Knowledge integration for analyzing ChIP-seq

To capture the genomic profiles for histone modification, chromatin immunoprecipitation (ChIP) is combined with next generation sequencing, which is called ChIP-seq. However, enriched regions generated from the ChIP-seq data are only evaluated on the limited knowledge acquired from manually examining the relevant biological literature. This paper proposes a novel framework, which integrates multiple knowledge sources such as biological literature, Gene Ontology, and microarray data. In order to precisely analyze ChIP-seq data for histone modification, knowledge integration is based on a unified probabilistic model. The model is employed to re-rank the enriched regions generated from peak finding algorithms. Through filtering the reranked enriched regions using some predefined threshold, more reliable and precise results could be generated. The combination of the multiple knowledge sources with the peaking finding algorithm produces a new paradigm for ChIP-seq data analysis. © (2012) Trans Tech Publications, Switzerland.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxiadependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

On-chip immunoprecipitation for protein purification

Immunoprecipitation (IP) is one of the most widely used and selective techniques for protein purification. Here, a miniaturised, polymer-supported immunoprecipitation (µIP) method for the on-chip purification of proteins from complex mixtures is described. A 4 µl PDMS column functionalised with covalently bound antibodies was created and all critical aspects of the µIP protocol (antibody immobilisation, blocking of potential non-specific adsorption sites, sample incubation and washing conditions) were assessed and optimised. The optimised µIP method was used to obtain purified fractions of affinity-tagged protein from a bacterial lysate.

Efficient purification of chromatin architectural proteins:histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells. This journal is © 2012 The Royal Society of Chemistry.