A longstanding unsettled question is whether pancreatic beta cells originate from exocrine duct cells. We have now used genetic labeling to fate map embryonic and adult pancreatic duct cells. We show that Hnf1beta+ cells of the trunk compartment of the early branching pancreas are precursors of acinar, duct, and endocrine lineages. Hnf1beta+ cells subsequent form the embryonic duct epithelium, which gives rise to both ductal and endocrine lineages, but not to acinar cells. By the end of gestation, the fate of Hnf1beta+ duct cells is further restrained. We provide compelling evidence that the ductal epithelium does not make a significant contribution to acinar or endocrine cells during neonatal growth, during a 6 month observation period, or during beta cell growth triggered by ligation of the pancreatic duct or by cell-specific ablation with alloxan followed by EGF/gastrin treatment. Thus, once the ductal epithelium differentiates it has a restricted plasticity, even under regenerative settings.

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 Lysinespecific 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 Kras G12D 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 nonclassical 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 tumorsuppressive role of KDM6A deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition.

Summary Despite the central role of chromosomal context in gene transcription, human noncoding DNA variants are generally studied outside of their genomic location. This limits our understanding of disease-causing regulatory variants. INS promoter mutations cause recessive neonatal diabetes. We show that all INS promoter point mutations in 60 patients disrupt a CC dinucleotide, whereas none affect other elements important for episomal promoter function. To model CC mutations, we humanized an ∼3.1-kb region of the mouse Ins2 gene. This recapitulated developmental chromatin states and cell-specific transcription. A CC mutant allele, however, abrogated active chromatin formation during pancreas development. A search for transcription factors acting through this element revealed that another neonatal diabetes gene product, GLIS3, has a pioneer-like ability to derepress INS chromatin, which is hampered by the CC mutation. Our in vivo analysis, therefore, connects two human genetic defects in an essential mechanism for developmental activation of the INS gene.

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SUMMARYUnderstanding genomic regulatory mechanisms of pancreas differentiation is relevant to the pathophysiology of diabetes mellitus, and to the development of replacement therapies. Numerous transcription factors promote β cell differentiation, although less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrine gene programs in the embryonic pancreas. However, pancreatic Rest knock-out mice failed to show increased numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we now observe a marked increase in the formation of pancreatic endocrine cells. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts, and induced β-cell specific genes in human adult duct-derived organoids. Finally, we define REST genomic programs that suppress pancreatic endocrine differentiation. These results establish a crucial role of REST as a negative regulator of pancreatic endocrine differentiation.

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 the histone demethylase UTX, carries somatic mutations in PDAC. Here, we show that pancreas-specific Hnf1a null mutations phenocopy Utx deficient mutations, and both synergize with Kras G12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic and biochemical studies to show that HNF1A recruits UTX to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates a differentiation program, and indirectly suppresses oncogenic and epithelial-mesenchymal transition genes. Finally, we identify a subset of non-classical PDAC samples that exhibit the HNF1A/UTX-deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A-deficiency promotes PDAC. They also connect the tumor suppressive role of UTX deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition. IntroductionPancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality 1 . The incidence of PDAC is rising, yet current chemotherapies are generally ineffective 2 . Genomic analysis of PDAC has identified almost universal driver mutations in KRAS, TP53, SMAD4 and CDKN2A, among a long list of loci that show recurrent somatic mutations and structural variations 3-7 . A small subset of tumors is caused by germ-line mutations in DNA-repair genes 5, 8 , whereas GWAS have identified dozens of common variants that impact PDAC susceptibility 9, 10 . Genetic studies have therefore uncovered leads that promise to define molecular targets for future precision therapies.Up to 18% of PDAC tumors carry mutations in KDM6A 5 , which encodes UTX, a component of the MLL/COMPASS transcriptional co-regulatory complex 11 . UTX catalyzes demethylation of histone H3K27me3, a modification associated with Polycomb-mediated repression [12][13][14] . Most somatic pathogenic UTX mutations are likely to result in a loss of function, and mouse genetic studies have shown that Utx and Kras mutations cooperate to promote PDAC 15, 16 . How UTX is recruited to its genomic targets in pancreatic cells, and the direct mechanisms through which it controls PDAC-relevant genetic programs, is still poorly understood 17 .There is increasing evidence that the transcriptional regulation of differentiated pancreatic exocrine cells is tightly linked to PDAC development and subtype definition [18][19][20][21][22] . Little is known, however, about the underlying molecular underpinnings. We have examined HNF1A, a homeodomain transcriptional regulator of liver, gut, kidney as well as pancreatic acinar and endocrine cells, which has been proposed to act as a candidate pancreatic tumor suppressor [22][23][24][25] . Human heterozygous HNF1A loss of function mu...

Despite the central role of chromosomal context in gene transcription, human noncoding DNA variants are generally studied outside of their endogenous genomic location. This poses major limitations to understand the true consequences of causal regulatory variants. We focused on a cis-regulatory mutation (c.-331C>G) in the INS gene promoter that is recurrently mutated in unrelated patients with recessive neonatal diabetes. We created mice in which a ~3.1 kb human INS upstream region carrying -331C or -331G alleles replaced the orthologous mouse Ins2 region. This human sequence drove cell-specific transcription in mice. It also recapitulated poised chromatin during pancreas development and active chromatin in differentiated bcells. The c.-331C>G mutation, however, blocked active chromatin formation in differentiated b-cells. We further show that another neonatal diabetes gene product, GLIS3, had a singular pioneer-like ability to activate INS chromatin in non-pancreatic cells, which was hampered by the c.-331C>G mutation. This in vivo analysis of human regulatory defects, therefore, uncovered cis and trans components of a mechanism that is essential to activate the endogenous INS gene.

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