d RNA-FISH analysis of nascent transcription from the α-globin and β-globin genes at MFL 0 h and 30 h. c Nascent Hba transcription relative to 18s in the cell types and MFL timepoints indicated, from 3 biological replicates. b Representative cytospins of the MFL 0 h and 30 h cultures. Bottom images are representative cell pellets at the two timepoints demonstrating the presence of haemoglobin at MFL 30 h. a Representative FACS plots of MFL erythroblasts defined by CD44/cell size and Ter119, at 0 h and after a further 30 h differentiation in vitro, identifying distinct populations at the two timepoints. Yet the largely convergent boundary elements are occupied by CTCF and cohesin in both cell types 17, suggesting that CTCF/cohesin are not the primary mediators of this tissue-specific domain formation.ĭescription of the mouse foetal liver-derived erythroblast populations and of the active murine α-globin locus. Further, we detect no evidence of a strong self-interacting domain in mES cells, whereas such a structure is clearly present in differentiating erythroblasts 16.
In mES cells the α-globin promoters and five enhancers are not bound by transcription factors and the genes are silent 15. This locus is contained within a 70 kb self-interacting domain in which we have previously identified all cis-acting elements, including promoters, enhancers and CTCF/cohesin-binding sites (Fig. To establish chromatin architecture at sequential stages of differentiation and in the absence of key elements, we have utilised the well-characterised murine α-globin regulatory locus. What remains unclear is the three-dimensional form of the domain that must arise to support optimal enhancer–promoter interactions and what elements are important to mediate this structure. Hence a sub-TAD or loop domain can define the region of influence of an enhancer, which represents a fundamental mechanism for specificity and selectivity in gene regulation. TADs and sub-TADs can be anchored by CTCF/cohesin 5, and such boundary sites have been shown as necessary to prevent abnormal enhancer–promoter contacts 11, 12, 13. Chromatin loops are thought to occur within such domains, bringing gene promoters into proximity with their cognate cis-acting regulatory elements within the same domain 6, 7, 8, 9, 10.
Within that framework there are sub-TAD structures or domains that appear to undergo reorganisation between cellular states 3, 4, 5. Proximity ligation and deep sequencing techniques have identified megabase-scale topologically associating domains (TADs) throughout the genome 1, 2. The biological activity of our genomes is not determined by the linear DNA sequence alone a major current goal in biology is to characterise the three-dimensional (3D) architecture of chromatin in the interphase nucleus, to establish how it changes during development and differentiation and to elucidate the functional implications of that organisation. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within. However, absence of the major enhancers does alter internal domain interactions.
Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. Self-interacting chromatin domains encompass genes and their cis-regulatory elements however, the three-dimensional form a domain takes, whether this relies on enhancer–promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear.
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