Role of GADD45 during stress response and embryonic heart development
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Abstract
Active DNA demethylation plays a crucial role in shaping the epigenetic landscape to achieve spatiotemporal transcription regulation during mouse embryonic development. Recent work from our lab showed that GADD45 (Growth arrest and DNA damage-inducible 45) proteins bind R-loops, non-canonical nucleic acid structures comprising of a DNA: RNA hybrid and a displaced single-stranded DNA (ssDNA). GADD45 proteins recruit TET1 (Ten-eleven translocation) to the R-loop site, facilitating site-specific active DNA demethylation and transcription activation. However, the genome-wide prevalence and underlying mechanism of GADD45 and TET1-mediated DNA demethylation remains elusive. I have addressed this gap in our understanding by studying (I) stress response and (II) embryonic heart development, using mouse embryonic stem cells (mESCs) as an in-vitro model.
(I): Unpublished CUT&Tag sequencing datasets from our lab showed a significant enrichment for heat shock element (HSE) DNA motif at R-loop sites bound by GADD45a and TET1 in mESCs. The HSE motif is commonly observed in promoters of heat shock genes and is recognized by HSF1 to activate gene transcription during heat shock response (HSR). I hypothesized that GADD45 proteins bind the R-loops in heat shock gene promoters and recruit TET1 to aid with transcription activation during HSR. I uncovered thermal stress-specific nuclear translocation of GADD45 proteins during the early phase of HSR. Lack of GADD45 proteins sensitized the Gadd45a,b,g triple knockout (Gadd45 TKO) mESCs to thermal stress. Furthermore, I showed that HSF1 occupancy at heat shock gene promoters was significantly reduced in Gadd45 TKO mESCs. However, the reduced HSF1 occupancy did not lead to altered heat shock gene expression in Gadd45 TKO mESCs (with and without R-loop level manipulation). Intrigued by the lack of gene expression changes in Gadd45 TKO mESCs, I revisited our previous GADD45a mapping data and found that the previously identified GADD45a binding sites in mESCs were artefacts of antibody non-specificity. Therefore, I did not pursue this line of research work further.
(II): Unpublished work from our lab showed that GADD45 and TET proteins are required for normal embryonic heart development. However, the underlying mechanism by which GADD45 and TET proteins regulate embryonic heart development remained elusive. I hypothesized that GADD45 proteins balance TET functions (catalytic vs. non-catalytic) to regulate gene transcription during embryonic heart development. In line with my hypothesis, RNA-seq in Gadd45 TKO mESCs, with deletion of coding sequence (CDS) of all Gadd45 genes, showed reduced expression of heart development-associated genes. Furthermore, in-vitro cardiomyocyte differentiation of WT and Gadd45 TKO (CDS) mESCs revealed impaired cardiac differentiation of Gadd45 TKO (CDS) mESCs. These cardiac differentiation defects in Gadd45 TKO (CDS) mESCs were supported by pilot single-cell RNA sequencing (scRNA-seq) analysis. In collaboration with colleagues, we identified differential TET1 binding and differentially methylated sites between WT and Gadd45 TKO (CDS) mESCs. I showed that the reduced expression of heart development-associated genes was linked to increased TET1 occupancy in Gadd45 TKO (CDS) mESCs but did not correlate with DNA methylation changes. Instead, the downregulated heart development-associated genes with increased TET1 occupancy were enriched for Polycomb repressive complex (PRC) binding sites. These novel results suggest that GADD45 proteins supress TET1 non-catalytic functions and thereby promote expression of genes crucial for normal heart development.