic profiles revealed shared functions for subunits within complexes. We also discovered that the uncharacterized protein Dhm2 plays a crucial role in maintaining constitutive and facultative heterochromatin, while its absence caused phenotypes akin to DNA replication-deficient mutants. Collectively, our systematic approach unveiled a landscape of domain-specific heterochromatin regulators controlling distinct states and identified Dhm2 as a previously unknown factor linked to heterochromatin inheritance and replication fidelity.

genomics↗

bioRxiv2023

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

AbstractHeterochromatic 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. The fission yeast Ccr4-Not complex has been involved 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. Author SummaryEukaryotic genomes are partitioned into relaxed, gene-rich regions, and condensed, gene-poor domains called heterochromatin. The maintenance of heterochromatin is crucial for proper genome expression and integrity, and requires multiple factors regulating histone modifications and/or the levels of RNA molecules produced from these regions. Such effectors not only promote heterochromatin assembly but also ensure its propagation from specific nucleation sites to defined domain boundaries. However, while the mechanisms involved in initiation of heterochromatin formation have been well documented, the molecular and biochemical properties underlying its spreading remain largely elusive. By combining genetic and single-cell approaches, we report here that the fission yeast Ccr4-Not complex, a multisubunit complex conserved throughout eukaryotes, is essential for efficient heterochromatin spreading to repress expression of nucleation-distal RNAs. The two catalytic activities of the complex, RNA deadenylation and protein ubiquitinylation, are each critical, thereby defining a dual enzymatic requirement in the process.

genetics↗

bioRxiv2021

The histone chaperone FACT facilitates heterochromatin spreading through regulation of histone turnover and H3K9 methylation states

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT has been implicated in heterochromatin silencing, however, the mechanisms by which FACT engages in this process remain opaque. Here, we pin-pointed its function to the heterochromatin spreading process. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1 via nucleosome stabilization. Together, our study identifies FACT as a histone chaperone that specifically promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of epigenetic marks.

genetics↗

bioRxiv2021

Activation of Histone 3 Lysine 9 methyl writing and reading capabilities within the G9a-GLP heterodimer

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding ("reading") and deposition ("writing") activity of G9a and GLP, and why heterodimerization is essential in vivo remains opaque. Here, we examine the H3K9me "reading" and "writing" activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared to the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking [~] 10-fold increased turnover rate for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. Crosslinking Mass Spectrometry suggests that differences between the homodimers and the unique activity of the heterodimer may be encoded in altered ground state conformations, as each dimer displays different domain contacts. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are deposited by G9a-GLP, which may require a more active form of the enzyme.

biochemistry↗

bioRxiv2020

Local chromatin context dictates the genetic determinants of the heterochromatin spreading reaction

Heterochromatin spreading, the expansion of gene-silencing structures from DNA-encoded nucleation sites, occurs in distinct settings. Spreading re-establishes gene-poor constitutive heterochromatin every cell cycle, but also invades gene-rich euchromatin de novo to steer cell fate decisions. How chromatin context, i.e. euchromatic, heterochromatic, or different nucleator types, influences the determinants of this process remains poorly understood. By screening a nuclear function gene deletion library in fission yeast using a previously established heterochromatin spreading sensor system, we identified regulators that positively or negatively alter the propensity of a nucleation site to spread heterochromatin. We find that different chromatin contexts are dependent on unique sets of genes for the regulation of heterochromatin spreading. Further, we find that spreading in constitutive heterochromatin requires Clr6 histone deacetylase complexes containing the Fkh2 transcription factor, while the Clr3 deacetylase is globally required for silencing. Fkh2 acts by recruiting Clr6 to nucleation-distal chromatin sites. Our results segregate the pathways that control lateral heterochromatin spreading from those that instruct DNA-directed assembly in nucleation.

molecular biology↗

bioRxiv2019

Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While hetero- and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary, using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Interestingly, this effect can be gene orientation dependent. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. At least two independent pathways direct this Set1 activity-inhibition of catalysis by Suv39/Clr4 and disruption of nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

genetics↗

bioRxiv2017

A memory element imposes epigenetic behavior on intrinsically labile RNAi-induced heterochromatin spread.

ABTRACTThe heterochromatin spreading reaction is a central contributor to the formation of gene-repressive structures, which are re-established with high fidelity following replication. The high fidelity of this process is not obviously encoded in the primary spreading reaction. To resolve origins of stable inheritance of repression, we probed the intrinsic fidelity of spreading events in fission yeast using a system that quantitatively describes the spreading reaction in live single cells. We show that spreading from RNAi-nucleated elements is stochastic, multimodal, and fluctuates dynamically across time. In contrast, a second form of spreading, nucleated by the cis-acting element REIII, is deterministic, has high memory capacity and acts as the source of locus fidelity. REIII enables fidelity in part by endowing the locus with resistance to perturbations. Together, our results suggest that epigenetic capacity may not be intrinsically encoded in the spreading reaction, but rather requires collaboration with specialized memory elements.

genetics↗