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- anterior position along embryo posterior -

- anterior position along embryo posterior -

FIGURE 18-22 Regulation of eve stripe 2. (a) The 500 bp enhancer contains a total of twelve binding Sites for the Bicoid, Hunchback, Krüppel, and Giant proteins The distribu tons of these regulatory proteins in the tarty Drosophila embryo is summarized in the diagram shown in (b). There fire high levels of the Bicoid and Hunchback proteins in the cefe that express eve stripe 2. The borders of the sir,pes are formed by the Giant and Krüppel repressors. (Giant is expressed in anterior and posterior regions. Only the anterior pattern is shown; the posterior pattern. which >s regulated by t iur dlbiick, ¡s rot shown.) (Source: Adapted from Alberts B. et aä. 2002. Meieruhr biology of the cell, 4th edition (a) p 409, f7-55, (b) p 4)0,17 56. Copyright © 2002. Reproduced by permission of Routiedge/Taylor 8 Francis Books, Inc.)

Krüppel mediates transcriptional repression through two distinct mechanisms. One is competition, which is similar to the strategy employed by many prokaryotic repressors (discussed in Chapter 16). There are ihree Krüppel binding sites in the stripe 2 enhancer (Figure 16-23), Two of these sites directly overlap Bicoid activator sites, and so it appears that the binding of Krüppel to these sites precludes the binding of the activator. The third Krüppel repressor

Box 18-6 Bioinformatics Methods for the Identification of Complex Enhancers

A variety of computer programs have been developed to identify regulatory DNAs within genomes that have been completely sequenced, known as "whole-genome assemblies" These programs take advantage of the fact thai regulatory DNAs contain dense clusters of DMA-binding sites. For example, the ew stripe 2 enhancer is 500 bp and contains 12 separate binding sites for four different regulatory proteins: Bicoid, Hunchback, Krüppel, and Kmrps (see Figure 16-22). Thus, there is more than one binding site per 50 bp over the length of the enhancer. This density of binding sites ts typical of enhancers that direct localized patterns of gene expression in the early Drosophila embryo.

As we have discussed in this chapter, a number of regulatory proteins have been implicated in the regulation of pair-rule stripes of gene expresston in the Drosophila embryo. These include Bicoid, Hunchback, Krüppel, Giant, and Knirps. Unfortunately, an irtsuftiaera number of Giant binding sites have been identified to determine the range of sequences that this protein is likely to recognize. In contrast, there is extensive DNA binding information for the other four regulatory proteins, as well as for a home domain protein called Caudal, which is expressed in a broad gradient in the posterior half of the embryo where it functions as a transcriptional activator.

Bicoid, Caudal, Hunchback, Kruppel, and Knirps each bind DNA as a monomer and recognize relatively simple sequences that are present in extremely high copy number in the Drosophila genome. Bicoid, for example, recognizes a simple sequence that contains an ATTA-core motif with a few flanking G/C residues. On average, there is a potential Bicoid binding site every 1 kb tn the Drosophila genome. Therefore, the use of Bicoid binding sites for identifying segmentation enhancers would be futile because there are more than 100,000 such sites in the genome (nearly ten sites per gene). However, dustcring Bicoid binding sites, together with the binding sites of regulatory proteins that work together with Bicoid, provides a powerful fitter for elime natjng fortuitous binding sites (or "noise*)

Consider a 1 Mb region encompassing tlie ere locus (Box 18-6 Figure 1). There are thousands of Bicoid, Cauda!, Hunchback, Kruppel, and Knirps binding sites in this interval (Box 18-6 Figure la). There are, however, only three clusters that contain at least 13 binding sites in a window of 700 bp or less (a density of nearly one binding site per 50 bp, (Box 18-6 Figure lb). Remarkably, these three clusters map in the 5' and 3' regulatory region of the eve gene. One duster corresponds to the eve stripe 3/7 enhancer, another cluster coincides with the eve stripe 2 enhancer, and the third duster is located in the 3' regulatory region and coincides with the eve stripe 4/6 enhancer (Box 18-6 Figure J).

Clustering of DNA-bmding sites has proven to be a valuable tool for identifying enhancers in the Drosophiia genome. However, the current computer programs are not 100% accurate. In the best cases, only approximately onethird of the identified clusters correspond to actual enhancers, ft is conceivable that a higher hit rate will be obtained by placing spatial constraints on binding sites rather than relying solely on simple clustering of sites. We saw in Chapter 17, for example, that the interferon enhanceosorne contains binding sites with fixed spaong, including helical phasing between neighboring sites.

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