Polycomb group (PcG) proteins are epigenetic transcriptional repressors that orchestrate numerous developmental processes and have been implicated in the maintenance of embryonic stem (ES) cell state. More recent evidence suggests that a subset of PcG proteins engages in transcriptional activation in some cellular contexts, but how this property is exerted remains largely unknown. Here, we generated ES cells with single or combined disruption of polycomb group RING finger protein 3 (Pcgf3) and Pcgf5 with the CRISPR-Cas9 technique. We report that although these mutant cells maintained their self-renewal and colony-forming capacity, they displayed severe defects in mesoderm differentiation and Using RNA-seq to analyze transcriptional profiles of ES cells with single or combined Pcgf3/5 deficiencies, we found that in contrast to the canonical role of the related polycomb repressive complex 1 (PRC1) in gene repression, Pcgf3/5 mainly function as transcriptional activators driving expression of many genes involved in mesoderm differentiation. Proteomic approaches and promoter occupancy analyses helped to establish an extended Pcgf3/5 interactome and identified several novel Pcgf3/5 interactors. These included testis-expressed 10 (Tex10), which may directly contribute to transcriptional activation via the transcriptional co-activator p300. Furthermore, Pcgf3/5 deletion in ES cells substantially reduced the occupancy of Tex10 and p300 at target genes. Finally, we demonstrated that Pcgf3/5 are essential for regulating global levels of the histone modifier H2AK119ub1 in ES cells. Our findings establish Pcgf3/5 as transcriptional activators that interact with Tex10 and p300 in ES cells and point to redundant activity of Pcgf3/5 in pluripotency maintenance.

The polycomb group (PcG) proteins are key epigenetic regulators in stem cell maintenance. PcG proteins have been thought to act through one of two polycomb repressive complexes (PRCs), but more recent biochemical analyses have challenged this model in the identification of noncanonical PRC1 (nc-PRC1) complexes characterized by the presence of Rybp or Yaf2 in place of the canonical Chromobox proteins. However, the biological significance of these nc-PRC1s and the potential mechanisms by which they mediate gene repression are largely unknown. Here, we explore the functional consequences of Yaf2 disruption on stem cell regulation. We show that deletion of Yaf2 results in compromised proliferation and abnormal differentiation of mouse embryonic stem cells (mESCs). Genome-wide profiling indicates Yaf2 functions primarily as a transcriptional repressor, particularly impacting genes associated with ectoderm cell fate in a manner distinct from Rybp. We confirm that Yaf2 assembles into a noncanonical PRC complex, with deletion analysis identifying the region encompassing amino acid residues 102-150 as required for this assembly. Furthermore, we identified serine 166 as a Yaf2 phosphorylation site, and we demonstrate that mutation of this site to alanine (S166A) compromises Ring1B-mediated H2A monoubiquitination and in turn its ability to repress target gene expression. We therefore propose that Yaf2 and its phosphorylation status serve as dual regulators to maintain the pluripotent state in mESCs.

Zearalenone (ZEA) and imprinted long noncoding RNAs (lncRNAs) are both closely related to reproduction and development. However, whether they have connections in regulating reproduction and development is not clear yet. The aim of this research is to investigate their relationship. lncRNA microarray was performed to analyze differentially expressed genes, and real-time quantitative polymerase chain reaction (PCR) was used to verify the accuracy of microarray analysis. Meanwhile, the technologies of rapid amplification of cDNA ends, RNA fluorescence in situ hybridization and bioinformatics were adopted to characterize the selected lncRNA. Analysis of lncRNA microarray showed lncRNAs and messenger RNAs related to reproduction and development were significantly differently expressed, and Dio3os was probably the target lncRNA. Then, the experiment of real-time quantitative PCR verified the accuracy of microarray data. Characterization of Dio3os showed Dio3os, an antisense lncRNA with 2312 bp and 15 open reading frames, was enriched in the cytoplasm. Our findings suggest ZEA probably exerts toxic effects on reproduction and development by mediating Dio3os. Highlights 1. Lots of long noncoding RNAs (lncRNAs) are also important in reproduction and development, and Dio3os is one of them. 2. Zearalenone probably exerts toxic effects on reproduction and development by mediating Dio3os at the genetic level. 3. Dio3os may be a lncRNA related to reproduction and development, and it probably works by regulating Dio3. K E Y W O R D S Dio3os, fluorescence in situ hybridization, lncRNA microarray, rapid amplification of cDNA ends, zearalenone (ZEA)