The lncRNA Sweetheart regulates compensatory cardiac hypertrophy after myocardial injury in murine males.

After myocardial infarction in the adult heart the remaining, non-infarcted tissue adapts to compensate the loss of functional tissue. This adaptation requires changes in gene expression networks, which are mostly controlled by transcription regulating proteins. Long non-coding transcripts (lncRNAs) are taking part in fine-tuning such gene programs. We describe and characterize the cardiomyocyte specific lncRNA Sweetheart RNA (Swhtr), an approximately 10 kb long transcript divergently expressed from the cardiac core transcription factor coding gene Nkx2-5. We show that Swhtr is dispensable for normal heart development and function but becomes essential for the tissue adaptation process after myocardial infarction in murine males. Re-expressing Swhtr from an exogenous locus rescues the Swhtr null phenotype. Genes that depend on Swhtr after cardiac stress are significantly occupied and therefore most likely regulated by NKX2-5. The Swhtr transcript interacts with NKX2-5 and disperses upon hypoxic stress in cardiomyocytes, indicating an auxiliary role of Swhtr for NKX2-5 function in tissue adaptation after myocardial injury.

Fendrr synergizes with Wnt signalling to regulate fibrosis related genes during lung development via its RNA:dsDNA triplex element.

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.

Activation and Functions of Col6a1+ Fibroblasts in Colitis-Associated Cancer.

Cancer-associated fibroblasts (CAFs) comprise a group of heterogeneous subpopulations with distinct identities indicative of their diverse origins, activation patterns, and pro-tumorigenic functions. CAFs originate mainly from resident fibroblasts, which are activated upon different stimuli, including growth factors and inflammatory mediators, but the extent to which they also maintain some of their homeostatic properties, at least at the earlier stages of carcinogenesis, is not clear. In response to cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor (TNF), as well as microbial products, CAFs acquire an immunoregulatory phenotype, but its specificity and pathophysiological significance in individual CAF subsets is yet to be determined. In this study, we analyzed the properties of Col6a1-positive fibroblasts in colitis-associated cancer. We found that Col6a1+ cells partly maintain their homeostatic features during adenoma development, while their activation is characterized by the acquisition of a distinct proangiogenic signature associated with their initial perivascular location. In vitro and in vivo experiments showed that Col6a1+ cells respond to innate immune stimuli and exert pro-tumorigenic functions. However, Col6a1+-specific inhibition of TNF receptor 1 (TNFR1) or IL-1 receptor (IL-1R) signaling does not significantly affect tumorigenesis, suggesting that activation of other subsets acts in a compensatory way or that multiple immune stimuli are necessary to drive the proinflammatory activation of this subset. In conclusion, our results show that adenoma-associated CAF subsets can partly maintain the properties of homeostatic fibroblasts while they become activated to support tumor growth through distinct and compensatory mechanisms.

First genome-wide association study of esophageal atresia identifies three genetic risk loci at CTNNA3, FOXF1/FOXC2/FOXL1, and HNF1B.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is the most common congenital malformation of the upper digestive tract. This study represents the first genome-wide association study (GWAS) to identify risk loci for EA/TEF. We used a European case-control sample comprising 764 EA/TEF patients and 5,778 controls and observed genome-wide significant associations at three loci. On chromosome 10q21 within the gene CTNNA3 (p = 2.11 × 10-8; odds ratio [OR] = 3.94; 95% confidence interval [CI], 3.10-5.00), on chromosome 16q24 next to the FOX gene cluster (p = 2.25 × 10-10; OR = 1.47; 95% CI, 1.38-1.55) and on chromosome 17q12 next to the gene HNF1B (p = 3.35 × 10-16; OR = 1.75; 95% CI, 1.64-1.87). We next carried out an esophageal/tracheal transcriptome profiling in rat embryos at four selected embryonic time points. Based on these data and on already published data, the implicated genes at all three GWAS loci are promising candidates for EA/TEF development. We also analyzed the genetic EA/TEF architecture beyond the single marker level, which revealed an estimated single-nucleotide polymorphism (SNP)-based heritability of around 37% ± 14% standard deviation. In addition, we examined the polygenicity of EA/TEF and found that EA/TEF is less polygenic than other complex genetic diseases. In conclusion, the results of our study contribute to a better understanding on the underlying genetic architecture of ET/TEF with the identification of three risk loci and candidate genes.

