Chromosome Wide Prediction of Human Limb Specific Cis-Regulatory Modules Employing Computational and Statistical Approaches

Abstract

Gene function is defined not only by its product but also by its specific pattern of expression, which needs to be adjusted to a variety of conditions (developmental timing, cell type, environmental factors). A large number of cis-acting elements contribute to this regulation both transcriptionally and post‐ transcriptionally. Elements that act on gene regulation at the transcriptional level fall in different functional categories, including enhancers, silencers, insulators and other architectural elements. Because of their decisive influence on transcription, enhancers appear as major contributors in regulation of gene expression. Giving their importance for gene expression, considerable efforts have been devoted to identify these cis‐regulatory elements. Recruitment of sequence‐specific transcription factors (TFs) is an essential hallmark of enhancers. Transcription factors (TFs) are thought to act in a combinatorial way, by competing and collaborating to regulate common target genes. Enhancers possess binding sites for these transcription factors in same combinatorial way and inherit the spatiotemporal specificity too. Due to lack of proper language, mode of action and degeneracy of binding sites, they are very hard to investigate, however different techniques both experimental and computational are devised to understand these cis-regulatory elements. Keeping the transcription factor cooperativity as rationale, a pipeline to predict Human limb specific enhancers was devised. Transcription factors can have one or more binding sites which means that TFBSs are degenerate. Utilizing this we endeavored to characterize limb specific transcription factor code through extensive literature survey and statistical analysis. The devised pipeline effectively predicted putative 844 limb specific enhancers in non-coding, non-repetitive segments of human chromosome 3, 4 and 7 respectively. Predicted enhancers were then validated by means of experimentally verified histone modification marks, DNase hypersensitive sites and limb related clinical variants to assure their significance and reliability. Conservation analysis was also performed for these predicted enhancers which revealed that most of these enhancers are conserved till mammals, while a small number goes down to amphibians and fishes. Cost and time proficiency, literature evidence and validation make this strategy of prediction a comparable substitute of already available computational and experimental approaches used to identify cis-regulatory modules