Identification of cis-regulatory elements affecting pancreatic Islet maturation using chromatin accessibility variations
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE122747 | GEO | 2019/12/31
REPOSITORIES: GEO
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Project description:Pancreatic beta-cells are highly specialized cells that produce and release insulin in response to nutrients, hormones and neurotransmitters. Glucose-induced insulin secretion, a unique feature of fully differentiated beta-cells, is only acquired after birth and is preceded by a phase of intense beta-cell proliferation. These events occurring in the neonatal period are critical for the establishment of an appropriate functional beta-cell mass covering the insulin needs throughout life. However, key regulators of gene expression and cis-regulatory elements involved in the cellular reprogramming along maturation remain to be elucidated. This project addresses this issue by using ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) permitting a fine genome-wide mapping of chromatin accessibility. This approach is used to compare open chromatin regions in newborn and adult rat beta-cells. We obtained a genome-wide picture of chromatin accessible sites (100000) among which 20% were differentially accessible during maturation. Nearly 60% of these sites were in the proximity of significantly differentially expressed genes. An analysis of transcription factor binding sites revealed key known and unforeseen transcription factors which could explain these changes. We validated a transcriptional repressor named SCRT1, that depicted a significant effect on beta-cell proliferation and targeted several genes implicated in the acquisition of glucose-stimulated insulin secretion function. Thus, we were able to find several known and unforeseen key transcriptional regulators acting at cis-regulatory sites and promoters which depicted a differential accessibility and induced differential gene expression along maturation. These findings could be of interest to induce maturation of surrogate insulin-producing cells.
2020-05-01 | GSE130651 | GEO
Project description:Identification of cis-regulatory elements affecting pancreatic Islet maturation using chromatin accessibility variations
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Project description:There is little information on chromatin structure that allows access of trans-acting transcription factors. Logically, the target DNA elements become accessible by either exposing themselves towards the environment on the surface of the nucleosome, or making the regulatory region free of the nucleosome. Here, we demonstrate that curved DNA that mimics a negative supercoil can play both roles in the promoter region. By constructing 35 reporter plasmids and using in vivo assay systems, we scrutinized the relationships between upstream DNA geometry, nucleosome positioning and promoter activity. When the left-handedly curved DNA was linked to the herpes simplex virus thymidine kinase (HSV tk) promoter at a specific rotational phase and distance, the curved DNA attracted the nucleosome and the TATA box was thereby left in the linker DNA with its minor groove facing outwards, which led to the activation of transcription. Neither planar curving, nor right-handedly curved DNA nor straight DNA had this effect. Our results seem to provide a clue for solving the problem of why curved DNA is often located near transcriptional control regions.
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Project description:Factor XII (FXII) deficiency is a recessive Mendelian trait due to mutations in the F12 gene. There is no bleeding associated with FXII deficiency, but FXII deficiency has been reported to be associated with risk of thrombosis in some studies.We examined the functional effect of two naturally-occurring mutations in two Spanish FXII deficient families: a C/G substitution at position -8, and a C/T substitution at position -13. Both mutations were located on a putative HNF4 binding site of F12 gene promoter. We also analyzed the F12 C46T polymorphism (rs1801020), associated with a decrease in the FXII levels, which also segregated in both families. A fragment containing each one of both -8 and -13 mutations, was cloned 5' of a reporter gene. We compared the in vitro expression of these constructs to the wild type expression.Our analyses confirm that the -8C/G and the -13C/T mutations decreased expression levels, demonstrating that both mutations are involved in the observed FXII deficiency. In addition, electrophoretic shift analyses suggest that they alter the union of nuclear proteins to the promoter. Coinheritance of these mutations with the C46T polymorphism, result in a significant genotype-phenotype correlation.We have identified two naturally-occurring mutations in the F12 promoter that drastically reduce FXII levels. Knowing rare genetic alterations in the F12 gene, together with the C46T common variant, may yield further understanding about the genetic architecture of FXII levels, which may have a role in the risk of thrombosis.
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Project description:Glucose-induced insulin secretion, a hallmark of mature β-cells, is achieved after birth and is preceded by a phase of intense proliferation. These events occurring in the neonatal period are decisive for establishing an appropriate functional β-cell mass that provides the required insulin throughout life. However, key regulators of gene expression involved in functional maturation of β-cells remain to be elucidated. Here, we addressed this issue by mapping open chromatin regions in newborn versus adult rat islets using the ATAC-seq assay. We obtained a genome-wide picture of chromatin accessible sites (~ 100,000) among which 20% were differentially accessible during maturation. An enrichment analysis of transcription factor binding sites identified a group of transcription factors that could explain these changes. Among them, Scrt1 was found to act as a transcriptional repressor and to control β-cell proliferation. Interestingly, Scrt1 expression was controlled by the transcriptional repressor RE-1 silencing transcription factor (REST) and was increased in an in vitro reprogramming system of pancreatic exocrine cells to β-like cells. Overall, this study led to the identification of several known and unforeseen key transcriptional events occurring during β-cell maturation. These findings will help defining new strategies to induce the functional maturation of surrogate insulin-producing cells.
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Project description:Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length to control protein production. However, the genome-wide relationship of poly(A) tail changes to mRNA translation during vertebrate oocyte maturation is not known. We used Tail-seq and polyribosome analysis to measure poly(A) tail and translational changes during oocyte maturation in Xenopus laevis We identified large-scale poly(A) and translational changes during oocyte maturation, with poly(A) tail length changes preceding translational changes. Proteins important for completion of the meiotic divisions and early development exhibited increased polyadenylation and translation during oocyte maturation. A family of U-rich sequence elements was enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. We propose that changes in mRNA polyadenylation are a conserved mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of cis-acting sequence elements.
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Project description:Transcription factor (TF) binding is determined by sequence as well as chromatin accessibility. Although the role of accessibility in shaping TF-binding landscapes is well recorded, its role in evolutionary divergence of TF binding, which in turn can alter cis-regulatory activities, is not well understood. In this work, we studied the evolution of genome-wide binding landscapes of five major TFs in the core network of mesoderm specification, between Drosophila melanogaster and Drosophila virilis, and examined its relationship to accessibility and sequence-level changes. We generated chromatin accessibility data from three important stages of embryogenesis in both Drosophila melanogaster and Drosophila virilis and recorded conservation and divergence patterns. We then used multivariable models to correlate accessibility and sequence changes to TF-binding divergence. We found that accessibility changes can in some cases, for example, for the master regulator Twist and for earlier developmental stages, more accurately predict binding change than is possible using TF-binding motif changes between orthologous enhancers. Accessibility changes also explain a significant portion of the codivergence of TF pairs. We noted that accessibility and motif changes offer complementary views of the evolution of TF binding and developed a combined model that captures the evolutionary data much more accurately than either view alone. Finally, we trained machine learning models to predict enhancer activity from TF binding and used these functional models to argue that motif and accessibility-based predictors of TF-binding change can substitute for experimentally measured binding change, for the purpose of predicting evolutionary changes in enhancer activity.
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