Biology subjects

Garnham, A. L.

Publications and source records attributed to Garnham, A. L..

7 recordsLinked to original sources

bioRxiv2026

Regulation of Human Erythroferrone Expression

Erythroferrone (ERFE) is an erythroblast-secreted hormone that suppresses hepatic hepcidin expression to increase iron availability for erythropoiesis, ensuring recovery from anaemia. ERFE excess drives iron overload in disorders of ineffective erythropoiesis. Despite its pivotal role in systemic iron homeostasis and diseases of erythropoiesis, ERFEs molecular regulation has remained undefined. Here, we applied a genomic approach to characterise the molecular mechanisms governing ERFE expression. Using the HUDEP-2 human erythroid progenitor model, integrative ATAC-seq, CUT&RUN and micro capture-C analysis we identified a stage-specific accessible chromatin region within the ERFE 3 UTR that interacts with the promotor. We also identified enhancer-associated chromatin marks including H3K4me1 and H3K27ac in this region, and demonstrate that this cis-regulatory element is bound by key erythroid transcription factors KLF1, GATA1, TAL1 and STAT5. Functional dissection using CRISPR-Cas9-mediated deletion of the central 3 UTR enhancer element led to marked reduction in ERFE mRNA expression, and we show a corresponding reduction in nascent mRNA, confirming a key role for this region in transcriptional regulation. We define the transcriptional regulatory mechanism by which maturing human erythroblasts activate ERFE, the endocrine signal that coordinates erythropoietic demand with systemic iron mobilisation.

molecular biology↗

bioRxiv2022

Iron homeostasis governs erythroid phenotype in Polycythemia Vera

Polycythemia Vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients hematocrit and hemoglobin concentration, placing them at risk of life-threatening thrombotic events. Our GWAS of 440 PV cases and 403,351 controls utilising UK Biobank data found that SNPs in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed over-representation of homozygous HFE mutations in PV patients. HFE influences expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of PV mouse models, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Further, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signalling via GP130 coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

cancer biology↗

bioRxiv2021

The minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers

Minor splicing is a second splicing system required for the correct expression of [~]700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38. Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3. We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

cancer biology↗

bioRxiv2020

Suv39h-catalysed H3K9me3 is critical for euchromatic genome organisation and the maintenance of gene transcription

H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyse heterochromatic H3K9me3 deposition. Unexpectedly, we reveal a predominant repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (DNaseI-accessible, H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) regions. Examination of the 3D nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3-dimensional space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3-dimensional genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organisation and the maintenance of gene transcription for healthy cellular function.

genomics↗

bioRxiv2020

Activation-induced re-organization of chromatin in human T cells

Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in genome organization orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analysed chromatin accessibility, chromosome conformation and gene expression after activation of human T cells. T cell activation led to widespread changes in chromatin interactions and accessibility that were mostly shared between CD4+ and CD8+ T cells. Differential chromatin interactions were associated with upregulation or downregulation of linked target genes. Moreover, activation was associated with the formation of shorter chromatin interactions, partitioning of topologically associating domains (TADs) and acquisition of new TAD boundaries characterized by higher nucleosome occupancy, and lower chromatin accessibility and gene expression. These findings render an integrated and multiscale characterization of activation-induced re-organization of chromatin architecture underlying gene transcription in human T cells.

genetics↗

bioRxiv2018

Impaired social behaviour and molecular mediators of associated neural circuits during chronic Toxoplasma gondii infection in female mice

Toxoplasma gondii (T. gondii) is a neurotropic parasite that is associated with various neuropsychiatric disorders. Rodents infected with T. gondii display a plethora of behavioural alterations, and Toxoplasma infection in humans has been strongly associated with disorders such as schizophrenia, in which impaired social behaviour is an important feature. Elucidating changes at the cellular level relevant to neuropsychiatric conditions can lead to effective therapies. Here, we compare changes in behaviour during an acute and chronic T. gondii infection in female mice. Further, we notice that during chronic phase of infection, mice display impaired sociability when exposed to a novel conspecific. Also, we show that T. gondii infected mice display impaired short-term social recognition memory. However, object recognition memory remains intact. Using c-Fos as a marker of neuronal activity, we show that infection leads to an impairment in neuronal activation in the medial prefrontal cortex, hippocampus as well as the amygdala when mice are exposed to a social environment and a change in functional connectivity between these regions. We found changes in synaptic proteins that play a role in the process of neuronal activation such as synaptophysin, PSD-95 and changes in downstream substrates of cell activity such as cyclic AMP, phospho-CREB and BDNF. Our results point towards an imbalance in neuronal activity that can lead to a wider range of neuropsychiatric problems upon T. gondii infection.

neuroscience↗

bioRxiv2017

No kissing in the nucleus: Unbiased analysis reveals no evidence of trans chromosomal regulation of mammalian immune development

It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions.

genomics↗