Functional analysis of CTCF sites associated with cytokine-sensing mammary-specific enhancers in the complex casein locus [ChIP-seq]

The zinc finger protein CTCF has been invoked in establishing functional boundaries between genes and thereby controlling enhancer activities. However, there is limited genetic evidence to support such a concept. We have now addressed this question in a locus containing five mammary-specific genes controlled by enhancers. We have identified four CTCF binding sites in the casein locus, two at the outside boundaries and two associated with a super-enhancer. Individual deletions of these sites from the mouse genome did not alter expression of the five casein genes and Odam. However, deletion of the border CTCF site separating the Csn1s1 enhancer from non-mammary genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Csn1s1 enhancer and the Sult1d1 promoter but not with the silent Sult1e1 gene. Our study suggests that most CTCF sites associated with cell-specific enhancers and super-enhancers have no measurable in vivo activity. Only the loss of one CTCF site led to the induction of a juxtaposed active non-target promoter. Our study also demonstrated that cell-specific enhancers are unable to activate juxtaposed silent non-target promoters. Overall design: ChIP-seq for mutant mice ?A were done for CTCF, GR, H3K27ac, Pol2, SMC1 and STAT5A in mammary tissue at lactation day 1 (L1). ChIP-seq for ?C was done for CTCF in mammary tissue at L1.锌指蛋白CTCF（zinc finger protein CTCF）此前被认为可介导基因间功能边界的建立，进而调控增强子活性。然而，支持这一假说的遗传学证据相对匮乏。我们针对一个受增强子调控的包含5个乳腺特异性基因的基因座开展了相关研究。我们在酪蛋白基因座中鉴定出4个CTCF结合位点：2个位于外侧边界区域，另外2个与超级增强子（super-enhancer）相关联。在小鼠基因组中单独缺失这些位点，并未改变5个酪蛋白基因与Odam的表达水平。然而，缺失分隔Csn1s1增强子与非乳腺基因的边界CTCF位点后，会在95 kb以外的区域激活Sult1d1的表达，但不会影响距离更近的Sult1e1基因。该CTCF位点的缺失会使Csn1s1增强子与Sult1d1启动子之间产生新生的染色质相互作用，但不会与处于沉默状态的Sult1e1基因发生此类相互作用。我们的研究表明，绝大多数与细胞特异性增强子及超级增强子相关的CTCF位点，在体内并未表现出可检测到的活性。仅当缺失某一个CTCF位点时，才会诱导相邻的活性非靶标启动子的表达。本研究还证实，细胞特异性增强子无法激活相邻的处于沉默状态的非靶标启动子。整体实验设计：于泌乳第1天（L1）的乳腺组织中，针对突变小鼠ΔA开展了针对CTCF、GR、H3K27ac、Pol2、SMC1及STAT5A的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）实验；针对突变小鼠ΔC的ChIP-seq实验则仅检测CTCF的结合情况，同样于泌乳第1天的乳腺组织中开展。