Biology subjects

Higgs, D.

Publications and source records attributed to Higgs, D..

5 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

Ancient genomic linkage couples metabolism with erythroid development

Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the -globin genes for >500 million years4, and harbours the majority of the -globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the -globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, -globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.

cell biology↗

bioRxiv2023

Loop extrusion by cohesin plays a key role in enhancer-activated gene expression during differentiation

Enhancers and their target promoters often come into close physical proximity when activated. This proximity may be explained by a variety of mechanisms; most recently via cohesin-mediated chromatin loop extrusion. Despite this compelling hypothesis, acute depletion of cohesin does not cause widespread changes in gene expression. We have tested the role of cohesin-mediated loop extrusion on gene expression at the mouse alpha-globin locus during erythropoiesis. Acute depletion of cohesin downregulates alpha-globin expression at early but not late stages of differentiation. When single or multiple CTCF sites are placed between the alpha-globin enhancers and promoters, alpha-gene expression is downregulated. Importantly, the orientation of the CTCF site plays a critical role, suggesting that within this activated domain, cohesin predominantly but not exclusively translocates from the enhancers to the promoters. We find that loop extrusion does play an important role in establishing enhancer-promoter proximity and consequent expression of inducible genes during differentiation.

molecular biology↗

bioRxiv2021

On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse -globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of -globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as -globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

molecular biology↗

bioRxiv2019

ATR16 Syndrome: Mechanisms Linking Monosomy to Phenotype

BackgroundSporadic deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individuals clinical phenotype is challenging.\n\nMethodsHere, as an example of this common phenomenon, we analysed 34 patients with simple deletions of [~]177 to [~]2000 kb affecting one allele of the well characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised precise deletion extent and screened for genetic background effects, telomere position effect and compensatory up regulation of hemizygous genes.\n\nResultsWe find the risk of developmental and neurological abnormalities arises from much smaller terminal chromosome 16 deletions ([~]400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes, however, genetic background effects substantially modify phenotypic abnormalities.\n\nConclusionsUsing ATR-16 as a general model of disorders caused by sporadic copy number variations, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but also depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling.

genetics↗