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The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express in adult mouse β-cells using transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
© 2018 by the American Diabetes Association.
OBJECTIVE - Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult β-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far.
METHODS - In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions.
RESULTS - ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult β-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes.
CONCLUSIONS - Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a β-cell developmental program.
Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.
Power/sample size (power) analysis estimates the likelihood of successfully finding the statistical significance in a data set. There has been a growing recognition of the importance of power analysis in the proper design of experiments. Power analysis is complex, yet necessary for the success of large studies. It is important to design a study that produces statistically accurate and reliable results. Power computation methods have been well established for both microarray-based gene expression studies and genotyping microarray-based genome-wide association studies. High-throughput sequencing (HTS) has greatly enhanced our ability to conduct biomedical studies at the highest possible resolution (per nucleotide). However, the complexity of power computations is much greater for sequencing data than for the simpler genotyping array data. Research on methods of power computations for HTS-based studies has been recently conducted but is not yet well known or widely used. In this article, we describe the power computation methods that are currently available for a range of HTS-based studies, including DNA sequencing, RNA-sequencing, microbiome sequencing and chromatin immunoprecipitation sequencing. Most importantly, we review the methods of power analysis for several types of sequencing data and guide the reader to the relevant methods for each data type.
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.
Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology.
© 2017 Wiley Periodicals, Inc.
During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.
SET domain-containing proteins play a vital role in regulating gene expression during development through modifications in chromatin structure. Here we show that SET domain-containing 5 (Setd5) is divergently transcribed with Gt(ROSA26)Sor, is necessary for mammalian development, and interacts with the PAF1 co-transcriptional complex and other proteins. Setd5-deficient mouse embryos exhibit severe defects in neural tube formation, somitogenesis and cardiac development, have aberrant vasculogenesis in embryos, yolk sacs and placentas, and die between embryonic day 10.5 and 11.5. Setd5-deficient embryonic stem cells have impaired cellular proliferation, increased apoptosis, defective cell cycle progression, a diminished ability to differentiate into cardiomyocytes and greatly perturbed gene expression. SETD5 co-immunoprecipitates with multiple components of the PAF1 and histone deacetylase-containing NCoR complexes and is not solely required for major histone lysine methylation marks. In the absence of Setd5, histone acetylation is increased at transcription start sites and near downstream regions. These findings suggest that SETD5 functions in a manner similar to yeast Set3p and Drosophila UpSET, and that it is essential for regulating histone acetylation during gene transcription.
© 2016. Published by The Company of Biologists Ltd.
Diabetes mellitus arises from insufficient insulin secretion from pancreatic islet β-cells. In type 2 diabetes (T2D), β-cell dysfunction is associated with inactivation and/or loss of transcription factor (TF) activity, including Pdx1. Notably, this particular TF is viewed as a master regulator of pancreas development and islet β-cell formation, identity and function. TFs, like Pdx1, recruit coregulators to transduce activating and/or repressing signals to the general transcriptional machinery for controlling gene expression, including modifiers of DNA, histones and nucleosome architecture. These coregulators impart a secondary layer of control that can be exploited to modulate TF activity. In this review, we describe Pdx1-recruited coregulators that impact chromatin structure, consequently influencing normal β-cell function and likely Pdx1 activity in pathophysiological settings.
© 2016 John Wiley & Sons Ltd.
The regulation of β-catenin turnover is the central mechanism governing activation of the Wnt signaling pathway. All components of the pathway are present in the early embryo of Xenopus laevis, and Xenopus egg extracts have been used to recapitulate complex biological reactions such as microtubule dynamics, DNA replication, chromatin assembly, and phases of the cell cycle. Herein, we describe a biochemical method for analyzing β-catenin degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extracts. We show that in such a biochemical system, cytoplasmic β-catenin degradation is regulated by soluble components of the Wnt pathway as well as small molecules.
The ubiquitin-proteasome system (UPS) influences gene transcription in multiple ways. One way in which the UPS affects transcription centers on transcriptional activators, the function of which can be stimulated by components of the UPS that also trigger their destruction. Activation of transcription by the yeast activator Gcn4, for example, is attenuated by mutations in the ubiquitin ligase that mediates Gcn4 ubiquitylation or by inhibition of the proteasome, leading to the idea that ubiquitin-mediated proteolysis of Gcn4 is required for its activity. Here we probe the steps in Gcn4 activity that are perturbed by disruption of the UPS. We show that the ubiquitylation machinery and the proteasome control different steps in Gcn4 function and that proteasome activity is required for the ability of Gcn4 to bind to its target genes in the context of chromatin. Curiously, the effect of proteasome inhibition on Gcn4 activity is suppressed by mutations in the ubiquitin-selective chaperone Cdc48, revealing that proteolysis per se is not required for Gcn4 activity. Our data highlight the role of Cdc48 in controlling promoter occupancy by Gcn4 and support a model in which ubiquitylation of activators-not their destruction-is important for function.
© 2016 Howard and Tansey. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).