A screen to identify regulators of FIRRE.

Xist activation during transformation in humans resulting from and associated with tumor suppressor loss and mutations is complex and frequently manifests as complete silencing rather than robust activation, indicating the presence of a gene or genes that function in concert with Xist as an imprinting switch, with a small number of genes controlling the expression of large number of targets in seemingly contrary fashion:a "mirror" effect. Xist non-coding RNA regulation and activation is controlled at least in part through a separate non-coding RNA encoded at a locus known as Dxz4, a ncRNA termed FIRRE. A basic and essential relationship exists between Xist activation and repression, control of Xist mobilization by FIRRE, and mirror effects generated at discrete genomic loci during imprinting-based epigenetic gene regulation. 

Xist-associated imprinting during embryonic development, like during methylation-associated imprinting erasure during germline development, is described as closely linked to DNA methylation, performed by the DNA methyltransferases DNMT1, DNMT3A and DNMT3B. Here we measured gene expression in cells partially or completely deficient in DNA methyltransferase function (also known as DNMT triple knockout cells) using whole transcriptome technologies to identify nuclear factors and other molecules functioning together with Xist during imprinting system activation and the DNA methylation machinery to modulate autosomal gene regulation by Xist, factors that will assist in deconvolution or reconciling contrary regulation modes by Xist at common targets. 

This expression screen identified PHF21A (also known as BHC80), a PHD-finger containing molecule we recently described as a component of the Xist RNP complex in transformation and which functions as a specific regulator (inhibitor) of LSD1 (also known as BHC110) demethylase function mediated by LSD1-interactions with CoREST, KMT2E, SLX1B and EME2, structure specific endonucleases that function in Holliday junction resolution and crosslink repair, as well as a second zinc finger nuclear factor, ZIC1 as candidate molecules with potential Xist-associated antagonistic-type regulatory properties. Two factors likely functioned in germ cell reprogramming in males: KMT2E (MLL5) a histone methyltransferase family gene important for development of spermatagonia, DZIP1, a factor expressed in and important for stem cells of the male germline. We identified a collection of factors that function in concert with Xist and the Dxz4-encoded ncRNA FIRRE in DNA methylation and imprinting-based epigenetic reprogramming that features a ‘mirror’ or recurrent transcriptional contrast.