Heterarchy of Transcription Factors Driving Basal and Luminal Cell Phenotypes in Human Urothelium

Cell differentiation is effected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq were used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor PPARG as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq as having altered accessibility during differentiation were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including FOXA1, P63, GRHL2, CTCF and GATA3. In addition, co-occurrence of GATA3 motifs was observed within sub-sets of differentiation-specific peaks containing P63 or FOXA1 after induction of differentiation. Changes in abundance of GRHL2, GATA3, and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally-regulated programme, urothelial differentiation operates as a heterarchy wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes

Functional transcription factor target discovery via compendia of binding and expression profiles

Genome-wide experiments to map the DNA-binding locations of transcription-associated factors (TFs) have shown that the number of genes bound by a TF far exceeds the number of possible direct target genes. Distinguishing functional from non-functional binding is therefore a major challenge in the study of transcriptional regulation. We hypothesized that functional targets can be discovered by correlating binding and expression profiles across multiple experimental conditions. To test this hypothesis, we obtained ChIP-seq and RNA-seq data from matching cell types from the human ENCODE resource, considered promoter-proximal and distal cumulative regulatory models to map binding sites to genes, and used a combination of linear and non-linear measures to correlate binding and expression data. We found that a high degree of correlation between a gene's TF-binding and expression profiles was significantly more predictive of the gene being differentially expressed upon knockdown of that TF, compared to using binding sites in the cell type of interest only. Remarkably, TF targets predicted from correlation across a compendium of cell types were also predictive of functional targets in other cell types. Finally, correlation across a time course of ChIP-seq and RNA-seq experiments was also predictive of functional TF targets in that tissue.Comment: 15 pages + 8 pages supplementary material; 6 figures, 6 supplementary figures, 5 supplementary table

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease

Prevention of organ rejection in renal and liver transplantation with extended release tacrolimus

Michael E Reschen, Christopher A O’Callaghan Henry Wellcome Building, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Abstract: Tacrolimus is the key immunosuppressant used to prevent allograft rejection in kidney and liver transplant recipients. Despite the efficacy of tacrolimus and adjunctive immunosuppressants, a substantial number of patients experience episodes of acute rejection and late graft loss. Nonadherence is an etiological factor in both acute rejection and graft loss. In 2007, a prolonged release version of tacrolimus became available that allows once daily administration, thus halving the pill burden compared to the standard twice-daily tacrolimus. An increasing number of studies in de novo transplantation and in treatment conversion have evaluated the pharmacokinetic profile, efficacy, and safety of prolonged-release tacrolimus. We have reviewed the literature on the use of prolonged-release tacrolimus and hope that this will be of value in the design of protocols for transplant immunosuppression.Keywords: immunosuppression, kidney, hepatic, allograft, adherenc

Genetic and environmental risk factors for atherosclerosis regulate transcription of phosphatase and actin regulating gene PHACTR1.

Background and aims Coronary artery disease (CAD) risk is associated with non-coding genetic variants at the phosphatase and actin regulating protein 1(PHACTR1) gene locus. The PHACTR1 gene encodes an actin-binding protein with phosphatase regulating activity. The mechanism whereby PHACTR1 influences CAD risk is unknown. We hypothesized that PHACTR1 would be expressed in human cell types relevant to CAD and regulated by atherogenic or genetic factors. Methods and results Using immunohistochemistry, we demonstrate that PHACTR1 protein is expressed strongly in human atherosclerotic plaque macrophages, lipid-laden foam cells, adventitial lymphocytes and endothelial cells. Using a combination of genomic analysis and molecular techniques, we demonstrate that PHACTR1 is expressed as multiple previously uncharacterized transcripts in macrophages, foam cells, lymphocytes and endothelial cells. Immunoblotting confirmed a total absence of PHACTR1 in vascular smooth muscle cells. Real-time quantitative PCR showed that PHACTR1 is regulated by atherogenic and inflammatory stimuli. In aortic endothelial cells, oxLDL and TNF-alpha both upregulated an intermediate length transcript. A short transcript expressed only in immune cells was upregulated in macrophages by oxidized low-density lipoprotein, and oxidized phospholipids but suppressed by lipopolysaccharide or TNF-alpha. In primary human macrophages, we identified a novel expression quantitative trait locus (eQTL) specific for this short transcript, whereby the risk allele at CAD risk SNP rs9349379 is associated with reduced PHACTR1 expression, similar to the effect of an inflammatory stimulus. Conclusions Our data demonstrate that PHACTR1 is a key atherosclerosis candidate gene since it is regulated by atherogenic stimuli in macrophages and endothelial cells and we identify an effect of the genetic risk variant on PHACTR1 expression in macrophages that is similar to that of an inflammatory stimulus.</p

