Ho-Ryun Chung

Histone modification levels are predictive for gene expression

Histones are frequently decorated with covalent modifications. These histone modifications are thought to be involved in various chromatin-dependent processes including transcription. To elucidate the relationship between histone modifications and transcription, we derived quantitative models to predict the expression level of genes from histone modification levels. We found that histone modification levels and gene expression are very well correlated. Moreover, we show that only a small number of histone modifications are necessary to accurately predict gene expression. We show that different sets of histone modifications are necessary to predict gene expression driven by high CpG content promoters (HCPs) or low CpG content promoters (LCPs). Quantitative models involving H3K4me3 and H3K79me1 are the most predictive of the expression levels in LCPs, whereas HCPs require H3K27ac and H4K20me1. Finally, we show that the connections between histone modifications and gene expression seem to be general, as we were able to predict gene expression levels of one cell type using a model trained on another one.

The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.

ChIP-exo signal associated with DNA-binding motifs provides insight into the genomic binding of the glucocorticoid receptor and cooperating transcription factors

The classical DNA recognition sequence of the glucocorticoid receptor (GR) appears to be present at only a fraction of bound genomic regions. To identify sequences responsible for recruitment of this transcription factor (TF) to individual loci, we turned to the high-resolution ChIP-exo approach. We exploited this signal by determining footprint profiles of TF binding at single-base-pair resolution using ExoProfiler, a computational pipeline based on DNA binding motifs. When applied to our GR and the few available public ChIP-exo data sets, we find that ChIP-exo footprints are protein- and recognition sequence-specific signatures of genomic TF association. Furthermore, we show that ChIP-exo captures information about TFs other than the one directly targeted by the antibody in the ChIP procedure. Consequently, the shape of the ChIP-exo footprint can be used to discriminate between direct and indirect (tethering to other DNA-bound proteins) DNA association of GR. Together, our findings indicate that the absence of classical recognition sequences can be explained by direct GR binding to a broader spectrum of sequences than previously known, either as a homodimer or as a heterodimer binding together with a member of the ETS or TEAD families of TFs, or alternatively by indirect recruitment via FOX or STAT proteins. ChIP-exo footprints also bring structural insights and locate DNA:protein cross-link points that are compatible with crystal structures of the studied TFs. Overall, our generically applicable footprint-based approach uncovers new structural and functional insights into the diverse ways of genomic cooperation and association of TFs.

Deletions of chromosomal regulatory boundaries are associated with congenital disease

BackgroundRecent data from genome-wide chromosome conformation capture analysis indicate that the human genome is divided into conserved megabase-sized self-interacting regions called topological domains. These topological domains form the regulatory backbone of the genome and are separated by regulatory boundary elements or barriers. Copy-number variations can potentially alter the topological domain architecture by deleting or duplicating the barriers and thereby allowing enhancers from neighboring domains to ectopically activate genes causing misexpression and disease, a mutational mechanism that has recently been termed enhancer adoption.ResultsWe use the Human Phenotype Ontology database to relate the phenotypes of 922 deletion cases recorded in the DECIPHER database to monogenic diseases associated with genes in or adjacent to the deletions. We identify combinations of tissue-specific enhancers and genes adjacent to the deletion and associated with phenotypes in the corresponding tissue, whereby the phenotype matched that observed in the deletion. We compare this computationally with a gene-dosage pathomechanism that attempts to explain the deletion phenotype based on haploinsufficiency of genes located within the deletions. Up to 11.8% of the deletions could be best explained by enhancer adoption or a combination of enhancer adoption and gene-dosage effects.ConclusionsOur results suggest that enhancer adoption caused by deletions of regulatory boundaries may contribute to a substantial minority of copy-number variation phenotypes and should thus be taken into account in their medical interpretation.

Antagonistic action of Bicoid and the repressor Capicua determines the spatial limits of Drosophila head gene expression domains.

