Assessment of the histone mark-based epigenomic landscape in human myometrium at term pregnancy

Abstract

The myometrium plays a critical role during pregnancy as it is responsible for both the structural integrity of the uterus and force generation at term. Emerging studies in mice indicate a dynamic change of the myometrial epigenome and transcriptome during pregnancy to ready the contractile machinery for parturition. However, the regulatory systems underlying myometrial gene expression patterns throughout gestation remain largely unknown. Here, we investigated human term pregnant nonlabor myometrial biopsies for transcriptome, enhancer histone mark cistrome, and chromatin conformation pattern mapping. More than thirty thousand putative enhancers with H3K27ac and H3K4me1 double positive marks were identified in the myometrium. Enriched transcription factor binding motifs include known myometrial regulators AP-1, STAT, NFkB, and PGR among others. Putative myometrial super enhancers are mostly colocalized with progesterone receptor-occupying sites and preferentially associated with highly expressing genes, suggesting a conserved role of PGR in regulating the myometrial transcriptome between species. In human myometrial specimens, inferred PGR activities are positively correlated with phospholipase C like 2 (PLCL2) mRNA levels, supporting that PGR may act through this genomic region to promote PLCL2 expression. PGR overexpression facilitated PLCL2 gene expression in myometrial cells. Using CRISPR activation, we assessed the functionality of a PGR putative enhancer 35 kilobases upstream of the contractile-restrictive gene PLCL2. In summary, the results of this study serve as a resource to study gene regulatory mechanisms in the human myometrium at the term pregnancy stage for further advancing women's health research.

Keywords: Progesterone; cell biology; chromatin; genetics; genomics; human; myometrium.

Figure 1.. Putative enhancers in term pregnant human myometrial tissues.

(A) Distinct and common putative enhancers in term pregnant myometrial biopsies from three subjects. Genomic regions with H3K27ac and H3K4me1 double positive histone marks are defined as putative active enhancers. (B) Association of commonly shared putative enhancers with active genes. The association between an interval and an active gene is defined by locating within 100 kb vicinity of each other. (C) Over-Represented transcription factor binding motifs in putative enhancers. A subset of enriched motifs that are relevant to myometrial homeostasis in the 13,090 H3K4me1/H3K27ac-positive putative enhancer regions is displayed.

Figure 1—figure supplement 1.. Subject variations on H3K27ac-positive histone marks.

Figure 2.. Putative super enhancers in term pregnant human myometrial tissues.

(A) Number of super enhancers mapped in tissues of each individual human subject. (B) Association of commonly shared super enhancers with active genes in human myometrium. (C) Selected enriched gene ontology terms in the 346 active genes that are associated with super enhancers.

Figure 2—figure supplement 1.. PGR occupancy in myometrial enhancers.

Percentages of enhancers and super enhancers that manifest PGR occupancy. PGR genome occupancy data was previously published in NCBI GEO accession numbers GSM4081683 and GSM4081684.

Figure 3.. Identification of enhancers for the PLCL2 gene.

(A) UCSC Genome Browser track view of the human PLCL2 locus marked with gRNA targeting locations. (B, C) Relative PLCL2 mRNA levels measured by qRT-PCR in hTERT-HM cells (B) or T-HESC cells (C) that express denoted gRNAs with the CRISPR activator (N=3 with technical duplicates). Statistical significance was determined using unpaired t-test for comparisons between two groups, or one-way ANOVA for comparisons between >2 groups. Significance levels are denoted as follows: ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05. Error bars are shown as mean with SEM.

Figure 4.. PGR regulation of PLCL2 expression.

(A) PGR mRNA abundance in hTERT-HM cells with the PGR-promoter targeting (PGR640) or non-targeting gRNAs under the CRISPR activation system. (N=3 with technical qPCR duplicates). (B) Protein abundance in hTERT-HM cells with the PGR-promoter targeting (PGR640 and P6SM) or non-targeting gRNAs under the CRISPR activation system. Protein extracts from unmanipulated MCF7 and HEK293T cells serve as positive and negative control for the PGR presence. (C) PLCL2 mRNA abundance in hTERT-HM cells with the PGR-promoter targeting (PGR640) or non-targeting gRNAs under the CRISPR activation system. (N=3 with technical qPCR duplicates). (D) Pearson correlation between PLCL2 mRNA levels and inferred PGR activities (T-Scores) in 43 human myometrial specimens. **, p<0.01; *, p<0.05 by Mann-Whitney test (A) and unpaired t-test (C). Error bars are shown as mean with SEM.