Pax6 associates with H3K4-specific histone methyltransferases Mll1, Mll2, and Set1a and regulates H3K4 methylation at promoters and enhancers

Background: Pax6 is a key regulator of the entire cascade of ocular lens formation through specific binding to promoters and enhancers of batteries of target genes. The promoters and enhancers communicate with each other through DNA looping mediated by multiple protein-DNA and protein-protein interactions and are marked by specific combinations of histone posttranslational modifications (PTMs). Enhancers are distinguished from bulk chromatin by specific modifications of core histone H3, including H3K4me1 and H3K27ac, while promoters show increased H3K4me3 PTM. Previous studies have shown the presence of Pax6 in as much as 1/8 of lens-specific enhancers but a much smaller fraction of tissue-specific promoters. Although Pax6 is known to interact with EP300/p300 histone acetyltransferase responsible for generation of H3K27ac, a potential link between Pax6 and histone H3K4 methylation remains to be established.

Results: Here we show that Pax6 co-purifies with H3K4 methyltransferase activity in lens cell nuclear extracts. Proteomic studies show that Pax6 immunoprecipitates with Set1a, Mll1, and Mll2 enzymes, and their associated proteins, i.e., Wdr5, Rbbp5, Ash2l, and Dpy30. ChIP-seq studies using chromatin prepared from mouse lens and cultured lens cells demonstrate that Pax6-bound regions are mostly enriched with H3K4me2 and H3K4me1 in enhancers and promoters, though H3K4me3 marks only Pax6-containing promoters. The shRNA-mediated knockdown of Pax6 revealed down-regulation of a set of direct target genes, including Cap2, Farp1, Pax6, Plekha1, Prox1, Tshz2, and Zfp536. Pax6 knockdown was accompanied by reduced H3K4me1 at enhancers and H3K4me3 at promoters, with little or no changes of the H3K4me2 modifications. These changes were prominent in Plekha1, a gene regulated by Pax6 in both lens and retinal pigmented epithelium.

Conclusions: Our study supports a general model of Pax6-mediated recruitment of histone methyltransferases Mll1 and Mll2 to lens chromatin, especially at distal enhancers. Genome-wide data in lens show that Pax6 binding correlates with H3K4me2, consistent with the idea that H3K4me2 PTMs correlate with the binding of transcription factors. Importantly, partial reduction of Pax6 induces prominent changes in local H3K4me1 and H3K4me3 modification. Together, these data open the field to mechanistic studies of Pax6, Mll1, Mll2, and H3K4me1/2/3 dynamics at distal enhancers and promoters of developmentally controlled genes.