Biology subjects

Rogerson, C.

Publications and source records attributed to Rogerson, C..

8 recordsLinked to original sources

bioRxiv2024

Oriented nucleosome binding by the pioneer factor ELF2

Pioneer transcription factors are DNA-binding proteins that can bind to nucleosomes in closed chromatin regions, exposing enhancers and promoters of genes for transcription. The action of these factors underpin stem cell pluripotency, cell reprogramming and differentiation. ELF2 is an ETS family pioneer factor that has a strong preference for oriented binding on nucleosomes at a composite head-to-tail dimeric sequence motif with a 2 bp spacing between the GGAA core elements. In this study, we investigated the interaction between ELF2 and a nucleosome using single-particle cryo-electron microscopy (cryo-EM). The ELF2-nucleosome structure shows two ELF2 proteins bound to the nucleosome cooperatively at superhelical location +4. The recognition [a]4 helices of both ELF2 monomers dock into the major groove at the core GGAA motifs and unwrap approximately four helical turns of the DNA from the surface of the nucleosome. The unwrapping almost completely exposes one histone H2A:H2B dimer, which dissociates in a subset of particles to form an ELF2-bound hexasome. ChIP-seq combined with genome-wide motif mapping indicates that all unmethylated high-affinity ELF2 double motifs are occupied, indicating that ELF2 is able to use its binding energy to function as a pioneer factor both in vitro and in vivo. ELF2 binding is highly enriched downstream of transcription start sites (TSS) of highly expressed genes, with the motifs oriented in such a way that the TSS becomes accessible upon ELF2 binding. This mechanism is essential for cells, as mutation of ELF2 motifs result in decreased cell proliferation. Taken together, our results indicate that ELF2 can use its binding energy to open chromatin in an oriented fashion, facilitating nucleosome positioning and transcription initiation at transcription start sites.

biochemistry↗

bioRxiv2022

FOXA2 controls the antioxidant response in FH-deficient cells independent of NRF2

Hereditary Leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The activation of the anti-oxidant response is key for cellular survival in FH-deficient cells, yet the extent to which chromatin remodelling shapes the anti-oxidant response is currently unknown. Here, we explored the global effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the highly remodelled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor which directly regulates anti-oxidant response genes and subsequent metabolic rewiring. Moreover, we also find that FOXA2 regulates anti-oxidant genes independent of the canonical anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides new insights into the molecular mechanisms behind cell responses to fumarate accumulation, and potentially provides new avenues for therapeutic intervention for HLRCC.

cancer biology↗

bioRxiv2022

HIRA loss transforms FH-deficient cells

Fumarate Hydratase (FH) is a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle. Germline mutations of FH lead to HLRCC, a cancer syndrome characterised by a highly aggressive form of renal cancer(1). Although HLRCC tumours metastasise rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas (2). How Fh1-deficient cells overcome these tumour suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR/Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of HIRA enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss enables the activation of MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

cancer biology↗

bioRxiv2020

Repurposing of KLF5 activates a cell cycle signature during the progression from Barrett's oesophagus to oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is one of the most common causes of cancer deaths and yet compared to other common cancers, we know relatively little about the underlying molecular mechanisms. Barretts oesophagus (BO) is the only known precancerous precursor to OAC, but our understanding about the specific events leading to OAC development is limited. Here, we have integrated gene expression and chromatin accessibility profiles of human biopsies of BO and OAC and identified a strong cell cycle gene expression signature in OAC compared to BO. Through analysing associated chromatin accessibility changes, we have implicated the transcription factor KLF5 in the transition from BO to OAC. Importantly, we show that KLF5 expression is unchanged during this transition, but instead, KLF5 is redistributed across chromatin in OAC cells to directly regulate cell cycle genes specifically in OAC. Our findings have potential prognostic significance as the survival of patients with high expression of KLF5 target genes is significantly lower. We have provided new insights into the gene expression networks in OAC and the mechanisms behind progression to OAC, chiefly the repurposing of KLF5 for novel regulatory activity in OAC.

cancer biology↗

bioRxiv2019

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 minutes precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. Single cell RNA sequencing of differentiating keratinocytes identified gene changes correlated with lamin dispersal, which we propose are due to changes in lamina-associated domains upon Lamin A/C dispersal. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.

cell biology↗

bioRxiv2018

Identification of a primitive intestinal transcription factor network shared between oesophageal adenocarcinoma and its pre-cancerous precursor state.

