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Chromosome conformation capture (3C) sequencing approaches, like Hi-C or micro-C, allow for an unbiased view of chromatin interactions. Most analysis methods rely on so-called interaction matrices, which are derived from counting read pairs in bins of fixed size. Here, we propose the Voronoi diagram, as implemented in Voronoi for chromosome conformation capture data visualization ( v3c-viz ) to visualize 3C data. The Voronoi diagram corresponds to an adaptive-binning strategy that adapts to the local densities of points. In this way, visualization of data obtained by moderate sequencing depth pinpoint many, if not most, interesting features such as high frequency contacts. The favorable visualization properties of the Voronoi diagram indicate that the Voronoi diagram as density estimator can be used to identify high frequency contacts at a resolution approaching the typical size of enhancers and promoters. v3c-viz is available at https://github.com/imbbLab/v3c-viz .

We present the software Condition-specific Regulatory Units Prediction (CRUP) to infer from epigenetic marks a list of regulatory units consisting of dynamically changing enhancers with their target genes. The workflow consists of a novel pre-trained enhancer predictor that can be reliably applied across cell types and species, solely based on histone modification ChIP-seq data. Enhancers are subsequently assigned to different conditions and correlated with gene expression to derive regulatory units. We thoroughly test and then apply CRUP to a rheumatoid arthritis model, identifying enhancer-gene pairs comprising known disease genes as well as new candidate genes.

Most facial bones, including frontal bones, are derived from neural crest cells through intramembranous ossification. Fibroblast growth factor receptor 1 (Fgfr1) plays a pivotal role in craniofacial bone development, and loss of Fgfr1 leads to cleft palate and facial cleft defects in newborn mice. However, the potential role of the Fgfr1 gene in neural crest cell-mediated craniofacial development remains unclear. To investigate the role of Fgfr1 in neural crest cells, we analyzed Wnt1-Cre;Fgfr1flox/flox mice. Our results show that specific knockout of Fgfr1 in neural crest cells induced heterotopic chondrogenesis and osteogenesis at the interface of the anterior portions of frontal bones. We observed that heterotopic bone formation continued through postnatal day 28, whereas heterotopic chondrogenesis lasted only through the embryonic period. In summary, our results indicate that loss of Fgfr1 in neural crest cells leads to heterotopic chondrogenesis and osteogenesis.

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. We report the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogénesis, br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. We found that this pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. We demonstrate that this similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes. In human and other eukaryotic cells, DNA is packaged around proteins called histones to form a structure known as chromatin. Chemical tags added to the histones alter how the DNA is packaged and the activity of the genes encoded by that DNA. For example, many active genes are packaged around histone H3 proteins that have “Lysine 4 tri-methyl” tags attached to them. Another protein that is associated with chromatin is called PHF13 and it has several roles, including repairing damaged DNA. However, it was not known whether PHF13 binds to chromatin via the chemical tags, or in another way. Ho-Ryun, Xu, Fuchs et al. used several biochemical techniques in mouse and human cells to explore how PHF13 specifically interacts with chromatin. These experiments showed that PHF13 binds specifically to DNA and to two types of methyl tags (lysine 4-tri-methyl or lysine 4-di-methyl). These chemical tags are predominantly found at active promoters as well as at a small subset of less active promoters known as bivalent promoters. PHF13 interacted with other proteins on the chromatin that are known to either drive or repress gene activity and it’s depletion affected the activity of many genes. Whether PHF13 increased or decreased gene activity depended on whether it was bound to active or bivalent promoters. The active promoters targeted by PHF13 had higher numbers of the tri-methyl tags whereas the di-methyl tags were more common on the bivalent promoters. These findings provide preliminary evidence that a protein binding to different methyl tags in the same place on histone H3 can have opposite effects on gene activity. Ho-Ryun, Xu, Fuchs et al. now intend to find out more about the other proteins that interact with PHF13 on chromatin.

Although it is widely appreciated that a typical developmental control gene is regulated by multiple enhancers, coordination of enhancer activities remains poorly understood. We propose a mechanism for such coordination in Drosophila oogenesis, when the expression of the transcription factor Broad (BR) evolves from a uniform to a two-domain pattern that prefigures the formation of two respiratory eggshell appendages. This change reflects sequential activities of two enhancers of the br gene, early and late, both of which are controlled by the epidermal growth factor receptor (EGFR) pathway. The late enhancer controls br in the appendage-producing cells, but the function of the early enhancer remained unclear. We found that the early enhancer is essential for the activity of the late enhancer and induction of eggshell appendages. This requirement can be explained by a mechanism whereby the BR protein produced by the early enhancer protects the late enhancer from EGFR-dependent repression. We illustrate this complex mechanism using a computational model that correctly predicts the wild-type dynamics of BR expression and its response to genetic perturbations.

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis -regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. We report the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogenesis. br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. We found that this pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. We demonstrate that this similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo.

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used homology directed repair (HDR)-mediated genome editing to add a single GR binding site directly upstream of the transcriptional start site of four genes. To our surprise, we found that the addition of a single GR binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two out of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Further, by introducing GR binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

Embryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.