P = perfect

S = strong (1-2 changes) W = weak (3 or more changes) A = absent

Figure 8.4

Evolutionary dynamics of transcription factor-binding site evolution in a conserved c/s-regulatory element

(a) Binding sites for the Kr├╝ppel (Kr), Giant (Gt), Bicoid (Bcd), and Hunchback (Hb) proteins in the 670-bp D. melanogastereve stripe 2 c/s-regulatory element are shown. (b) The conserved binding sites in five different Drosophila species are tabulated. The degree of sequence conservation within each site is indicated (P, S, W, A). Note that certain sites such as Kr5, Kr6, and Bcd5 are perfectly conserved, whereas other sites such as Hb1 and Bcd3 are absent from certain species.

Sources: Data from Stanojevic D, Small S, Levine M, et al. Regulation of a segmentation stripe by overlapping activators and repressors in the Drosophila embryo. Science 1991; 254: 1385-1387; Ludwig MZ, Patel NH, Kreitman M. Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change. Development 1998; 125: 949-958.

dynamics of sequence turnover in the element. Because multiple sites exist for each regulatory protein, as well as both positive and negative inputs, slightly deleterious substitutions may accumulate if compensatory mutations are also occurring that maintain the overall function of the element.

The sequence variation seen in the eve elements is similar to those observed in other complex elements. A survey of eight cis-regulatory elements for early developmental genes in several Drosophila species revealed that among 104 transcription factor binding sites found in D. melanogaster, approximately 30-40% were not present in orthologous elements in D. virilus. The rate of turnover of binding sites was approximately 1% (gain or loss) of sites per million years.

Similarly, a survey of 51 human cis-regulatory elements found that approximately 32-40% of the human transcription factor binding sites are not present in the homologous elements of rodents, also indicating a high turnover rate in mammalian cis-regulatory elements. Together, these data suggest that binding site turnover is a general feature of cis-regulatory sequence evolution, and that the balancing effects of mutation and selection are likely to apply, in general, to the functional conservation of cis-regulatory elements.

Indeed, in some cases, it appears that cis-regulatory element function can be maintained despite nearly complete turnover of transcription factor binding sites. The regulation of the Endo16 of the sea urchin Strongylocentrotus purpuratus has been analyzed in extensive detail, revealing a modular architecture of 5' cis-regulatory elements (comprising seven modules named A-G). In Lytechinus variegatus, a species some 35 million years diverged from S. pupuratus, only the A module is conserved, the remaining elements cannot be identified by sequence similarity. Yet, the pattern of transcription of the Endo16 gene during development is largely conserved. Experimental evidence suggests that the set of transcription factors that interact with the upstream elements of Endo16 has also diverged. These findings suggest that orthologous genes expressed in homologous positions in two well-diverged species may not be regulated by homologous cis-regulatory sequences or transcription factors. This demonstrates both the remarkable potential fluidity of cis-regulatory DNA sequences, as well as the challenge of understanding the mechanisms of regulatory divergence between genes of long-diverged taxa.

Co-evolution of cis-regulatory elements and the transcriptional factors that bind them

The high rate of transcription factor binding site turnover suggests that compensation is not restricted to changes in cis-regulatory elements. Another means of compensation is the co-evolution of transcription factor specificities. Detailed examination of the evolution of elements regulated by the Bicoid protein has uncovered evidence for significant functional sequence evolution in the Bicoid protein in Diptera, apparently driven by the continuous process of enhancer restructuring.

One of the best characterized targets of the Bicoid protein is the P2 promoter of the hunchback gene (see Chapter 3, pp. 64-65). There are seven Bcd binding sites upstream of the hb P2 promoter in D. melanogaster (Fig. 8.5). The number, spacing, sequence, and orientation of Bcd sites differs in other Dipterans (Fig. 8.5), as does the sequence of the Bcd home-odomain. The differences in the hb enhancer and Bcd protein are of functional significance, because components combined from different species fail to interact as effectively as those

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