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. 2020 Jul 15;18(7):e3000710.
doi: 10.1371/journal.pbio.3000710. eCollection 2020 Jul.

The pregnant myometrium is epigenetically activated at contractility-driving gene loci prior to the onset of labor in mice

Virlana M Shchuka  1 , Luis E Abatti  1 , Huayun Hou  2   3 , Nawrah Khader  1 , Anna Dorogin  4 , Michael D Wilson  2   3 , Oksana Shynlova  4   5 , Jennifer A Mitchell  1
Affiliations

The pregnant myometrium is epigenetically activated at contractility-driving gene loci prior to the onset of labor in mice

Virlana M Shchuka et al. PLoS Biol. .

Abstract

During gestation, uterine smooth muscle cells transition from a state of quiescence to one of contractility, but the molecular mechanisms underlying this transition at a genomic level are not well-known. To better understand these events, we evaluated the epigenetic landscape of the mouse myometrium during the pregnant, laboring, and postpartum stages. We generated gestational time point-specific enrichment profiles for histone H3 acetylation on lysine residue 27 (H3K27ac), histone H3 trimethylation of lysine residue 4 (H3K4me3), and RNA polymerase II (RNAPII) occupancy by chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq), as well as gene expression profiles by total RNA-sequencing (RNA-seq). Our findings reveal that 533 genes, including known contractility-driving genes (Gap junction alpha 1 [Gja1], FBJ osteosarcoma oncogene [Fos], Fos-like antigen 2 [Fosl2], Oxytocin receptor [Oxtr], and Prostaglandin G/H synthase 2 (Ptgs2), for example), are up-regulated at day 19 during active labor because of an increase in transcription at gene bodies. Labor-associated promoters and putative intergenic enhancers, however, are epigenetically activated as early as day 15, by which point the majority of genome-wide H3K27ac or H3K4me3 peaks present in term laboring tissue is already established. Despite this early exhibited histone signature, increased noncoding enhancer RNA (eRNA) production at putative intergenic enhancers and recruitment of RNAPII to the gene bodies of labor-associated loci were detected only during labor. Our findings indicate that epigenetic activation of the myometrial genome precedes active labor by at least 4 days in the mouse model, suggesting that the myometrium is poised for rapid activation of contraction-associated genes in order to exit the state of quiescence.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Quiescent and term laboring myometrial transcriptomes exhibit differential expression profiles.
(A) Gestational schematic outlining days and time points at which myometrial tissues were collected for transcriptome- or genome-wide sequencing analyses. Collection days include gestational d15, d19 TNIL, d19 LAB, and pp. (B) RNA-seq volcano plot highlighting transcriptional status of genes exhibiting differential expression levels between d15 and d19 LAB myometrial tissues. Data associated with this figure can be found in S1 Table. (C) Hierarchical clustering of gene groups based on RNA expression changes between d15 and d19 LAB samples (n = 5 per gestational day). Data associated with this figure can be found in S2 Data. (D) Total RNA-seq reads (RPM) at the labor-associated Fos gene locus for d15 and d19 LAB samples mapped to the mm10 mouse genome assembly. chr12, chromosome 12; d, day; FC, fold-change; LAB, active labor; NS, nonsignificant; pp, postpartum; RNA-seq, RNA-sequencing; RPM, reads per million; TNIL, term-not-in-labor.
Fig 2
Fig 2. Up-regulation of labor-associated genes involves an increase in their primary transcript levels.
(A) Exon- and intron-specific RNA-seq reads (RPM) at the labor-associated Fos gene locus from d15 and d19 LAB samples, mapped to the mm10 mouse genome assembly. (B) iRNA-seq volcano plot highlighting expression changes based on intron reads between d15 and d19 LAB samples. (C) Venn diagram displaying the number of genes significantly up-regulated during labor relative to d15 that show enrichment in exon and intron reads (region of overlap) or enrichment in either exon or intron reads (regions of nonoverlap). (D) Confirmation of laboring time point–specific up-regulation of primary transcript expression of contractility-promoting genes by RT-qPCR. Groups labeled with different letters show significant difference, with p < 0.05. Data associated with this figure can be found in S3 Data. chr12, chromosome 12; d, day; FC, fold-change; iRNA-seq, intron RNA-seq; LAB, active labor; NS, nonsignificant; P(adj), adjusted p-value; RNA-seq, RNA-sequencing; RPM, reads per million; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction; TNIL, term-not-in-labor.
Fig 3
Fig 3. Gene expression activation–associated histone marks are enriched at promoters of contractility-driving genes in advance of labor onset.
