Mucosal transcriptomics highlight lncRNAs implicated in ulcerative colitis, Crohn's disease, and celiac disease.

Ulcerative colitis (UC), Crohn's disease (CD), and celiac disease are prevalent intestinal inflammatory disorders with nonsatisfactory therapeutic interventions. Analyzing patient data-driven cohorts can highlight disease pathways and new targets for interventions. Long noncoding RNAs (lncRNAs) are attractive candidates, since they are readily targetable by RNA therapeutics, show relative cell-specific expression, and play key cellular functions. Uniformly analyzing gut mucosal transcriptomics from 696 subjects, we have highlighted lncRNA expression along the gastrointestinal (GI) tract, demonstrating that, in control samples, lncRNAs have a more location-specific expression in comparison with protein-coding genes. We defined dysregulation of lncRNAs in treatment-naive UC, CD, and celiac diseases using independent test and validation cohorts. Using the Predicting Response to Standardized Pediatric Colitis Therapy (PROTECT) inception UC cohort, we defined and prioritized lncRNA linked with UC severity and prospective outcomes, and we highlighted lncRNAs linked with gut microbes previously implicated in mucosal homeostasis. HNF1A-AS1 lncRNA was reduced in all 3 conditions and was further reduced in more severe UC form. Similarly, the reduction of HNF1A-AS1 ortholog in mice gut epithelia showed higher sensitivity to dextran sodium sulfate-induced colitis, which was coupled with alteration in the gut microbial community. These analyses highlight prioritized dysregulated lncRNAs that can guide future preclinical studies for testing them as potential targets.

Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

The HASTER lncRNA promoter is a cis-acting transcriptional stabilizer of HNF1A.

The biological purpose of long non-coding RNAs (lncRNAs) is poorly understood. Haploinsufficient mutations in HNF1A homeobox A (HNF1A), encoding a homeodomain transcription factor, cause diabetes mellitus. Here, we examine HASTER, the promoter of an lncRNA antisense to HNF1A. Using mouse and human models, we show that HASTER maintains cell-specific physiological HNF1A concentrations through positive and negative feedback loops. Pancreatic ß cells from Haster mutant mice consequently showed variegated HNF1A silencing or overexpression, resulting in hyperglycaemia. HASTER-dependent negative feedback was essential to prevent HNF1A binding to inappropriate genomic regions. We demonstrate that the HASTER promoter DNA, rather than the lncRNA, modulates HNF1A promoter-enhancer interactions in cis and thereby regulates HNF1A transcription. Our studies expose a cis-regulatory element that is unlike classic enhancers or silencers, it stabilizes the transcription of its target gene and ensures the fidelity of a cell-specific transcription factor program. They also show that disruption of a mammalian lncRNA promoter can cause diabetes mellitus.

Genetic regulation of RNA splicing in human pancreatic islets.

BACKGROUND: Non-coding genetic variants that influence gene transcription in pancreatic islets play a major role in the susceptibility to type 2 diabetes (T2D), and likely also contribute to type 1 diabetes (T1D) risk. For many loci, however, the mechanisms through which non-coding variants influence diabetes susceptibility are unknown. RESULTS: We examine splicing QTLs (sQTLs) in pancreatic islets from 399 human donors and observe that common genetic variation has a widespread influence on the splicing of genes with established roles in islet biology and diabetes. In parallel, we profile expression QTLs (eQTLs) and use transcriptome-wide association as well as genetic co-localization studies to assign islet sQTLs or eQTLs to T2D and T1D susceptibility signals, many of which lack candidate effector genes. This analysis reveals biologically plausible mechanisms, including the association of T2D with an sQTL that creates a nonsense isoform in ERO1B, a regulator of ER-stress and proinsulin biosynthesis. The expanded list of T2D risk effector genes reveals overrepresented pathways, including regulators of G-protein-mediated cAMP production. The analysis of sQTLs also reveals candidate effector genes for T1D susceptibility such as DCLRE1B, a senescence regulator, and lncRNA MEG3. CONCLUSIONS: These data expose widespread effects of common genetic variants on RNA splicing in pancreatic islets. The results support a role for splicing variation in diabetes susceptibility, and offer a new set of genetic targets with potential therapeutic benefit.

HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer.

Defects in transcriptional regulators of pancreatic exocrine differentiation have been implicated in pancreatic tumorigenesis, but the molecular mechanisms are poorly understood. The locus encoding the transcription factor HNF1A harbors susceptibility variants for pancreatic ductal adenocarcinoma (PDAC), while KDM6A, encoding Lysine-specific demethylase 6A, carries somatic mutations in PDAC. Here, we show that pancreas-specific Hnf1a null mutant transcriptomes phenocopy those of Kdm6a mutations, and both defects synergize with KrasG12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic, and biochemical studies to show that HNF1A recruits KDM6A to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates differentiated acinar cell programs, and indirectly suppresses oncogenic and epithelial-mesenchymal transition genes. We also identify a subset of non-classical PDAC samples that exhibit the HNF1A/KDM6A-deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A deficiency promotes PDAC. They also connect the tumor-suppressive role of KDM6A deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition.

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production.

OBJECTIVE: Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated. METHODS: We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes. RESULTS: Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine. CONCLUSION: These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes.

Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer clusters or super-enhancers. So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in three-dimensional (3D) space. Furthermore, their target genes are often unknown. We have created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers to their target genes, often located hundreds of kilobases away. It also revealed >1,300 groups of islet enhancers, super-enhancers and active promoters that form 3D hubs, some of which show coordinated glucose-dependent activity. We demonstrate that genetic variation in hubs impacts insulin secretion heritability, and show that hub annotations can be used for polygenic scores that predict T2D risk driven by islet regulatory variants. Human islet 3D chromatin architecture, therefore, provides a framework for interpretation of T2D genome-wide association study (GWAS) signals.

