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Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2019)

The microrchidia (MORC) family of proteins are widely conserved eukaryotic and prokaryotic organisms. MORCs are GHKL (Gyrase, HSP90, Histidine Kinase, MutL) ATPases. Despite evidence that they are involved in gene silencing and genome compaction in multiple eukaryotic organisms, it is unknown what is their direct contribution to gene regulation in vivo or how they act on a molecular level. To elucidate how MORCs act to maintain gene silencing, I determined that MORCs are GHKL ATPases that form multimers and regulate a unique subset of genes. I show that MORCs with functional CW histone mark reader domains can be targeted to chromatin by recognition of histone tails, and validate MORC3 localization in vivo. From a structural analysis perspective, we also show that MORCs dimerize at the N terminus upon ATP binding, and that they are DNA binding proteins which show length-dependent binding preferences. MORCs prefer to bind to longer DNA templates over short DNA templates, and they also have little sequence specificity with regards to their binding specificity. Using single molecule studies, we show that C. elegans MORC-1 compacts DNA by loop trapping it; MORC-1 also compacts nucleosome templates. These single molecule studies also clarify the role of ATP metabolism in MORC action. We show that ATP stimulates the rate of compaction on DNA, and is further stimulated by an ATP non-hydrolysable analog, demonstrating that nucleotide binding impacts MORC-1 stability on DNA. We also show that MORC-1 phase separates in vitro and during DNA compaction, MORC-1 forms large foci that are topologically entrapped on DNA. The foci mirror the 1,6 hexanediol resistant puncta observed in vivo. Their resistance to 1,6 hexanediol treatment is likely because MORC-1 puncta are topologically entrapped and use DNA as a scaffold.

MORCs thus depend on alternative mechanisms in order to bind to specific regions of chromatin for the same mechanistic process, which culminates in genome compaction. Altogether, my thesis reveals novel mechanistic insights into the MORC family. I propose a model whereby MORCs are targeted in vivo by either their own CW domains or other protein factors, trap loops of DNA at that locus to compact and silence the gene, and multimerize on themselves to create a phase separated environment that enforces genome compaction by inducing DNA compaction.

  • 8 supplemental videos
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Cover page: Elucidating the mechanism of the microrchidia family of ATPases
  • Article
  • Peer Reviewed
UCLA Previously Published Works (2011)
Abstract Background We performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines. Results We find that heavily methylated non-CG sites are strongly conserved, especially when found within the motif TACAG. They are enriched in splice sites and are more methylated than other non-CG sites in genes. We next studied the relationship between allele-specific expression and allele-specific methylation. By combining bisulfite sequencing and whole transcriptome shotgun sequencing (RNA-seq) data we identified 1,020 genes that show allele-specific expression, and 14% of CG sites genome-wide have allele-specific methylation. Finally, we asked whether the methylation state of transcription factor binding sites affects the binding of transcription factors. We identified variations in methylation levels at binding sites and found that for several transcription factors the correlation between the methylation at binding sites and gene expression is generally stronger than in the neighboring sequences. Conclusions These results suggest a possible but as yet unknown functional role for the highly methylated conserved non-CG sites in the regulation of HESCs. We also identified a novel set of genes that are likely transcriptionally regulated by methylation in an allele-specific manner. The analysis of transcription factor binding sites suggests that the methylation state of cis-regulatory elements impacts the ability of factors to bind and regulate transcription.
  • 7 supplemental files
Cover page: A comparative analysis of DNA methylation across human embryonic stem cell lines
  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2019)
Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.
    Cover page: The Gene-Silencing Protein MORC-1 Topologically Entraps DNA and Forms Multimeric Assemblies to Cause DNA Compaction
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2012)
    In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var)3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.
      Cover page: The SET-Domain Protein SUVR5 Mediates H3K9me2 Deposition and Silencing at Stimulus Response Genes in a DNA Methylation–Independent Manner
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