Inês Cebola

Pulmonary arterial hypertension (PAH) is a severe, currently incurable lung disease characterized by endothelial injury and excessive repair, leading to arterial narrowing. However, the contributory mechanisms remain poorly understood. Here we show that Krüppel-like factor 6 (KLF6) is a feature of vascular pathology in PAH. KLF6 expression is elevated in human PAH and preclinical models of PAH and promotes endothelial repair and angiogenesis through transcriptomic remodelling, with effects distinct from those of KLF2 and KLF4. Endothelial KLF6 also stimulates vascular smooth muscle cell proliferation, which is attenuated by bosentan and imatinib. DisGeNET and spatial transcriptomic analyses of control and PAH lungs reveal elevated KLF6 in PAH endothelium, endothelial progenitor cells, and PAH with alveolar capillary dysplasia. In summary, KLF6 activation uniquely orchestrates endothelial repair and is a feature of the angio-proliferative vascular phenotype in PAH.

Background: Both rare and common variants in the SRY-Box Transcription Factor 17 ( SOX17 ) locus are associated with pulmonary arterial hypertension (PAH). SOX17 dysregulation leads to pulmonary artery endothelial cell (PAEC) dysfunction and the obstructive remodelling that characterises PAH. Hypothesis: Impaired SOX17 expression contributes to the pathogenesis of PAH. Restoring the function of SOX17 or its downstream targets using compounds that mimic its transcriptomic signature will rescue PAEC dysfunction and prevent PAH development. Methods and Results: We defined thousands of genes with direct SOX17 genomic binding sites and identified important potential binding partners, including ETS-transcription factors such as ERG by ChIP-seq in PAECs. Through the integration of three PAEC RNA-seq datasets involving overexpression and silencing of SOX17 , we defined a robust SOX17 transcriptomic signature. In PAH patients, circulating plasma protein levels of 10 SOX17 signature genes were associated with the SOX17 common risk variants. This included EFNB2 and UNC5B ; knockdown of these genes altered the viability and apoptosis of PAECs in response to TNFα treatment. The drug-transcriptome database Connectivity Map (CMap) was used to predict novel potential therapeutic compounds to correct the SOX17 transcriptomic signature. Five compounds were selected for in vitro testing and were able to partially reinstate SOX17 target gene expression in PAECs. One compound, BX-912, was selected for in vivo testing as it corrected the levels of multiple target genes, including suppressing Runt-related transcription factor-1 ( RUNX1 ). BX-912 blocked the development of pulmonary hypertension in mice lacking the SOX17 enhancer associated with human disease. Conclusion: We have demonstrated the therapeutic potential of targeting SOX17 in PAH through correction of its gene targets, identifying BX-912 as a lead compound with in vivo efficacy.

Transcription factor networks are crucial for the regulation of endothelial cell gene expression during vascular development and homeostasis. A recent analysis of 269 rare diseases in 77,539 individuals revealed an association between loss-of-function variants in ERG, encoding an ETS transcription factor, with primary lymphoedema. However, the pathogenicity of such variants and possible mechanisms of ERG-associated lymphatic vessel dysfunction remains to be established. Here, we have further identified and characterised lymphoedema-associated ERG genetic variants, revealing pathogenic mechanisms ranging from differential ERG subcellular localisation to altered DNA-binding and impaired transactivation. We confirm a role for ERG in regulating lymphangiogenesis using in vitro assays and a lymphatic endothelium-specific Erg deletion mouse model. Furthermore, we characterise the transcriptional and epigenomic landscape of human dermal lymphatic endothelial cells, identifying a unique role for ERG in regulating lymphatic gene programs, including the establishment of cooperative TF networks with PROX1 and GATA2. Our studies identify ERG as a master regulator of lymphatic endothelial cell transcriptional networks and uncover the mechanisms that underpin a novel causative gene for primary lymphoedema.

Type 2 diabetes (T2D) genome-wide association studies (GWASs) often overlook rare variants as a result of previous imputation panels’ limitations and scarce whole-genome sequencing (WGS) data. We used TOPMed imputation and WGS to conduct the largest T2D GWAS meta-analysis involving 51,256 cases of T2D and 370,487 controls, targeting variants with a minor allele frequency as low as 5 × 10⁻⁵. We identified 12 new variants, including a rare African/African American-enriched enhancer variant near the LEP gene (rs147287548), associated with fourfold increased T2D risk. We also identified a rare missense variant in HNF4A (p.Arg114Trp), associated with eightfold increased T2D risk, previously reported in maturity-onset diabetes of the young with reduced penetrance, but observed here in a T2D GWAS. We further leveraged these data to analyze 1,634 ClinVar variants in 22 genes related to monogenic diabetes, identifying two additional rare variants in HNF1A and GCK associated with fivefold and eightfold increased T2D risk, respectively, the effects of which were modified by the individual’s polygenic risk score. For 21% of the variants with conflicting interpretations or uncertain significance in ClinVar, we provided support of being benign based on their lack of association with T2D. Our work provides a framework for using rare variant GWASs to identify large-effect variants and assess variant pathogenicity in monogenic diabetes genes.

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic β‐cell identity and function. Elimination of Lkb1 from the β‐cell results in improved glucose‐stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of β cell‐selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from β‐cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors (TFs) important for β‐cell identity, such as FOXA, MAFA or RFX6, and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates β‐cell identity and function.

Background Pulmonary arterial hypertension (PAH) is a severe lung condition with an unmet clinical need. Endothelial damage is followed by excessive repair and narrowing of lung arteries, but the mechanisms are unclear. Methods We investigated the role of Krüppel-like transcription factor 6 (KLF6), known for its involvement in tissue injury response and cancer onset, in PAH through functional and expression analyses of human pulmonary artery endothelial cells (HPAECs) and PAH lung tissues. KLF6 activation patterns and transcriptional programs were compared with those regulated by KLF2 and KLF4, previously linked to PAH. Results KLF6 expression increased due to hypoxic and inflammatory triggers in early rodent PAH. KLF6 overexpression improved endothelial survival and induced angiogenesis in cultured HPAECs and human pulmonary arterial explants. Transcriptomic analysis of KLF6- overexpressing HPAECs revealed its role in regulating endothelial homeostasis and expression of arterial identity genes, such as SOX17, ERG, BMPR2, KDR, TEK, ENG, ACVRL1 and CDH5. DisGeNET analysis showed a significant association of KLF6-regulated genes with PAH. Spatial transcriptomic analysis of PAH lung vascular tissues (n=6/group) revealed significant enrichment of KLF6-regulated genes in angiogenesis, cell motility, VEGFR signalling, and extracellular matrix organization. KLF6⁺ Erg⁺ vWF⁺ cells accumulated in vascular channels of remodelled PAH lungs and. KLF6 expression was elevated in PAH blood-derived endothelial progenitor cells (n=5) and pulmonary arterial cells in PAH associated with Alveolar Capillary Dysplasia. Conclusions KLF6 uniquely orchestrates endothelial repair but its sustained activation promotes development of the apoptosis-resistant, angio-proliferative vascular phenotype in human PAH. Dysregulation of KLF6 signalling may have broader implications for pulmonary vascular disease.

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic β-cell identity and function. Elimination of Lkb1 from the β-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of β cell- selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from β-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors important for β-cell identity, such as FOXA, MAFA or RFX6 and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates β-cell identity and function.