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis.

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.

Fibroblast Reprogramming in Gastrointestinal Cancer.

Gastrointestinal cancers are a significant cause of cancer mortality worldwide and have been strongly linked with chronic inflammation. Current therapies focus on epithelial/cancer cells; however, the importance of the tumor microenvironment in the development and treatment of the disease is also now well established. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment, and are actively participating in tumor initiation, promotion and metastasis. They structurally and functionally affect cancer cell proliferation, tumor immunity, angiogenesis, extracellular matrix remodeling and metastasis through a variety of signaling pathways. CAFs originate predominantly from resident mesenchymal cells, which are activated and reprogrammed in response to cues from cancer cells. In recent years, chronic inflammation of the gastrointestinal tract has also proven an important driver of mesenchymal cell activation and subsequent CAF development, which in turn are capable of regulating the transition from acute to chronic inflammation and cancer. In this review, we will provide a concise overview of the mechanisms that drive fibroblast reprogramming in cancer and the recent advances on the downstream signaling pathways that regulate the functional properties of the activated mesenchyme. This new mechanistic insight could pave the way for new therapeutic strategies and better prognosis for cancer patients.

Human exome and mouse embryonic expression data implicate ZFHX3, TRPS1, and CHD7 in human esophageal atresia.

INTRODUCTION: Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development. METHODS: To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal. RESULTS: In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants. CONCLUSION: Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.

BRACHYURY directs histone acetylation to target loci during mesoderm development.

T-box transcription factors play essential roles in multiple aspects of vertebrate development. Here, we show that cooperative function of BRACHYURY (T) with histone-modifying enzymes is essential for mouse embryogenesis. A single point mutation (TY88A) results in decreased histone 3 lysine 27 acetylation (H3K27ac) at T target sites, including the T locus, suggesting that T autoregulates the maintenance of its expression and functions by recruiting permissive chromatin modifications to putative enhancers during mesoderm specification. Our data indicate that T mediates H3K27ac recruitment through a physical interaction with p300. In addition, we determine that T plays a prominent role in the specification of hematopoietic and endothelial cell types. Hematopoietic and endothelial gene expression programs are disrupted in TY88A mutant embryos, leading to a defect in the differentiation of hematopoietic progenitors. We show that this role of T is mediated, at least in part, through activation of a distal Lmo2 enhancer.

Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in Pericytes.

RATIONALE: Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. OBJECTIVE: Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. METHODS AND RESULTS: RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays (P<0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α (P<0.001), ATF6 (P<0.01), and soluble BiP (P<0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure (P<0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). CONCLUSIONS: Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.

Roles for long non-coding RNAs in physiology and disease.

While the vast majority of the genome is transcribed into RNA, only a small fraction of these transcripts have protein-coding potential. A large fraction of the transcribed RNA belongs to the class known as long non-coding RNAs (lncRNAs). Several recent studies have shown that at least some of these lncRNA transcripts represent functional RNA molecules. LncRNAs can utilize a wide range of mechanisms to regulate the RNA and/or the protein content of a cell on the transcriptional and the post-transcriptional levels. So far, many studies have identified differentially expressed lncRNAs in various physiological contexts, genetic disorders and human diseases. A steadily increasing number of studies could establish functional roles for some of these lncRNAs in developmental processes, cancer and tissue homeostasis. Taken together, these functions provide an additional layer of gene regulation and contribute to the high complexity of physiological and disease-related phenotypes.