A recent study published in Science and led by the groups of Dr. ZHU Bing and Dr. XU Ruiming from the Institute of Biophysics of the Chinese Academy of Sciences reported a reining function of BEND3 at its highly occupied bivalent genes in ES cells to prevent them from premature activation during differentiation.

In vertebrate genome, approximately 70% of gene promoters are associated with DNA elements called CpG islands (CGIs), which are characterized by high density of CpG dinucleotides and lack of DNA methylation. CGIs are not only associated with housekeeping genes, but also with bivalent genes. BANP, a DNA methylation-sensitive CGI binding protein, has been reported to bind CGCG element and activates CpG-island-regulated essential metabolic genes. It is intriguing to ask whether and how CGI-associated bivalent genes are regulated during development by sequence-specific transcription factors targeting CGIs.

A previous study of Dr. ZHU Bing's group identified BEND3 as a protein preferentially associating with unmethylated DNA in vitro, suggesting BEND3 may recognize non-methylated CGIs in vivo. However, little was known about the genomic distribution and physiological function of BEND3.

In this study, the researchers performed ChIP-seq experiments and found that BEND3 is enriched at regulatory elements associated with CGIs, including promoters and enhancers. They determined the binding motif of BEND3 and solved the cocrystal structure of the critical fourth BEN domain in complex with its target DNA.

To investigate the physiological roles of BEND3, they performed mouse experiments and found that mouse embryos depleted of Bend3 died around peri-gastrulation stage. Consistently, Bend3 KO ES cells exhibited severe defects in differentiation, both in teratoma and embryoid body (EB) formation assays.

To clarify the molecular mechanism underlying the developmental defect, the researchers performed RNA-seq experiments of EB differentiation process. Gene expression profiling revealed that hundreds of CGI-containing bivalent genes were activated prematurely in Bend3 null mutants. Furthermore, bivalent genes with high level of BEND3 binding and more BEND3 binding motifs showed the most significant transcriptional changes.

It was reported that BEND3 participates in the recruitment of PRC2 to major satellites in cells lacking DNA methylation. Therefore, the researchers performed ChIP-seq experiments for SUZ12 (a subunit of PRC2) and H3K27me3, and demonstrated that BEND3 is required for stable association of PRC2 complex and maintaining high level of H3K27me3 at some bivalent genes highly enriched with BEND3.

This study identified a novel CGI binding protein, BEND3, which is required for the optimal association of PRC2 at bivalent genes to prevent these genes from premature activation during differentiation. CGIs have long been considered as regulatory elements of transcription. This study offers interesting new insights into the critical roles of CGIs and CGI-binding proteins.

Gene bivalency is widely expected in priming for faster induction, largely due to the enrichment of H3K4me3. However, H3K4me3 removal by MLL2 deletion at bivalent genes didn't affect their induction kinetics. Bend3 deletion leads to reduced PRC2 and H3K27me3 levels at its highly enriched bivalent genes. This does not immediately activate the majority of these genes, but affects the kinetics of these bivalent gene induction. This study propose that gene bivalency can prevent premature gene activation during differentiation, which is an opposite effect of priming, and the researchers term it as reining.