Role of Jmjd2b in trophoblast stemness maintenance and differentiation

Abstract

The multipotent stem cell of trophectoderm is the source of trophoblast lineage. Proper maintenance of the stemness state and differentiation into specialized cell types are crucial for the formation of the functional placenta. Jmjd2 family of proteins had been shown essential for supporting the undifferentiated state of stem cells and directing differentiation in various developmental contexts. However, their roles in trophoblast lineage remain unexplored. Here, my study shows that Jmjd2b and Jmjd2c are indispensable for sustaining the stemness of trophoblast stem cells (TS cells). The inhibition of pan-Jmjd2 proteins activity by JIB-04 and specific knockdown of Jmjd2b or Jmjd2c by siRNA approach resulted in differentiation of TS cells, which is reflected by the altered gene expression and acquisition of the differentiated giant cell morphology. Genome-wide examination of Jmjd2b occupancy by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed its enrichment at the genomic loci of cell fate regulators and genes associated with placental development. It was observed that Jmjd2b binds to the promoter of key factors, such as Cdx2, Eomes, Elf5, Tfap2c, Ascl2, Hand1, Gcm1, and Syna, in TS cells. These demonstrate the epigenetic regulator role of Jmjd2b in the regulation of trophoblast cell fate. In contrast to the reported H3K9me3 demethylase function of Jmjd2 proteins, the negative correlation between Jmjd2b and H3K9me3 was rarely noticed in the context of trophoblast. Strikingly, my study illustrated that H3K9me3 is rarely associated with the repression of trophoblast lineage genes at the genome-wide level, which argues the previously reported transcriptional silencing function of H3K9me3 based on a limited number of stemness and developmental genes in trophoblast lineage. Importantly, comparison of the ChIP-seq profile between Jmjd2b, TS cell factors, and H3K36me3 revealed the potential molecular mechanisms that underline Jmjd2b-mediated trophoblast stemness. Immunoprecipitation assay and ChIP-seq analysis proposed that Tfap2c and Lsd1 proteins are the co-factors of Jmjd2b in driving TS cell stemness. Furthermore, it was found that the promoter and putative enhancer of critical cell fate regulators (e.g. Cdx2 and Eomes) are bound by Jmjd2b-Tfap2c-Lsd1 and Tfap2c-Lsd1, respectively, which suggests that these proteins mediate enhancer-promoter interaction for active gene expression. In addition, it was observed that depletion of Jmjd2b resulted in the accumulation of gene body H3K36me3 and induction of transcriptionally silent genes, implying that Jmjd2b prevents inappropriate gene expression by H3K36me3 removal. In summary, my study identified Jmjd2b and Jmjd2c as the novel regulators of trophoblast stemness. Jmjd2b is also potentially regulating trophoblast differentiation. The comprehensive prediction of the molecular mechanisms provides insight into the epigenetic regulations that coordinate trophoblast cell fate, which furthers our understanding of placental development.