Biology subjects

Bickmore, W.

Publications and source records attributed to Bickmore, W..

5 recordsLinked to original sources

bioRxiv2022

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional condensates, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harboring distinct ecDNA species (EGFR, MYC, PDGFR). We found no evidence that ecDNA cluster with one another or closely interact with transcriptional condensates. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy was similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with the cellular transcriptional machinery.

genetics↗

bioRxiv2020

Q-STARZ: Quantitative Spatial and Temporal Assessment of Regulatory element activity in Zebrafish

Noncoding regions of the genome harbouring cis-regulatory elements (CREs) or enhancers drive spatial and temporal gene expression. Mutations or single nucleotide polymorphisms (SNPs) in enhancers have been widely implicated in human diseases and disease-predispositions. However, our ability to assay the regulatory potential of genetic variants in enhancers is currently very limited, in part because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type (Wt) and disease-associated, mutant (Mut) human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. Using this single transgenic cassette, the functional activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing the simultaneous visualisation of the activity of both alleles. This can reveal where and when in embryonic development the wild-type allele is active and how this activity is altered by the disease-associated mutation.

genetics↗

bioRxiv2019

Developmentally regulated Shh expression is robust to TAD perturbations

Topologically Associating Domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD - creating internal deletions, deleting CTCF sites including those at TAD boundaries, as well as larger deletions and inversions of TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used the investigate changes in chromatin conformation that result from these manipulations. Our data suggest that the substantial alteration of TAD structure has no readily detectable effect on Shh expression patterns during development - except where enhancers are deleted - and results in no detectable phenotypes. Only in the case of a larger deletion of one TAD boundary could some ectopic influence of the Shh limb enhancer be detected on a gene - Mnx1 in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.

developmental biology↗

bioRxiv2018

Nuclear pore density controls heterochromatin reorganization during senescence

Oncogene induced senescence (OIS) is a cell cycle arrest program triggered by oncogenic signalling. An important characteristic of OIS is activation of the senescence associated secretory phenotype (SASP)1 which can reinforce cell cycle arrest, lead to paracrine senescence but also promote tumour progression2-4. Concomitant with cell cycle arrest and the SASP activation, OIS cells undergo a striking nuclear chromatin reorganization, with loss of heterochromatin from the nuclear periphery and the appearance of internal senescence-associated heterochromatin foci (SAHF)5. The mechanisms by which SAHF are formed, and their role in cell cycle arrest and expression of the SASP, remain poorly understood. Here we show that nuclear pore density increases during OIS and is responsible for SAHF formation. In particular, we show that the nucleoporin TPR is required for both SAHF formation and maintenance. The TPR-induced loss of SAHF does not affect cell cycle arrest but completely abrogates the SASP. Our results uncover a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei and in senescence, which uncouples the cell cycle arrest from the SASP.

cell biology↗

bioRxiv2017

PARP mediated chromatin unfolding is coupled to long-range enhancer activation

Enhancers are critical regulators of gene expression and can be located far from their target gene. It is widely assumed that mechanisms of enhancer action involve reorganization of three-dimensional chromatin architecture, but this is poorly understood. Here we identify a novel mechanism of long-range enhancer associated chromatin reorganization. At the Sonic hedgehog (Shh) locus we observe large-scale decompaction of chromatin between Shh and its brain enhancers in neural progenitor cells. We show that the chromatin unfolding is dependent on activation of the enhancers, not the promoter, is impeded by chromatin-bound proteins located between the enhancer and promoter, and is mediated by the recruitment of Poly (ADP-Ribose) Polymerase 1. We suggest that large-scale chromatin decompaction, analogous to the inducible puffs in Drosophila polytene chromosomes, represents a new mechanism of chromatin reorganization coupled to long-range gene activation from mammalian enhancers and that seems incompatible with a chromatin-looping model of enhancer-promoter communication

genomics↗