Biology subjects

Kassouf, M. T.

Publications and source records attributed to Kassouf, M. T..

4 recordsLinked to original sources

bioRxiv2023

The characteristics of CTCF binding sequences contribute to enhancer blocking activity

While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterised. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the amount of CTCF or cohesin bound at the natural genomic sites of these elements; the degree of evolutionary conservation; or their resemblance to the consensus core sequences. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify CTCF elements in a uniform and unbiased way.

molecular biology↗

bioRxiv2022

Multipartite super-enhancers function in an orientation-dependent manner

Transcriptional enhancers regulate gene expression in a developmental-stage and cell-specific manner. They were originally defined as individual regulatory elements that activate expression regardless of distance and orientation to their cognate genes. Genome-wide studies have shown that the mammalian enhancer landscape is much more complex, with different classes of individual enhancers and clusters of enhancer-like elements combining in additive, synergistic and redundant manners, possibly acting as single, integrated regulatory elements. These so-called super-enhancers are largely defined as clusters of enhancer-like elements which recruit particularly high levels of Mediator and often drive high levels of expression of key lineage-specific genes. Here, we analysed 78 erythroid-specific super-enhancers and showed that, as units, they preferentially interact in a directional manner, to drive expression of their cognate genes. Using the well characterised -globin super-enhancer, we show that inverting this entire structure severely downregulates -globin expression and activates flanking genes 5 of the super-enhancer. Our detailed genetic dissection of the -globin locus clearly attributes the clusters functional directionality to its sequence orientation, demonstrating that, unlike regular enhancers, super-enhancers act in an orientation-dependent manner. Together, these findings identify a novel emergent property of super-enhancers and revise current models by which enhancers are thought to contact and activate their cognate genes.

molecular biology↗

bioRxiv2020

A functional overlap between actively transcribed genes and chromatin boundary elements

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 -like globin genes and their five enhancers (5-R1-R2-R3-Rm-R4-1-2-3). 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 -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 2-globin gene rather than the CTCF sites at the 3-end of the sub-TAD. Of interest, the almost identical 1- and 2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal 1-globin gene which behaves as a partial boundary between the enhancers and the distal 2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements. Significance StatementMammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers/promoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.

genomics↗

bioRxiv2017

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions

A variety of self-interacting domains, defined at different levels of resolution, have been described in mammalian genomes. These include Chromatin Compartments (A and B)1, Topologically Associated Domains (TADs)2,3, contact domains4,5, sub-TADs6, insulated neighbourhoods7 and frequently interacting regions (FIREs)8. Whereas many studies have found the organisation of self-interacting domains to be conserved across cell types389, some do form in a lineage-specific manner6710. However, it is not clear to what degree such tissue-specific structures result from processes related to gene activity such as enhancer-promoter interactions or whether they form earlier during lineage commitment and are therefore likely to be prerequisite for promoting gene expression. To examine these models of genome organisation in detail, we used a combination of high-resolution chromosome conformation capture, a newly-developed form of quantitative fluorescence in-situ hybridisation and super-resolution imaging to study a 70 kb self-interacting domain containing the mouse -globin locus. To understand how this self-interacting domain is established, we studied the region when the genes are inactive and during erythroid differentiation when the genes are progressively switched on. In contrast to many current models of long-range gene regulation, we show that an erythroid-specific, decompacted self-interacting domain, delimited by convergent CTCF/cohesin binding sites, forms prior to the onset of robust gene expression. Using previously established mouse models we show that formation of the self-interacting domain does not rely on interactions between the -globin genes and their enhancers. As there are also no tissue-specific changes in CTCF binding, then formation of the domain may simply depend on the presence of activated lineage-specific cis-elements driving a transcription-independent mechanism for opening chromatin throughout the 70 kb region to create a permissive environment for gene expression. These findings are consistent with a model of loop-extrusion in which all segments of chromatin, within a region delimited by CTCF boundary elements, can contact each other. Our findings suggest that activation of tissue-specific element(s)within such a self-interacting region is sufficient to influence all chromatin within the domain.

cell biology↗