Project description:HSF1 binds DNA via the DBD domain, causing gene upregulation during HS. We assessed the effect of RD-mediated phase separation on chromatin targeting of HSF1 using Cut&Tag followed by high-throughput sequencing to map genome-wide binding of LLPS-competent versus LLPS-incompetent HSF1 mutants under both HS and NHS conditions. Comparing with WT HSF1 under NHS, both WT under HS and M1 under NHS showed increased and broad binding to enhancers and distal intergenic region, with binding most enriched in expected motifs of HSF-related transcription factors. To further assess the role for IDR-induced LLPS in chromatin targeting of HSF1, we used several additional strategies. First, the treatment of 1,6-hexanediol markedly decreased chromatin occupancy both of WT under HS and M1 under NHS conditions. Second, we interrogate chromatin binding of LLPS-deficient mutant M3. Cut&Tag analysis revealed that M3 showed decreased chromatin binding compared to WT under HS and M1 under NHS. The decreased chromatin binding of M3 to chromatin was not due to the loss of its DNA binding ability, as the EMSA assay revealed that M3 was still capable of binding HSE. Instead, the decreased chromatin binding reflects the loss of inter-molecular interaction between HSF1 that holds LLPS. Furthermore, M3 in heat shocked cells shows similar reduced genomic targeting and shallow binding pattern as NHS cells . Third, the enrichment of transcriptional apparatus RNA Pol II, CYCT1, BRD4 to HSF1 target genes also depend on whether HSF1 can phase separate at these sites. Lastly, we conducted live cell single molecule imaging to evaluate chromatin binding kinetics of LLPS-deficient mutant M3 relative to WT HSF1. Measurements of single molecule displacement and diffusion coefficient showed M3 to be significantly more mobile than WT under HS, which suggests M3 was less confined within phase-separated puncta compared with LLPS-competent HSF1. Consistent with this result, super resolution imaging of M3 also showed decreased cluster formation at HSP gene foci but maintained nSBs formation. Altogether, LLPS-forming capability of HSF1 is essential for the efficient recruitment of HSF1 and transcriptional apparatus to HSP gene loci.

Project description:Type 2 diabetes (T2D) is among the leading causes of death in the U.S. Ethnic differences in T2D prevalence are evident, including especially for Native Hawaiians (NHs) who remain disproportionately affected by it. This difference in T2D susceptibility involves an interplay between genetic and environmental factors, of which epigenetic mechanisms, including DNA methylation (DNAm) provide a novel approach to investigating gene-environment interactions of health and disease. Monocytes, an innate immune cell intrinsic to the inflammatory response, are a fundamental immune cell component that likely underlies T2D pathogenesis, given their involvement in inflammation and inflammation-associated insulin resistance and metabolic dysfunction. From participants enrolled into the Multiethnic Cohort Study (MEC), who self-identified as NH (n=152), Japanese American (JA; n=119), or White (n=121), we investigated monocyte-specific DNAm patterns in participants at a baseline visit, when free of T2D, using the HumanMethylation850K (850K) to determine whether monocytes harbor an ethnic-specific epigenetic signature of T2D risk that precedes T2D diagnosis. Using an epigenome-wide association study (EWAS), we found 904 significantly (q < 0.01) differentially methylated loci (DML) at a 5% difference in DNAm between participants who remained T2D free at a 15-year follow-up (i.e., controls) and those that would be diagnosed with T2D by follow-up (i.e., incident T2D) after adjusted for age, sex, and education level. These methylation differences were enriched at regulatory regions of the genome, including intergenic and intragenic regions. Notably, these DML were able to distinctly stratify NHs by T2D risk groups, however, this signature was inapparent in Whites and JAs. Likewise, NHs in the incident T2D group displayed a higher degree of DNAm variability. Sensitivity analysis with traditional risk factors (fasting glucose and body mass index [BMI]) and neighborhood socioeconomic status (nSES) found these risk factors had minimal effect on T2D risk-associated DML. Next, using a similar approach we found ethnic-specific DML in monocytes that was able to uniquely stratify NHs, JAs, and Whites, however, in NHs this epigenetic landscape displayed a higher degree of DNAm variability. Similarly, these DML were enriched at regulatory regions of the genome. In both cases, we found genes associated with biological functions and pathways relevant to monocyte functionality, including immune activation and cellular metabolism. Due to the higher degree of epigenetic variability in NHs, including especially NHs with T2D risk, we investigated differentially variable CpGs associated with T2D risk and ethnic-differences. We found differentially variable CpGs between T2D risk groups were able to stratify NHs with and without T2D risk, whereas it was unresolvable in JAs and Whites. Ethnic-specific DNAm variability clustered each ethnic population distinctly, and NH controls were further partitioned from NHs with T2D risk. Our findings suggest monocytes harbor a unique DNAm signature associated with T2D risk, which may be related to ethnic differences that underlie DNAm variability in monocytes. The increased epigenetic variability in monocytes from NHs may underlie epigenetic plasticity that could allow these cells to readily respond to adverse environmental conditions throughout the life course that may underlie long-term disease risk, whereas in other less susceptible populations with similar environmental exposure, this epigenetic plasticity may not be apparent in monocytes.

			Action	DRS
	SRR15114369_1.fastq.gz	Fastqsanger.gz
	SRR15114369_2.fastq.gz	Fastqsanger.gz

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