Diverse Heterochromatin-Associated Proteins Repress Distinct Classes of Genes and Repetitive Elements (RNA-seq)

Heterochromatin, typically marked by H3K9me3 or H3K27me3, is key to the repression of genes and repetitive elements. The mechanisms responsible for establishing and maintaining heterochromatin in a locus specific manner are unclear. Heterochromatin restricts cell identity by preventing activation of alternative lineage genes. This barrier can be partially overcome by inducing ectopic transcription factors (TFs) to activate an alternative transcriptional program. However, TFs are impeded by highly restrictive heterochromatin leading to low reprogramming rates and incomplete activation of the target transcriptome. Previously we utilized the heterochromatin property of sonication resistance to identify 172 proteins associated with sonication resistant heterochromatin (srHC) by mass spectrometry (Becker, et al., 2017). We hypothesized that by functionally assessing the srHC proteins and identifying the genes they repress during reprogramming we could improve our understanding of heterochromatin maintenance and learn how to disrupt heterochromatin to enhance reprogramming. Focusing on reprogramming from fibroblasts to induced human hepatocytes (hiHeps) we conducted an siRNA screen of 97 srHC proteins without reprogramming factors (noTF) and with hiHep reprogramming and assessed the impact on the expression of genes located in srHC domains by RNA-seq. We identified enhancer of rudimentary homolog (ERH) as a key regulator of H3K9me3 heterochromatin maintenance. ERH was found to associate broadly with H3K9me3 domains and when depleted caused global changes in H3K9me3 and srHC. Collectively our screen of srHC protein repressive function demonstrates specific functionality of groups of srHC proteins and increases our understanding of H3K9me3 gene repression. Overall design: This study was designed to assess the role of 97 sonication resistant heterochromatin associated proteins in impeding the activation of sonication resistant heterochromatin embedded genes. RNA seq was performed on Human BJ Fibroblasts that were treated with two rounds of siRNAs. This was assessed both in cells expressing the hepatic transcription factors (FOXA3/HNF1A/HNF4A) for 7 days ("hepatic TF" protocol) , and cells expressing no ectopic transcription factors ("noTF" protocol). There were a total of ___ samples analyzed, including controls with three independent non-targetting siRNAs, labelled as NT siRNAs. There were a total of ___ siRNAs used, with two biological replicates for each samples in each of the two experimental conditions.

异染色质（Heterochromatin）通常以H3K9me3或H3K27me3作为标志性修饰，在基因与重复序列元件的沉默调控中发挥核心作用。然而，以位点特异性方式建立并维持异染色质的分子机制仍有待阐明。异染色质通过阻断替代谱系基因的激活，限制细胞身份的转换。该抑制屏障可通过诱导异位转录因子（Transcription Factors, TFs）激活替代转录程序得以部分破除，但转录因子会受到高度致密异染色质的阻碍，导致重编程效率低下且靶标转录组的激活不完全。
此前本团队利用异染色质的抗超声破碎特性，通过质谱技术鉴定出172种与抗超声破碎异染色质（sonication resistant heterochromatin, srHC）相关的蛋白质（Becker等，2017）。我们提出假说：通过功能层面评估srHC蛋白质，并鉴定其在重编程过程中沉默的靶基因，可加深对异染色质维持机制的理解，并探索破坏异染色质以提升重编程效率的可行策略。
本研究聚焦于成纤维细胞向诱导型人肝细胞（induced human hepatocytes, hiHeps）的重编程过程，针对97种srHC蛋白质开展了小干扰RNA（small interfering RNA, siRNA）筛选实验，分别设置无重编程因子（noTF）以及添加hiHep重编程因子的两组条件，并通过RNA测序（RNA-seq）评估其对srHC结构域内基因表达的影响。最终我们鉴定出rudimentary同源增强子（enhancer of rudimentary homolog, ERH）作为H3K9me3型异染色质维持的关键调控因子。研究发现ERH可广泛结合H3K9me3结构域，当其被敲低时会引发H3K9me3修饰与srHC结构的全局改变。综上，本团队针对srHC蛋白质沉默功能的筛选实验，揭示了srHC蛋白质群组的特异性功能，并加深了对H3K9me3介导基因沉默的理解。
总体实验设计：本研究旨在评估97种srHC相关蛋白质在阻碍抗超声破碎异染色质嵌入基因激活过程中的作用。实验以人BJ成纤维细胞为材料，使用两轮siRNA进行处理。分别对表达肝脏转录因子（FOXA3/HNF1A/HNF4A）并培养7天的细胞（"hepatic TF"方案），以及未表达异位转录因子的细胞（"noTF"方案）进行RNA测序分析。本研究共分析了___个样本，其中包括3组独立的非靶向siRNA对照（标记为NT siRNAs）。本研究共使用___种siRNA，两种实验条件下的每个样本均设置2次生物学重复。