Bicoid (Bcd) is the anterior determinant in Drosophila. Accordingly, loss of Bcd causes loss of head and thorax and their replacement with posterior structures. bcd mRNA is maternally deposited at the anterior pole and Bcd forms an anterior-to-posterior (AP) concentration gradient. The expression of a series of zygotic head genes is thought to be differentially regulated by distinct threshold concentrations of the Bcd gradient. Thereby Bcd functions as a morphogen, instructing fields of cells to take on specific fates. Here, we show that spatial limits of anterior genes are also set in the absence of a Bcd gradient and depend on factors of the maternal terminal system. The receptor tyrosine kinase Torso (Tor), a key component of this system, is active in the pole regions of the embryo. Its activity downregulates the maternally deposited repressor Capicua (Cic), leaving high Cic activity in the central regions and decreasingly lower Cic activities toward the poles. We show that the positions of posterior boundaries of Bcd target genes are dependent not only on Bcd, but also on Tor-mediated Cic activity. The results indicate that Cic can mediate repression through distinct binding sites within a Bcd responsive enhancer and that gene activation by Bcd is antagonized by Cic. The activating and repressive effects of Bcd and Cic, respectively, are integrated by the Bcd target gene enhancer. We conclude that the spatial domains of head gene expression are determined by Bcd in concert with Tor-dependent repressors.

Sequence-dependent Nucleosome Positioning

A new methodology is proposed to estimate 3D displacement fields from pairs of images obtained from X-Ray Computed Micro Tomography (XCMT). Contrary to local approaches, a global approach is followed herein that evaluates {\em continuous} displacement fields. Although any displacement basis could be considered, the procedure is specialized to finite element shape functions. The method is illustrated with the analysis of a compression test on a polypropylene solid foam (independently studied in a companion paper). A good stability of the measured displacement field is obtained for cubic element sizes ranging from 16 voxels to 6 voxels.

Methylation and deamination of CpGs generate p53-binding sites on a genomic scale

The formation of transcription-factor-binding sites is an important evolutionary process. Here, we show that methylation and deamination of CpG dinucleotides generate in vivo p53-binding sites in numerous Alu elements and in non-repetitive DNA in a species-specific manner. In light of this, we propose that the deamination of methylated CpGs constitutes a universal mechanism for de novo generation of various transcription-factor-binding sites in Alus.

Genomic expansion and clustering of ZAD-containing C2H2 zinc-finger genes in Drosophila

C2H2 zinc‐finger proteins (ZFPs) constitute the largest family of nucleic acid binding factors in higher eukaryotes. In silico analysis identified a total of 326 putative ZFP genes in the Drosophila genome, corresponding to ∼2.3% of the annotated genes. Approximately 29% of the Drosophila ZFPs are evolutionary conserved in humans and/or Caenorhabditis elegans. In addition, ∼28% of the ZFPs contain an N‐terminal zinc‐finger‐associated C4DM domain (ZAD) consisting of ∼75 amino acid residues. The ZAD is restricted to ZFPs of dipteran and closely related insects. The evolutionary restriction, an expansion of ZAD‐containing ZFP genes in the Drosophila genome and their clustering at few chromosomal sites are features reminiscent of vertebrate KRAB‐ZFPs. ZADs are likely to represent protein–protein interaction domains. We propose that ZAD‐containing ZFP genes participate in transcriptional regulation either directly or through site‐specific modification and/or regulation of chromatin.

Gain-of-Function Screen for Genes That Affect Drosophila Muscle Pattern Formation

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.

Conserved selenoprotein synthesis is not critical for oxidative stress defence and the lifespan of Drosophila

Selenoprotein synthesis is conserved from bacteria to man. It involves the differential decoding of the UGA stop codon as selenocysteine. The proteomes of both prokaryotes and eukaryotes, with the exception of yeast, contain only few selenoproteins. This low number is explained by a counterselection of readily oxidized selenocysteine after the introduction of oxygen into the atmosphere and the need to conserve selenoenzymes that control redox homeostasis of cells. Lack of selenoprotein synthesis in vertebrates impairs the oxidative stress defence and causes lethality. Here we show that Drosophila mutants that lack the translation elongation factor SelB/eEFsec fail to decode the UGA codon as selenocysteine, but they are viable and fertile. Oxidative stress responses and the lifespan of these flies are not affected. Protecting cells from oxidative stress can therefore not account for the selection pressure that conserves selenoprotein biosynthesis during the course of evolution.