Biology subjects

Moazed, D.

Publications and source records attributed to Moazed, D..

7 recordsLinked to original sources

bioRxiv2026

Genetic compensation in β-actin mutants occurs independently of mutations that destabilize β-actin mRNA

Proper maintenance of gene expression in response to mutations or environmental fluctuations is critical for cell development and survival. Recently, a novel genetic compensation mechanism was described wherein mutant mRNA decay triggers increased transcription of paralogous genes. This effect was reported for several genes, including {beta}-actin (Actb) in mouse embryonic stem cells, where Actb mRNA with a premature termination codon enhances transcription of its paralog, {gamma}-actin (Actg1), and partially rescues cytoskeletal defects. Here we show that, in both mouse and human embryonic stem cells, mutations in the ACTB gene, regardless of mutant mRNA expression, trigger genetic compensation. Furthermore, transgenic expression of mutant ACTB mRNA with a premature stop codon fails to induce genetic compensation. Depletion of the SRF or MRTF-A transcription factors, which are known to increase ACTB transcription in response to low ACTB protein levels, diminishes the genetic compensation response in ACTB mutants. These results suggest that genetic compensation in ACTB mutants is primarily mediated by a transcriptional feedback loop via SRF/MRTF-A, independent of the expression or degradation of mutant ACTB mRNA.

molecular biology↗

bioRxiv2025

Catalytic pocket of Clr4 (Suv39h) methyltransfer-ase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

Histone H3 lysine 9 (H3K9) methylation must be regulated to prevent inappropriate heterochromatin formation. Regulation of the conserved fission yeast H3K9 methyltransferase Clr4 (Suv39h) involves an automethylation-induced conformational switch and interaction of its catalytic SET domain with mono-ubiquitinated histone H3 lysine 14 (H3K14ub), a modification catalyzed by the Cul4 subunit of the CLRC complex. Using reconstituted CLRC, we show that Clr4 catalytic pocket serves as a substrate receptor for Cul4-dependent H3K14 ubiquitination. H3K14ub activates Clr4 to catalyze cis methylation of H3K9 on the same histone tail, while Clr4 auto-methylation enables H3K14ub-bound Clr4 to methylate H3K9 on an unmodified H3 tail in trans. Crosslinking and structural modeling reveal interactions between Clr4 chromo and SET domains, and between the chromo-domain and H3K14ub, suggesting that the chromodomain reads H3K9me3 and H3K14ub to allosterically regulate Clr4 activity. H3K14 ubiquitination therefore regulates Clr4 by promoting its recruitment and by positioning H3K9 in the active site.

biochemistry↗

bioRxiv2023

A simple mechanism for epigenetic inheritance of silent chromatin.

Mechanisms enabling genetically identical cells to differentially regulate gene expression are complex and central to organismal development and evolution. While gene silencing pathways involving sequence-specific recruitment of histone-modifying enzymes are prevalent in nature, examples of sequence-independent heritable gene silencing are scarce. Studies of Schizosaccharomyces pombe indicate that sequence-independent propagation of heterochromatin can occur but requires numerous multisubunit protein complexes and their various activities. Such complexity has precluded a coherent articulation of the minimal requirements for heritable gene silencing by conventional approaches. Here, we take an unconventional approach to defining these requirements by engineering sequence-independent silent chromatin inheritance in Saccharomyces cerevisiae. The memory-conferring mechanism is remarkably simple and requires only two proteins, one that recognizes histone H3 methylation and deacetylates histone H4, and another that recognizes unmodified H4 and catalyzes H3 methylation. These bilingual "read-write" proteins form an interdependent positive feedback loop capable of transmitting sequence-independent silent information over multiple generations.

molecular biology↗

bioRxiv2023

Requirements for establishment and epigenetic stability of mammalian heterochromatin

Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing transposons and genes, but the mechanisms that establish and maintain these domains are not fully understood. Here we use an inducible heterochromatin formation system combined with a CRISPR-based genetic screen to investigate the requirements for the establishment and maintenance of heterochromatin in mouse embryonic stem cells (mESCs). We show that DNA sequence-independent and histone H3 lysine 9 methylation (H3K9me)-dependent heterochromatin can be inherited for a limited number of cell divisions in mESCs but becomes stable upon differentiation. We provide evidence that the increased stability of heterochromatin in differentiated cells results from the downregulation of one or more enzymes that erase H3K9me and DNA methylation. Moreover, we show that in addition to components of the H3K9 and DNA methylation pathways, heterochromatin maintenance requires DHX9 and other RNA processing proteins. DHX9 is an RNA/DNA helicase with previously described roles in preventing genomic instability resulting from transcription-associated replication stress. We found that deletion of DHX9 results in defective heterochromatin inheritance and is associated with increased transcription of major satellite repeats, accumulation of R-loops, and loss of H3K9me. Our findings define the requirements for the establishment and epigenetic inheritance of mammalian heterochromatin and suggest that R-loops and replication stress lead to epigenetic instability.

developmental biology↗

bioRxiv2023

Three rules for epigenetic inheritance of human Polycomb silencing

Heritable expression of developmental genes is mediated by autoregulating transcription factor networks (TFNs) and Polycomb repressive chromatin silencing complexes (PRCs). Whether the mammalian Polycomb pathway mediates DNA sequence-independent epigenetic memory at genes being activated by TFNs remains unknown. Here, we show that, in human cells, newly induced Polycomb silencing at initially active developmental genes can be inherited for many cell divisions. However, when Polycomb silencing is similarly induced near ubiquitously expressed housekeeping genes, it is not heritable. Inheritance requires the recognition of CG-rich DNA by a Polycomb accessory protein, MTF2/PCL2, the histone H3 lysine 27 trimethylation (H3K27me3) and histone H2A lysine 119 mono-ubiquitination (H2AK119ub1) activities of PRC1 and PRC2, and their associated positive feedback (read-write). In the absence of H3K27me3, H2AK119ub1 can mediate inheritance of silencing by a mechanism that requires the recognition of H2AK119ub1 by the variant Polycomb Repressive Complex 1. These findings indicate that inheritance of Polycomb silencing in human cells (1) depends on sequence features of developmental gene loci (2) requires PRC1 and H2AK119ub1, and (3) can be mediated by PRC1-H2AK119ub1 independently of H3K27me3.

developmental biology↗

bioRxiv2019

An RNA degradation complex required for silencing of Polycomb target genes

Polycomb Repressive Complex (PRC) 1 and 2 are histone-modifying and chromatin-binding complexes that are required for silencing of developmental regulatory genes and genes that control cellular proliferation. Their gene silencing functions are thought to involve chromatin compaction and condensate formation but whether other mechanisms contribute to silencing is unknown. Here we show that the rixosome, a conserved RNA degradation complex with roles in ribosomal RNA processing and heterochromatic RNA degradation in fission yeast, associates with human PRC complexes, is recruited to promoters of Polycomb target genes in differentiated cell lines and embryonic stem cells, and is required for efficient silencing of Polycomb target genes. These findings reveal an unanticipated role for RNA degradation in Polycomb-mediated silencing.

molecular biology↗

bioRxiv2019

An RNA Degradation Complex Required for Spreading and Epigenetic Inheritance of Heterochromatin

Heterochromatin assembly requires the methylation of histone H3 lysine 9 by the Clr4(Suv39h) methyltransferase and both the spreading and epigenetic inheritance of heterochromatin involve the recognition of H3K9me-containing nucleosomes by Clr4 and catalysis of H3K9me on adjacent nucleosomes. How this read-write mechanism overcomes obstacles posed by RNA polymerase II and nascent RNA in its path is not fully understood. Here we identify a role for the highly conserved and essential Rix1-containing complex (here referred to as the rixosome), with known RNA endonuclease and polynucleotide kinase activities required for ribosomal RNA (rRNA) processing, in spreading and epigenetic inheritance of heterochromatin. Viable mutations in rixosome subunits that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing away from nucleation sites into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.

molecular biology↗