Impact of the COVID-19 pandemic on emergency department attendances and acute medical admissions

Background To better understand the impact of the COVID-19 pandemic on hospital healthcare, we studied activity in the emergency department (ED) and acute medicine department of a major UK hospital. Methods Electronic patient records for all adult patients attending ED (n = 243,667) or acute medicine (n = 82,899) during the pandemic (2020–2021) and prior year (2019) were analysed and compared. We studied parameters including severity, primary diagnoses, co-morbidity, admission rate, length of stay, bed occupancy, and mortality, with a focus on non-COVID-19 diseases. Results During the first wave of the pandemic, daily ED attendance fell by 37%, medical admissions by 30% and medical bed occupancy by 27%, but all returned to normal within a year. ED attendances and medical admissions fell across all age ranges; the greatest reductions were seen for younger adults in ED attendances, but in older adults for medical admissions. Compared to non-COVID-19 pandemic admissions, COVID-19 admissions were enriched for minority ethnic groups, for dementia, obesity and diabetes, but had lower rates of malignancy. Compared to the pre-pandemic period, non-COVID-19 pandemic admissions had more hypertension, cerebrovascular disease, liver disease, and obesity. There were fewer low severity ED attendances during the pandemic and fewer medical admissions across all severity categories. There were fewer ED attendances with common non-respiratory illnesses including cardiac diagnoses, but no change in cardiac arrests. COVID-19 was the commonest diagnosis amongst medical admissions during the first wave and there were fewer diagnoses of pneumonia, myocardial infarction, heart failure, cellulitis, chronic obstructive pulmonary disease, urinary tract infection and other sepsis, but not stroke. Levels had rebounded by a year later with a trend to higher levels of stroke than before the pandemic. During the pandemic first wave, 7-day mortality was increased for ED attendances, but not for non-COVID-19 medical admissions. Conclusions Reduced ED attendances in the first wave of the pandemic suggest opportunities for reducing low severity presentations to ED in the future, but also raise the possibility of harm from delayed or missed care. Reassuringly, recent rises in attendance and admissions indicate that any deterrent effect of the pandemic on attendance is diminishing

Process of care and activity in a clinically inclusive ambulatory emergency care unit: progressive effect over time on clinical outcomes and acute medical admissions

Clinically relevant outcomes for same-day emergency care provided by ambulatory emergency care units (AECs) are largely unknown. We report the activity and outcomes for a large UK adult AEC operating an ambulatory-care-by-default model without specific exclusion criteria. The AEC consultant triaged all acute medical referrals to either the AEC or the standard non-ambulatory ‘take’ pathway during AEC opening hours. The proportion of acute medical referrals seen in the AEC increased to 42% (mean 700 referrals seen per month) in the last 6 months of the study and numbers seen in the non-ambulatory pathway fell. The most common diagnoses were for chest pain, pneumonia, cellulitis, heart failure and urinary system disorders. Seventy-four point eight per cent of patients completed their care in a single visit. In the last calendar year, the conversion rate from AEC to inpatient admission was 12%, and the 30-day readmission rate was 6.9% and 18% for the AEC and non-ambulatory pathways, respectively. Across the whole study period, the 30-day mortality was 1.6% and 6.9% for the AEC and non-ambulatory pathway, respectively. This ambulatory approach is safe and effective.</p

Genetic fine-mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease