PUBLICATION

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Authors

Punnamoottil, B., Herrmann, C., Pascual Anaya, J., D'Aniello, S., Garcia-Fernàndez, J., Akalin, A., Becker, T.S., and Rinkwitz, S.

ID

ZDB-PUB-100211-31

Date

2010

Source

Developmental Biology 340(2): 269-282 (Journal)

Registered Authors

Becker, Thomas S., Punnamoottil, Beena, Rinkwitz, Silke

Keywords

Hox, cis-regulation, CNE, zebrafish, GFP, transgenesis, amphioxus, sequence conservation, cluster

MeSH Terms

Genes, Reporter
Gene Duplication
Green Fluorescent Proteins/genetics
Green Fluorescent Proteins/metabolism
Enhancer Elements, Genetic
Animals, Genetically Modified
Animals
Genes, Homeobox*
Regulatory Sequences, Nucleic Acid*
Zebrafish/embryology
Zebrafish/genetics
Zebrafish/metabolism
Evolution, Molecular
Multigene Family
DNA/genetics
DNA/isolation & purification
Embryo, Nonmammalian/metabolism
Humans
Phylogeny
Conserved Sequence/genetics
Molecular Sequence Data
Genome
Base Sequence
In Situ Hybridization
Sequence Alignment
Immunohistochemistry
Sequence Analysis, DNA

PubMed

20144609 Full text @ Dev. Biol.

Abstract

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.

Genes / Markers

Figures