H3K9 trimethylation in active compartments optimizes stimulus-regulated transcription by restricting usage of CTCF sites in SINE-B2 repeats [RNA-Seq]. H3K9 trimethylation in active compartments optimizes stimulus-regulated transcription by restricting usage of CTCF sites in SINE-B2 repeats [RNA-Seq]

Six methyltransferases divide labor in establishing genomic profiles of histone H3 lysine 9 methylation (H3K9me), an epigenomic modification involved in the formation of constitutive heterochromatin, gene repression and silencing of retroelements. Among them, SETDB1 is recruited to active chromatin compartments to silence the expression of endogenous retroviruses. In the context of experiments aimed at determining the role of SETDB1 in stimulus-inducible gene expression in macrophages, we unexpectedly found that upon SETDB1 depletion, loss of H3K9me3 in active compartments was associated with increased recruitment of CTCF to >1,600 DNA-binding motifs contained within SINE-B2 repeats, a previously unidentified target of SETDB1-mediated repression. CTCF is an essential regulator of chromatin folding that restrains DNA looping by cohesin, thus creating boundaries among adjacent topological domains. Increased CTCF binding to SINE-B2 repeats generated novel boundaries within topological domains containing lipopolysaccharide-inducible genes, correlating with their impaired regulation in response to stimulation. These data indicate a role of H3K9me3 in restraining genomic distribution and activity of CTCF, with impact on chromatin organization and gene regulation. Overall design: RNAseq of mouse bone marrow-derived macrophages (BMDM) were infected with purified lentiviruses expressing two non-targeting (NT) shRNAs or two distinct shRNAs specific for SETDB1.

六种甲基转移酶协同分工，共同构建组蛋白H3赖氨酸9甲基化（histone H3 lysine 9 methylation, H3K9me）的基因组图谱。该表观基因组修饰参与组成型异染色质形成、基因阻遏与逆转录元件的沉默调控。
其中，SETDB1会被招募至活跃染色质区域，以沉默内源性逆转录病毒的基因表达。
在旨在探究SETDB1在巨噬细胞刺激诱导型基因表达中作用的实验中，我们意外发现：当SETDB1被敲低后，活跃染色质区域内H3K9me3的缺失，与CTCF结合至SINE-B2重复序列中包含的1600余个DNA结合基序的招募量增加存在关联——而SINE-B2重复序列是此前未被鉴定的SETDB1介导沉默的靶点。
CTCF是染色质折叠的关键调控因子，可通过黏连蛋白（cohesin）抑制DNA环化，从而在相邻拓扑结构域之间形成边界。
CTCF与SINE-B2重复序列结合的增强，会在含有脂多糖诱导型基因的拓扑结构域内部生成新的边界，这与这些基因在受刺激时的调控功能受损存在相关性。
上述实验数据表明，H3K9me3可通过限制CTCF的基因组分布与活性，进而影响染色质组织与基因调控过程。
实验整体设计：对感染纯化慢病毒的小鼠骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDM）进行RNA测序（RNAseq），所用慢病毒分别携带两种非靶向（non-targeting, NT）短发夹RNA（short hairpin RNA, shRNA），或两种特异性靶向SETDB1的短发夹RNA。