Biology subjects

Kerry, J.

Publications and source records attributed to Kerry, J..

6 recordsLinked to original sources

bioRxiv2023

Autophagy-dependent alternative splicing event produces a more stable ribosomal protein S24 isoform that aids in hypoxic cell survival

Overlapping or convergent stress-activated molecular pathways exist to coordinate cell fate in response to stimuli such as hypoxia, oxidative stress, DNA damage, and unfolded proteins. Cells can remodel the splicing and translation machineries to mount a specialized gene expression response to certain stresses. Here, we show that hypoxic human cells in 2D and 3D culture models increase the relative abundance by 1.7- to 2.6-fold and 4.7- to 11.5-fold, respectively, of a longer mRNA variant of ribosomal protein S24 (RPS24L) compared to a shorter mRNA variant (RPS24S) by favoring the inclusion of a 22 bp cassette exon. Mechanistically, RPS24L and RPS24S are induced and repressed, respectively, by distinct parallel pathways in hypoxia: RPS24L is induced in an autophagy-dependent manner, while RPS24S is reduced by mTORC1 repression and in a HIF-dependent manner. RPS24L is a more stable mRNA in hypoxia and produces a more stable protein isoform compared to RPS24S. Cells overexpressing RPS24L display improved survival and growth in hypoxia relative to control cells and cells overexpressing RPS24S, which display impaired survival. Previous work from our group showed a correlation between RPS24L levels and tumor hypoxia in prostate cancer. These data highlight RPS24L as a stress-induced alternative splicing event that favors hypoxic cell survival, which could be exploited by cancer cells in the tumor microenvironment.

molecular biology↗

bioRxiv2020

Targeted high-resolution chromosome conformation capture at genome-wide scale.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at up to several hundred loci. All 3C methods are affected to varying degrees by inefficiency, bias and noise. As such, generation of reproducible high-resolution interaction profiles has not been achieved at scale. To overcome this barrier, we systematically tested and improved upon current methods. We show that isolation of 3C libraries from intact nuclei, as well as shortening and titration of enrichment oligonucleotides used in high-resolution methods reduces noise and increases on-target sequencing. We combined these technical modifications into a new method Nuclear-Titrated (NuTi) Capture-C, which provides a >3-fold increase in informative sequencing content over current Capture-C protocols. Using NuTi Capture-C we target 8,061 promoters in triplicate, demonstrating that this method generates reproducible high-resolution genome-wide 3C interaction profiles at scale.

genomics↗

bioRxiv2020

CaptureCompendium: a comprehensive toolkit for 3C analysis.

DNA folding within nuclei is a highly ordered process, with implications for gene regulation and development. An array of chromosome conformation capture (3C) methods have been developed to investigate how DNA is packaged within nuclei and to interrogate specific interactions. While these methods use different approaches to examine target loci (many-versus-all) or the entire genome (all-versus-all), they all rely on the core principle of endonuclease digestion and proximity-based ligation to re-arrange genomic order to reflect the three-dimensional nuclear conformation. This sequence reorganization creates novel chimeric DNA fragments which require specialist bioinformatic tools to analyze and visualize. Despite this need for specialist bioinformatic skills, the core biological importance of genome folding has seen widespread methodological uptake. To service the needs of experimentalists using the many-versus-all Capture-C family of methods we have developed CaptureCompendium; a toolkit of software to simplify the design, analysis and presentation of 3C experiments.

bioinformatics↗

bioRxiv2019

BET inhibition disrupts transcription but retains enhancer-promoter contact

In higher eukaryotes, enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. The formation of phase condensates is thought to be an essential component of enhancer function. Here, we show that pharmacological targeting of cells with inhibitors of BET (Bromodomain and Extra-Terminal domain) proteins can have a strong impact on transcription but very little impact on enhancer-promoter interactions. Treatment with 1,6-hexanediol, which dissolves phase condensate structures and reduces BET and Mediator protein binding at enhancers, can also have a strong effect on gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further suggest that enhancer-promoter interactions are not dependent on high levels of BRD4 (Bromodomain-containing protein 4) and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and loop extrusion by CTCF/cohesin.

molecular biology↗

bioRxiv2019

An integrated platform to systematically identify causal variants and genes for polygenic human traits.

Genome-wide association studies (GWAS) have identified over 150,000 links between common genetic variants and human traits or complex diseases. Over 80% of these associations map to polymorphisms in non-coding DNA. Therefore, the challenge is to identify disease-causing variants, the genes they affect, and the cells in which these effects occur. We have developed a platform using ATAC-seq, DNaseI footprints, NG Capture-C and machine learning to address this challenge. Applying this approach to red blood cell traits identifies a significant proportion of known causative variants and their effector genes, which we show can be validated by direct in vivo modelling.

genetics↗

bioRxiv2019

Dissection of the 4D chromatin structure of the α-globin locus through in vivo erythroid differentiation with extreme spatial and temporal resolution

Mammalian gene expression patterns are controlled by regulatory elements, which interact within Topologically Associating Domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. We developed Tiled-C, a low-input Chromosome Conformation Capture (3C) approach, to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of matched chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. In contrast to previous reports, our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development.

genomics↗