A Chemical Genetic Method for Monitoring Genome-Wide Dynamics of O-GlcNAc Turnover on Chromatin-Associated Proteins.

Advances in DNA sequencing are enabling new experimental modalities for studying chromatin. One emerging area is to use high-throughput DNA sequencing to monitor dynamic changes occurring to chromatin. O-Linked N-acetylglucosamine (O-GlcNAc) is a reversible protein modification found on many chromatin-associated proteins. The mechanisms by which O-GlcNAc regulates gene transcription are of high interest. Here we use DNA precipitation methods to enable monitoring time-dependent turnover of O-GlcNAc modified proteins associated with chromatin. Using an antibody-free chemical reporter strategy to map O-GlcNAc to the genome, we performed time course metabolic feeding experiments with wild-type Drosophila larvae alongside larvae lacking O-GlcNAc hydrolase (OGA), which are accordingly unable to remove O-GlcNAc. Analysis of resulting next-generation DNA sequencing data revealed that O-GlcNAc on chromatin-associated proteins at most genomic loci is processed with a half-life in hours. Notably, loss of OGA only increases this half-life by ∼3-fold. Interestingly, a small set of genomic loci are particularly sensitive to loss of OGA. In addition to these observations and new strategies to permit monitoring turnover of O-GlcNAc on chromatin, we also detail methods for coded blinding of samples alongside new normalization strategies to enable time-resolved, genome-wide analyses using chemical genetic methods. We envision these general methods will be applicable to diverse protein and nucleic acid modifications.

Software for rapid time dependent ChIP-sequencing analysis (TDCA).

BACKGROUND: Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and associated methods are widely used to define the genome wide distribution of chromatin associated proteins, post-translational epigenetic marks, and modifications found on DNA bases. An area of emerging interest is to study time dependent changes in the distribution of such proteins and marks by using serial ChIP-seq experiments performed in a time resolved manner. Despite such time resolved studies becoming increasingly common, software to facilitate analysis of such data in a robust automated manner is limited. RESULTS: We have designed software called Time-Dependent ChIP-Sequencing Analyser (TDCA), which is the first program to automate analysis of time-dependent ChIP-seq data by fitting to sigmoidal curves. We provide users with guidance for experimental design of TDCA for modeling of time course (TC) ChIP-seq data using two simulated data sets. Furthermore, we demonstrate that this fitting strategy is widely applicable by showing that automated analysis of three previously published TC data sets accurately recapitulates key findings reported in these studies. Using each of these data sets, we highlight how biologically relevant findings can be readily obtained by exploiting TDCA to yield intuitive parameters that describe behavior at either a single locus or sets of loci. TDCA enables customizable analysis of user input aligned DNA sequencing data, coupled with graphical outputs in the form of publication-ready figures that describe behavior at either individual loci or sets of loci sharing common traits defined by the user. TDCA accepts sequencing data as standard binary alignment map (BAM) files and loci of interest in browser extensible data (BED) file format. CONCLUSIONS: TDCA accurately models the number of sequencing reads, or coverage, at loci from TC ChIP-seq studies or conceptually related TC sequencing experiments. TC experiments are reduced to intuitive parametric values that facilitate biologically relevant data analysis, and the uncovering of variations in the time-dependent behavior of chromatin. TDCA automates the analysis of TC ChIP-seq experiments, permitting researchers to easily obtain raw and modeled data for specific loci or groups of loci with similar behavior while also enhancing consistency of data analysis of TC data within the genomics field.

Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster.

N-Acetylglucosamine ß-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

Evolutionarily emerged G tracts between the polypyrimidine tract and 3' AG are splicing silencers enriched in genes involved in cancer.

BACKGROUND: The 3' splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position "N" can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes. RESULTS: Taking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3' splice sites of annotated introns. This identified several thousand such 3'SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3' splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3'SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer. CONCLUSION: We call these elements together with CaRRE1 regulatory RNA elements between the Py and 3'AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions.