Biology subjects

Francis, H. S.

Publications and source records attributed to Francis, H. S..

4 recordsLinked to original sources

bioRxiv2023

Active regulatory elements recruit cohesin to establish cell-specific chromatin domains.

As the structure of the genome is analysed at ever increasing resolution it is becoming clear that there is considerable variation in the 3D chromatin architecture across different cell types. It has been proposed that this may, in part, be due to increased recruitment of cohesin to activated cis-elements (enhancers and promoters) leading to cell-type specific loop extrusion underlying the formation of new subTADs. Here we show that cohesin correlates well with the presence of active enhancers and this varies in an allele-specific manner with the presence or absence of polymorphic enhancers which vary from one individual to another. Using the alpha globin cluster as a model, we show that when all enhancers are removed, peaks of cohesin disappear from these regions and the erythroid specific subTAD is no longer formed. Re-insertion of the major alpha globin enhancer (R2) is associated with the appearance of a new peak of cohesin at the site of insertion. In complementary experiments insertion of R2 into a "neutral" region of the genome recruits cohesin, induces transcription and creates a new large (75kb) erythroid specific domain. Together these findings support the proposal that active enhancers recruit cohesin, stimulate loop extrusion and promote the formation of cell specific subTADs.

genomics↗

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↗

bioRxiv2022

Systematic analysis of YFP gene traps reveals common discordance between mRNA and protein across the nervous system

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation. Brief outlineA novel high resolution and sensitive approach to systematically co-visualise the distribution of mRNAs and proteins in the intact nervous system reveals that post-transcriptional regulation of gene expression is very common. The rich data landscape is provided as a browsable resource (link), using Zegami, a cloud-based data exploration platform (link). Our solution provides a paradigm for the characterisation of post-transcriptional regulation of most genes and model systems. HighlightsO_LI196/200 (98%) Drosophila genes show discordant RNA and protein expression in at least one nervous system region C_LIO_LI137/200 (68.5%) mRNAs are present in at least one synaptic compartment C_LIO_LINovel localised mRNA and protein discovered in periphery of glial processes C_LIO_LINew paradigm for analysis of post-transcriptional regulation and data exploration C_LI

cell biology↗