(A) Anti-H3K4me3 and anti-H3K27ac ChIP-seq reads (RPM) mapped at promoter of labor-associated gene Fos in d15, d19 TNIL, d19 LAB, and pp samples. (B) Genome-wide enrichment of H3K4me3 and H3K27ac at gene promoters throughout the genome. Signal +/− 2 kb of TSSs is displayed, with genes ordered at each indicated time point according to decreasing enrichment (red → white) profile in d15 samples. (C) Plots exhibiting log2-fold H3K4me3 or H3K27ac signal, normalized to input, at promoters of genes whose expression is enriched in laboring samples relative to d15 samples based on intron reads. Data associated with this figure can be found in S6 Data. (D) Violin plots displaying log2-fold H3K4me3 or H3K27ac signal (RPM), normalized to input, at promoters of genes whose intron read–based expression is enriched in laboring samples relative to d15 samples. Groups labeled with different letters show significant difference, with p < 0.05. Data associated with this figure can be found in S7 and S8 Datas. ChIP-seq, chromatin immunoprecipitation with massively parallel sequencing; chr12, chromosome 12; d, day; Fos, FBJ osteosarcoma oncogene; H3K4me3, H3 trimethylation of lysine residue 4; H3K27ac, H3 acetylation on lysine residue 27; LAB, active labor; pp, postpartum; RPM, reads per million; TNIL, term-not-in-labor; TSSs, transcription start site.
Fig 4
Fig 4. Active labor is associated with recruitment of RNAPII to labor-driving genes and eRNA expression at gene-adjacent intergenic regions with H3K27ac peaks.
(A) Metagene plot exhibiting log2-fold RNAPII signal, normalized to input, at bodies of genes whose intron read–based expression levels are enriched in d19 LAB samples relative to d15 samples. Data associated with this figure can be found in S6 Data. (B) Violin plots displaying log2-fold RNAPII signal (RPM), normalized to input, at promoters of genes whose intron read–based expression levels are enriched in d19 LAB samples relative to d15 samples. Groups labeled with different letters show significant difference, with p < 0.05. Data associated with this figure can be found in S10 Data. (C) RNA-seq, RNAPII, and H3K27ac ChIP-seq reads (RPM) mapped at the Fos locus in d15 or d19 LAB samples. Regions containing RNAPII (red bars) or H3K27ac peaks (blue bars) indicated in d15 and d19 LAB samples. RNA-seq profile at labor–up-regulated eRNA-exhibiting region (situated between the dashed lines) downstream of Fos is presented at a different scale compared with the rest of the locus. (D) eRNA volcano plot highlighting intergenic H3K27ac peaks in genomic regions that exhibit significant differences in eRNA levels between d15 and d19 LAB samples. (E) All enriched transcription factor motifs identified at genomic regions that feature intergenic H3K27ac peaks and exhibit a significant increase in eRNA levels in d19 LAB samples compared with d15 samples. (F) The top 10 enriched motifs identified at promoters for genes that exhibit a significant increase in intronic RNA levels for d19 laboring samples compared with d15 samples. ChIP-seq, chromatin immunoprecipitation with massively parallel sequencing; chr12, chromosome 12; d, day; eRNA, enhancer RNA; FC, fold-change; Fos, FBJ osteosarcoma oncogene; H3K27ac, H3 acetylation on lysine residue 27; LAB, active labor; NS, nonsignificant; RNAPII, RNA polymerase II; RNA-seq, RNA-sequencing; RPM, reads per million; P(adj), adjusted p-value; TES, transcription end site; TSS, transcription start site.
Fig 5
Fig 5. Model of epigenetic priming and transcriptional regulation mechanisms that initiate increased gene expression during labor.
Display of the chromatin landscape around typical labor–up-regulated genes at quiescent (above) and term laboring (below) stages of gestation. Quiescent: during pregnancy, H3K27ac and H3K4me3—histone markers typically associated with active genes—are already present at labor-associated gene promoters and putative intergenic enhancers, thereby priming the epigenome for contractility-driving transcriptional events in advance of term. The aforementioned regions also contain an enrichment of AP-1 transcription factor motifs that may allow for potential binding of homodimerized JUN proteins, which are already known to be expressed in the quiescent stage. Laboring: conversely, the progression of gestation toward term induces transcription events at premarked intergenic regions and results in eRNA accumulation within key gene loci. Putative regulatory region–bound JUN proteins may be replaced by FOS:JUN proteins alongside phosphorylated RELA. This hypothetical switch in dimerized transcription factor binding events may underlie the labor-specific recruitment of RNAPII to labor-associated gene promoters that we have observed and, consequently, enhance labor-specific transcription through the bodies of these genes. Molecular events depicted in this figure that have not been experimentally validated in our or previous studies are marked by “?”. Ac, acetylation; AP-1, activator protein 1; CTD, C-terminal domain; FOS, FBJ osteosarcoma oncogene; H3K4me3, H3 trimethylation of lysine residue 4; H3K27ac, H3 acetylation on lysine residue 27; JUN, Jun proto-oncogene; Me3, trimethylation; P, phosphorylation site; RELA, Nuclear factor kappa beta p65 subunit; RNAPII, RNA polymerase II.
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