Human stem cell models: lessons for pancreatic development and disease.

A comprehensive understanding of mechanisms that underlie the development and function of human cells requires human cell models. For the pancreatic lineage, protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine and exocrine cells through intermediates resembling in vivo development. In recent years, this differentiation system has been employed to decipher mechanisms of pancreatic development, congenital defects of the pancreas, as well as genetic forms of diabetes and exocrine diseases. In this review, we summarize recent insights gained from studies of pancreatic hPSC models. We discuss how genome-scale analyses of the differentiation system have helped elucidate roles of chromatin state, transcription factors, and noncoding RNAs in pancreatic development and how the analysis of cells with disease-relevant mutations has provided insight into the molecular underpinnings of genetically determined diseases of the pancreas.

Inferring time series chromatin states for promoter-enhancer pairs based on Hi-C data.

BACKGROUND: Co-localized combinations of histone modifications ("chromatin states") have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points ("chromatin state trajectories") have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs. RESULTS: We present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex . CONCLUSIONS: TimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.

RNA polymerase II pausing during development.

The rapid expansion of genomics methods has enabled developmental biologists to address fundamental questions of developmental gene regulation on a genome-wide scale. These efforts have demonstrated that transcription of developmental control genes by RNA polymerase II (Pol II) is commonly regulated at the transition to productive elongation, resulting in the promoter-proximal accumulation of transcriptionally engaged but paused Pol II prior to gene induction. Here we review the mechanisms and possible functions of Pol II pausing and their implications for development.

Molecular evolution of the Yap/Yorkie proto-oncogene and elucidation of its core transcriptional program.

Throughout Metazoa, developmental processes are controlled by a surprisingly limited number of conserved signaling pathways. Precisely how these signaling cassettes were assembled in early animal evolution remains poorly understood, as do the molecular transitions that potentiated the acquisition of their myriad developmental functions. Here we analyze the molecular evolution of the proto-oncogene yes-associated protein (Yap)/Yorkie, a key effector of the Hippo signaling pathway that controls organ size in both Drosophila and mammals. Based on heterologous functional analysis of evolutionarily distant Yap/Yorkie orthologs, we demonstrate that a structurally distinct interaction interface between Yap/Yorkie and its partner TEAD/Scalloped became fixed in the eumetazoan common ancestor. We then combine transcriptional profiling of tissues expressing phylogenetically diverse forms of Yap/Yorkie with ChIP-seq validation to identify a common downstream gene expression program underlying the control of tissue growth in Drosophila. Intriguingly, a subset of the newly identified Yorkie target genes are also induced by Yap in mammalian tissues, thus revealing a conserved Yap-dependent gene expression signature likely to mediate organ size control throughout bilaterian animals. Combined, these experiments provide new mechanistic insights while revealing the ancient evolutionary history of Hippo signaling.

A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation.

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin+ cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.

A Network of microRNAs Acts to Promote Cell Cycle Exit and Differentiation of Human Pancreatic Endocrine Cells.

Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.

Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis.

BACKGROUND: Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to date, and illustrates the fundamental role of enhancers in controlling patterning, cell fate specification, and morphogenesis during development. Histone acetylation such as H3K27ac is an excellent marker for active enhancers, but it is challenging to obtain precise locations for enhancers as the highest levels of this modification flank the enhancer regions. How to best identify tissue-specific enhancers in a developmental system de novo with a minimal set of data is still unclear. RESULTS: Using DV patterning as a test system, we develop a simple and effective method to identify tissue-specific enhancers de novo. We sample a broad set of candidate enhancer regions using data on CREB-binding protein co-factor binding or ATAC-seq chromatin accessibility, and then identify those regions with significant differences in histone acetylation between tissues. This method identifies hundreds of novel DV enhancers and outperforms ChIP-seq data of relevant transcription factors when benchmarked with mRNA expression data and transgenic reporter assays. These DV enhancers allow the de novo discovery of the relevant transcription factor motifs involved in DV patterning and contain additional motifs that are evolutionarily conserved and for which the corresponding transcription factors are expressed in a DV-biased fashion. Finally, we identify novel target genes of the regulatory network, implicating morphogenesis genes as early targets of DV patterning. CONCLUSIONS: Taken together, our approach has expanded our knowledge of the DV patterning network even further and is a general method to identify enhancers in any developmental system, including mammalian development.

Poised RNA polymerase II changes over developmental time and prepares genes for future expression.

Poised RNA polymerase II (Pol II) is predominantly found at developmental control genes and is thought to allow their rapid and synchronous induction in response to extracellular signals. How the recruitment of poised RNA Pol II is regulated during development is not known. By isolating muscle tissue from Drosophila embryos at five stages of differentiation, we show that the recruitment of poised Pol II occurs at many genes de novo and this makes them permissive for future gene expression. A comparison with other tissues shows that these changes are stage specific and not tissue specific. In contrast, Polycomb group repression is tissue specific, and in combination with Pol II (the balanced state) marks genes with highly dynamic expression. This suggests that poised Pol II is temporally regulated and is held in check in a tissue-specific fashion. We compare our data with findings in mammalian embryonic stem cells and discuss a framework for predicting developmental programs on the basis of the chromatin state.