Project description:Comparison of Escherichia coli proteomics of different DNA sequence binding proteins and identification of heterologous expressed protein
Project description:Study purpose: to explore the entire spectrum of proteomic and genomic changes (amongst others) involved in diseases and in healthy/control populations. The Study is designed to discover biomarkers, develop and validate diagnostic assays, instruments and therapeutics as well as other medical research. Specifically, researchers may analyze proteins, RNA, DNA copy number changes, including large and small (1,000-100,000 kb) scale rearrangements, transcription profiles, epigenetic modifications, sequence variation, and sequence in both diseased tissue and case-matched germline DNA from Subjects.
Project description:DNA binding protein are generally thought to bind specific DNA sequences through selective interactions with DNA bases. However, it is now becoming more widely appreciated that DNA shape, which may not be specified by a unique base sequence, also contributes to site-specific binding. Here we elucidate how DNA sequence and shape confer site specificity on a genomic scale, and relate this to specificity imparted indirectly through occlusion of sequences by the in vivo environment. For simplicity, we focus on the set of General Regulatory Factors (GRFs) that do not rely on other factors for binding. They also serve a related function in organizing chromatin. Remarkably, we find that GRFs will not bind to their cognate motif if the DNA surrounding that sequence lacks a specific shape. While proper DNA sequence/shape properties tend to be restricted to promoter regions, weaker sites that are still binding-competent reside in gene bodies, but are prevented from binding by resident chromatin. Thus, site-specificity is achieved across a genome in vivo by the combined action of favorable DNA sequence and shape interactions, and occlusion by chromatin.
Project description:Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity: how these binding modes are integrated to determine select transcriptional outputs remains unresolved. We designed mutants of the MYC transcription factor bearing substitutions in residues that contact either the DNA backbone or specific bases within the consensus motif (E-box), and profiled their DNA-binding and gene-regulatory properties in cultured cells. Our data reveal that non-specific DNA binding is required for MYC to target genomic regulatory regions, and a prerequisite for sequence recognition; beyond merely stabilizing MYC onto select target loci, sequence-specific binding contributes to its precise positioning and – most unexpectedly – to transcriptional activity per se. Hence, seemingly promiscuous genome interaction profiles encompass diverse binding modalities and drive defined, sequence-dependent transcriptional responses.