LEDGF and HDGF2, histone H3K36 methyl-binding proteins that assist RNA polymerase II transcription through nucleosomes

FACT (facilitates chromatin transcription) is a protein complex that allows RNAPII to overcome the nucleosome-induced barrier to transcription. While abundant in stem cells and many cancer cells, FACT it is not very abundant in most tissues. Therefore, we screened for additional factors that might replace FACT in such tissues. Here we report the identification of two such proteins, LEDGF and HDGF2, each containing two HMGA-like AT-hooks and a PWWP methyl-lysine reading domain known to bind to H3K36me2 and H3K36me3. LEDGF and HDGF2 localize with H3K36me2/3 at genomic regions containing active genes, usually adjacent to H3K27me3 domains. In myoblasts, where FACT expression is low, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, HDGF2 is recruited to the majority of myotube up-regulated genes and is required for their expression. We propose that LEDGF and HDGF2 substitute for FACT in differentiated tissues and that their distribution on the genome helps maintain transcriptional programs unique to particular cell types. Overall design: Characterization of two new proteins having FACT-like activity ChIP-seq and RNA-Seq for studying the role of LEDGF and HDGF2 in 293TRex and two differentiation systems PRO-Seq of WT, HDGF2KO, and 'HDGF2KO rescued by WT' myoblast cells

FACT（facilitates chromatin transcription，染色质转录辅助因子）是一种蛋白质复合物，可使RNA聚合酶II（RNA polymerase II，RNAPII）克服核小体对转录造成的阻碍。尽管FACT在干细胞与多种癌细胞中含量丰富，但在大多数组织中其表达水平较低。为此，我们筛选了可在这类组织中替代FACT的其他因子。本研究成功鉴定出两种此类蛋白质——LEDGF与HDGF2，二者均含有两个类高迁移率族蛋白A（HMGA）的AT钩结构域，以及一个可结合H3K36me2与H3K36me3的PWWP甲基赖氨酸识别结构域。LEDGF与HDGF2可与H3K36me2/3共同定位在携带活跃基因的基因组区域，且通常紧邻H3K27me3结构域。在FACT表达水平较低的成肌细胞中，LEDGF与HDGF2富集于大多数活跃基因区域。当细胞分化为肌管细胞后，HDGF2会被招募至多数肌管细胞的上调基因区域，且对这些基因的表达至关重要。我们提出，LEDGF与HDGF2可在分化组织中替代FACT，且它们在基因组上的分布模式有助于维持特定细胞类型独有的转录程序。实验整体设计：对两种具备FACT类似活性的新型蛋白质进行功能表征；通过染色质免疫沉淀测序（ChIP-seq）与RNA测序（RNA-Seq）研究LEDGF与HDGF2在293TRex细胞及两种分化系统中的功能；对野生型（WT）、HDGF2基因敲除型（HDGF2KO）以及"HDGF2KO rescued by WT"成肌细胞开展PRO测序（PRO-Seq）实验。