Biology subjects

Spracklin, G.

Publications and source records attributed to Spracklin, G..

4 recordsLinked to original sources

bioRxiv2023

Tunable DNMT1 degradation reveals cooperation of DNMT1 and DNMT3B in regulating DNA methylation dynamics and genome organization

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present untransformed and cancer cell models that allow inducible and reversible global modulation of DNAme through DNMT1 depletion. By dynamically assessing the effects of induced passive demethylation through cell divisions at both the whole genome and locus-specific level, we reveal a cooperative activity between DNMT1 and DNMT3B to maintain and control DNAme. Moreover, we show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin abundance, compartmentalization, and peripheral localization. DNA methylation loss coincided with a gradual reduction of cell fitness due to G1 arrest, but with minor level of mitotic failure. Altogether, this powerful system allows DNMT and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNA methylation dysfunction and human disease.

molecular biology↗

bioRxiv2021

Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1 and HP1{beta} correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1/{beta} is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1/{beta} heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1/{beta}, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1/{beta} heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion. HighlightsO_LIThree inactive chromatin states are distinguishable by long-range contact frequencies in HCT116, respectively associated with H3K9me3, H3K27me3 and a H3K9me2 state with neutral contact preferences. C_LIO_LIH3K9me3-HP1/{beta} heterochromatin has a high degree of homotypic affinity and is permissive to loop extrusion but depleted in extrusion barriers. C_LIO_LIDisrupting DNA methylation causes widespread loss of H3K9me3-HP1/{beta} and dramatic remodeling of genome compartmentalization. C_LIO_LIH3K9me3-HP1/{beta} is replaced by the neutral inactive state, which gains CTCF loop extrusion barriers and associated contact frequency patterns. C_LIO_LIDNA methylation suppresses CTCF binding via two distinct mechanisms. C_LI

genomics↗

bioRxiv2020

Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negate the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high resolution genomic feature studies.

genetics↗

bioRxiv2017

Transgenerational Epigenetic Inheritance Factors Localize to Spatiallyand Temporally Ordered Liquid Droplet Assemblages

Epigenetic information can be inherited for multiple generations (termed transgenerational epigenetic inheritance or TEI) 1,2. Non-coding RNAs have emerged as important mediators of TEI, although the mechanism(s) by which non-coding RNAs mediate TEI remains poorly understood. dsRNA-mediated gene silencing (RNAi) in C. elegans is a robust example of RNA-directed TEI3-5. To further our understanding of RNA-directed TEI, we conducted a genetic screen in C. elegans to identify genes required for RNAi inheritance. Our screen identified the conserved RNA helicase/Zn finger protein ZNFX-1 and the Argonaute protein WAGO-4. We find that WAGO-4 and ZNFX-1 act cooperatively in inheriting generations to maintain small interfering (si)RNA expression over generational time. ZNFX-1/ WAGO-4 localize to a liquid droplet organelle termed the P granule in early germline blastomeres. Later in development, ZNFX-1/WAGO-4 appear to separate from P granules to form independent foci that are adjacent to, yet remain distinct, from P granules. ZNFX-1/WAGO-4 labeled foci exhibit properties reminiscent of liquid droplets and we name these foci Z granules. In the adult germline, Z granules assemble into ordered tri-droplet assemblages with P granules and another germline droplet-like foci termed the Mutator foci. This work identifies a conserved RNA-binding protein that drives RNA-directed TEI in C. elegans, defines a new germline foci that we term the Z granule, demonstrates that liquid droplet formation is under developmental control, and shows that liquid droplets can assemble into spatially ordered multi-droplet structures. We speculate that temporal and spatial ordering of liquid droplets helps cells organize and coordinate the complex RNA processing pathways underlying gene regulatory systems, such as RNA-directed TEI.

genetics↗