Long Non-coding RNAs as Local Regulators of Pancreatic Islet Transcription Factor Genes.

The transcriptional programs of differentiated cells are tightly regulated by interactions between cell type-specific transcription factors and cis-regulatory elements. Long non-coding RNAs (lncRNAs) have emerged as additional regulators of gene transcription. Current evidence indicates that lncRNAs are a very heterogeneous group of molecules. For example, selected lncRNAs have been shown to regulate gene expression in cis or trans, although in most cases the precise underlying molecular mechanisms is unknown. Recent studies have uncovered a large number of lncRNAs that are selectively expressed in pancreatic islet cells, some of which were shown to regulate ß cell transcriptional programs. A subset of such islet lncRNAs appears to control the expression of ß cell-specific transcription factor (TF) genes by local cis-regulation. In this review, we discuss current knowledge of molecular mechanisms underlying cis-regulatory lncRNAs and discuss challenges involved in using genetic perturbations to define their function. We then discuss known examples of pancreatic islet lncRNAs that appear to exert cis-regulation of TF genes. We propose that cis-regulatory lncRNAs could represent a molecular target for modulation of diabetes-relevant genes.

Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury.

Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair.

Human Pancreatic ß Cell lncRNAs Control Cell-Specific Regulatory Networks.

Recent studies have uncovered thousands of long non-coding RNAs (lncRNAs) in human pancreatic ß cells. ß cell lncRNAs are often cell type specific and exhibit dynamic regulation during differentiation or upon changing glucose concentrations. Although these features hint at a role of lncRNAs in ß cell gene regulation and diabetes, the function of ß cell lncRNAs remains largely unknown. In this study, we investigated the function of ß cell-specific lncRNAs and transcription factors using transcript knockdowns and co-expression network analysis. This revealed lncRNAs that function in concert with transcription factors to regulate ß cell-specific transcriptional networks. We further demonstrate that the lncRNA PLUTO affects local 3D chromatin structure and transcription of PDX1, encoding a key ß cell transcription factor, and that both PLUTO and PDX1 are downregulated in islets from donors with type 2 diabetes or impaired glucose tolerance. These results implicate lncRNAs in the regulation of ß cell-specific transcription factor networks.

Human ß cell transcriptome analysis uncovers lncRNAs that are tissue-specific, dynamically regulated, and abnormally expressed in type 2 diabetes.

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and ß cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the ß cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a ß cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to ß cell programming and diabetes pathophysiology.

The homeodomain-containing transcription factors Arx and Pax4 control enteroendocrine subtype specification in mice.

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3(+) endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3(+)) and in early differentiating (ChromograninA(-)) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.

Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development.

During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities.

Loss of enteroendocrine cells in mice alters lipid absorption and glucose homeostasis and impairs postnatal survival.

At least 10 enteroendocrine cell types have been identified, and the peptide hormones they secrete have diverse functions that include regulation of glucose homeostasis, food intake, and gastric emptying. Mice lacking individual enteroendocrine hormones, their receptors, or combinations of these have shed light on the role of these hormones in the regulation of energy homeostasis. However, because enteroendocrine hormones have partially overlapping functions, these loss-of-function studies produced only minor phenotypes, and none of the enteroendocrine hormones was shown to be essential for life. To examine the effect of loss of all enteroendocrine cells and hormones on energy homeostasis, we generated mice with intestinal-specific ablation of the proendocrine transcription factor neurogenin 3 (referred to herein as Ngn3Deltaint mice). Ngn3Deltaint mice were deficient for all enteroendocrine cells and hormones, and died with a high frequency during the first week of life. Mutant mice were growth retarded and had yellowish stool suggestive of steatorrhea. Subsequent analyses revealed that Ngn3Deltaint mice had impaired lipid absorption, reduced weight gain, and improved glucose homeostasis. Furthermore, intestinal epithelium of the mutant mice showed an enlarged proliferative crypt compartment and accelerated cell turnover but no changes to goblet and Paneth cell numbers. Enterocytes had shorter microvilli, but the expression of the main brush border enzymes was unaffected. Our data help unravel the role of enteroendocrine cells and hormones in lipid absorption and maintenance of the intestinal epithelium.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

Endocrine pancreatic tissue plasticity in obese humans is associated with cytoplasmic expression of PBX-1 in pancreatic ductal cells.

In vivo lineage tracing experiments in mice have recently cast doubt on the potential islet neogenesis from ductal precursors in adult mammals. We examined, in human obesity, a model for pancreatic endocrine tissue plasticity, the gene and protein expression of PBX-1-a transcription factor expressed in regenerating rat ductules and potentially implicated in the pancreatic development, alone or in association with PDX-1. When comparing gene expression, by quantitative real-time RT-PCR, in pancreatic exocrine tissue from obese non-diabetic subjects with increased islet mass, we found that Pbx-1 and Pdx-1 were up-regulated (5.9+/-1.2 and 2.4+/-0.6 versus non-obese). Immunohistochemistry confirmed PBX-1 over-expression and its cytoplasmic sequestration in ductal cells of obese subjects, associated with pronounced islet neogenesis (cytokeratin 19/chromogranin A double labeling). cDNA microarray analysis also showed up-regulation of other genes implicated in islet regeneration, including betacellulin, laminin, TGFa, NeuroD1, Pax6, substantiating the role of the islet neogenesis pathway in human obesity.