Characterisation of the mouse alpha-globin sub-TAD 3' boundary in erythroid cells [ChIP-seq]

Mammalian genomes are subdivided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an individual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a ~70 kb sub-TAD containing the duplicated mouse a-like globin genes and their five enhancers (5'-R1-R2-R3-Rm-R4-a1-a2-3'). The 5' border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the a-globin locus leaves the sub-TAD largely intact. The predominant 3' border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed a2-globin gene rather than the CTCF sites at the 3'-end of the sub-TAD. Of interest, the almost identical a1- and a2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal a1-globin gene which behaves as a partial boundary between the enhancers and the distal a2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements. Overall design: CTCF ChIP-seq on chromatin isolated from primary erythroid Ter119+ cells obtained from the spleens of phenylhydrazine treated mice containing deletions for HS+44/+48 (?44-48), or ?1 and/or ?2 (??1, ??2, ??1?2) CTCF binding sites. Experiments performed on biological triplicates.