Comparative Analysis of Insulin Gene Promoters: Implications for Diabetes Research

Colin W. Hay; Kevin Docherty

Disclosures

Diabetes. 2006;55(12):3201-3213. 

In This Article

Homology Between Insulin Promoters

It has been estimated from large-scale studies that the number of conserved intergenic sequences is similar to that of coding sequences,[35,36,37] and evolutionary changes in promoters together with their attendant alteration in transcriptional response to physiological and environmental demands have been documented.[38,39] This is facilitated by the fact that promoters of protein-encoding genes are laid out into functional modules,[40] allowing independent evolutionary selection of distinct characteristics of the overall transcription profile. Promoters are also considered to be more prone to genetic change than coding sequences[41,42] as the constraints typical of coding sequences are absent. In light of different regions of vertebrate genomes diverging at dissimilar rates[43] and this heterotachy being witnessed across different classes of mutation and lineages,[44] this study utilized a variety of comparative alignment and transcription factor binding site search techniques with parameters that were appropriate for the evolutionary distances between species in order to detect meaningful evolutionary conserved regions (ECRs). The computational tools included CLUSTAL W,[45] T-Coffee,[46] GraphAlign,[47] ECR Browser,[48] Mulan,[49] zPicture,[50] TRES,[51] and TRANSFAC.[52]

Calculations of homology between the different insulin promoters and the human version were carried out across the region spanning –600 to +1. The downstream 100 bp, which contains two cyclic AMP response element (CRE) sites in human (see the section on CREs below), is comprised mostly of the extremely poorly conserved first intron that unduly influences the overall results. Percentage identity plots (PIPs) comparing the human insulin promoter to those of the other species reveal that, not surprisingly, the most closely related chimpanzee and other great apes share the greatest homology to human, making discernment of conserved regions impossible. Mammals that are more distantly diverged from human display several regions of conservation within the first 350 bp upstream, which correspond to the major regulatory elements. There is a clear fall off in homology beyond –350 or –400 bp upstream from the start of transcription, which is especially apparent in rhesus macaque. While PIPs are useful for identifying ECRs, a detailed breakdown of identity values for specific regions can expose the overall relatedness of different insulin promoters ( Table 1 ). Interestingly, the degree of homology does not follow a simple direct correlation with time from divergence. For example, African green monkey and owl monkey diverged from humans 25 and 35 million years ago, but the main regulatory region of their promoters (–300 to +1) display 90 and 98% identity, respectively. Similarly, most nonprimate mammals have 65–69% identity in this region and 49–55% in the adjacent upstream 50 bp. Dog stands out in having much higher homology with 69 and 75% identity for these two regions, respectively.

Together, these results are in agreement with the opinion that vertebrate genes and immediate upstream flanks are highly constrained and, more important, confirm the accepted demarcation of the insulin promoter. There is no discernable significant homology between human and either chicken or zebrafish insulin promoters, which is in keeping with the view that most human DNA is not alignable to species separated by more than 200 million years. Likewise, there is no homology between chicken and zebrafish insulin promoters.

Computational analysis of the insulin promoters for novel evolutionary conserved sequences uncovered a single short region immediately upstream of the A3 box (see ABOXES); however, this region does not appear to contain any currently known transcription factor consensus sequences.

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