Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters

Abstract

Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aalpha or Abeta duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.

Figure 1

Evolutionary relationships among the species included in this work. The divergence date between the lineage leading to Chondrichthyes (to which Heterodontus, the horn shark, belongs) and that leading to the clade of all other taxa on this tree is about 500 millions years. Actinopterygii (the ray-finned fishes) and Sarcopterygii (the tetrapods) diverged about 450 million years ago. Teleosts radiated more than 200 million years ago. The divergence between human and mouse is dated to about 80 million years (Pough et al. 1999). Horn shark, mouse, and human have a single HoxA cluster, while all fishes examined so far have two (see text for details). Among fishes, independent gene losses took place in zebrafish and pufferfish relative to tilapia. Solid boxes represent individual genes. Duplicated clusters are designated as α or β. Pseudogenes are marked with a cross. Question marks represent genomic regions that are not yet characterized.

Figure 2

Relative sizes of HoxA clusters. Boxes represent individual genes. The duplicated α and β clusters are shown only for zebrafish. The alignable portion of the pseudogenes HoxA7α of pufferfish, HoxA2α and HoxA10α of zebrafish are shown as well.

Figure 3

Relationship between genome size and length of the portion HoxA4 to HoxA9 of HoxA clusters. The length of HoxA clusters is correlated (P = 0.06) with genome size expressed as C value. The HoxAα cluster lengths are shown. To be able to include also striped bass (HoxA cluster sequence is available only from HoxA4α to HoxA10α) and zebrafish (HoxAα cluster lacks HoxA10α) into the analysis, only the length of the HoxA4 to HoxA9 portion of the cluster is shown.

Figure 4

Pip output of the comparison of tilapia HoxAα, striped bass HoxAα, pufferfish HoxAα, zebrafish HoxAα and Aβ, horn shark HoxA, human HoxA, and mouse HoxA clusters. The tilapia sequence has been used as reference sequence. Kilobase (kb) markings are based on the tilapia sequence. Blue background indicates coding regions, yellow indicates introns, red indicates conserved noncoding sequences (CNSs) previously described in literature, and the green background indicates heretofore undescribed CNSs. Horizontal arrows indicate the direction of transcription, tall black boxes showexons, short open boxes indicate a CpG/GpC ratio between 0.6 and 0.75, and short gray boxes indicate a CpG/GpC ratio over 0.75. Interspersed repeat elements are shown as triangles (e.g., in position 91 kb).

Figure 5

Total lengths of CNSs for each intergenic region. The intergenic regions located 3′ in the cluster are better conserved than those between genes located 5′ in the cluster. The graph shows the number of conserved bases (CNS as defined in text) per intergenic region. There is a significant relationship between the number of conserved bases per intergenic region and the position of the region in the cluster (P = 0.007).

Figure 6

Alignment of known RARE elements. The alignment shows the RARE element described as “box c” and “box d” (Odenwald et al. 1989) immediately upstream of the HoxA5 genes. In zebrafish, the RARE element is present only in HoxAα cluster (indicated as zebrafish α).

Figure 7

Alignment of known regulatory elements. (A) Sequence of Krx20 binding sites in different species. Krox20 binding sites are involved in Hox2 regulation and they are conserved in HoxA and B clusters from human, mouse, pufferfish, and HoxA from tilapia. Both Krx20 and the “box a” are widely conserved. The degree of identity is 67% among the species in this comparison. (B) Alignment of sequences of the “box a” motif.