Oesophageal adenocarcinoma (OAC) is one of the most frequent causes of cancer deaths and yet compared to other common cancers, we know relatively little about the molecular composition of this tumour type. To further our understanding of this cancer we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in OAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centred on HNF4A and GATA6. These transcription factors work together to control the OAC transcriptome. Importantly, we show that this network is activated in Barretts oesophagus, the putative precursor state to OAC thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone, is sufficient to drive chromatin opening and activation of a Barretts-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorise OAC at a genome scale and implicate HNF4A activation as a potential pivotal event in regulating its malignant transition from healthy cells.

cancer biology↗

bioRxiv2017

Classifying cells with Scasat - a tool to analyse single-cell ATAC-seq

MotivationThe assay for transposase-accessible chromatin using sequencing (ATAC-seq) reveals the landscape and principles of DNA regulatory mechanisms by identifying the accessible genome of mammalian cells. When done at single-cell resolution, it provides an insight into the cell-to-cell variability that emerges from identical DNA sequences by identifying the variability in the genomic location of open chromatin sites in each of the cells. Processing of single-cell ATAC-seq requires a number of steps and a simple pipeline to processes and analyse single-cell ATAC-seq is not yet available.\n\nResultsThis paper presents ScAsAT (single-cell ATAC-seq analysis tool), a complete pipeline to process scATAC-seq data with simple steps. The pipeline is developed in a Jupyter notebook environment that holds the executable code along with the necessary description and results. For the initial sequence processing steps, the pipeline uses a number of well-known tools which it executes from a python environment for each of the fastq files. While functions for the data analysis part are mostly written in R, it is robust, flexible, interactive and easy to extend. The pipeline was applied to a single-cell ATAC-seq dataset in order to identify different cell-types from a complex cell mixture. The results from Scasat showed that open chromatin location corresponding to potential regulatory elements can account for cellular heterogeneity and can identify regulatory regions that separates cells from a complex population.\n\nAvailabilityThe jupyter notebook with the complete pipeline applied to the dataset published with this paper are publicly available on the Github (https://github.com/ManchesterBioinference/Scasat). An additional notebook is also provided for analysis of a publicly available dataset. The fastq files are submitted at ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-6116.\n\nContactsyed.murtuzabaker@manchester.ac.uk and magnus.rattray@manchester.ac.uk\n\nSupplementary informationSupplementary data are available at bioRxiv online.

bioinformatics↗

bioRxiv2017

Rap1, AF6 and Pak4 cooperate with Par3 to promote ZA assembly and remodeling in the fly photoreceptor

The epithelial Zonula adherens (ZA) is a main adhesion compartment that enables organogenesis by allowing epithelial cells to assemble into sheets. How ZA assembly is regulated during epithelial cell morphogenesis is not fully understood. We show that during ZA morphogenesis, the function of the small GTPase Rap1 and the F-actin binding protein AF6/Cno are both linked to that of the P21-activated kinase Pak4/Mbt. We find that Rap1 and Mbt regulate each others localization at the ZA and cooperate in promoting ECadherin stabilization. During this process Cno regulates the recruitment of Baz at the ZA, a process that is also regulated by Arm phosphorylation by Mbt. Altogether, we propose that Rap1, Cno and Mbt regulate ZA morphogenesis by coordinating ECadherin stabilization and Baz recruitment and retention. In addition, our work uncovers a new link between two main oncogenes, Rap1 and Pak4/Mbt, in a model developing epithelial cell.

cell biology↗