EVX2, a human homeobox gene homologous to the even-skipped segmentation gene, is localized at the 5' end of HOX4 locus on chromosome 2.

We isolated and mapped three new human homeoboxes located on chromosome 2 upstream from the reported seven HOX4 homeobox sequences. Two of them, HOX41 and HOX4H, clearly belong to the HOX gene family, in particular to homology groups 1 and 2, and possibly represent the most 5' HOX4 homeoboxes. A third homeobox 13 kb upstream from HOX41 was identified. Sequencing data show that this is the human homolog of the murine Evx-2 homeobox. Both homeoboxes are closely related to the murine Evx-1 and to the frog Xhox-3 homeoboxes. The four genes represent vertebrate homologs of Drosophila even-skipped (eve), a segmentation gene of the pair-rule class. Human EVX2 sequences belong to an active gene because they are transcribed and properly processed in cells and tissues. We have identified for the first time a homeogene of a different class at a HOX locus. These findings are relevant to the understanding of the evolution of HOX gene clusters and their regulation.     

Isolation and mapping of EVX1, a human homeobox gene homologous to even-skipped, localized at the 5' end of HOX1 locus on chromosome 7.

We isolated and mapped the human homeobox gene EVX1. This gene encodes a protein of 407 amino acid residues containing a homeodomain closely related to the Drosophila even-skipped (eve) segmentation gene of the pair-rule class. EVX1 belongs to a small family of vertebrate eve-related homeobox genes including human EVX1 and EVX2 genes, their murine homologs, Evx 1 and Evx 2, and the frog Xhox-3 gene. We previously reported that EVX2 is localized at the 5' end of the HOX4 locus on chromosome 2. We show here that EVX1 is localized at the 5' end of the HOX1 locus on chromosome 7, 48 kb upstream from the most 5' of the eleven HOX1 genes, namely HOX1J. Both EVX genes are transcribed in an opposite orientation as compared to that of adjacent HOX genes. Human HOX1 and HOX4 complex loci appear to be both closely linked to a homeobox gene of the EVX family.     

Organization of human class I homeobox genes

We report the genomic organization of 20 human class I homeoboxes and the predicted primary sequence of the encoded homeodomains. These homeoboxes are clustered in four complex HOX loci on chromosomes 2, 7, 12, and 17. The homeoboxes of one HOX locus can be aligned to the homeoboxes of the other HOX loci so that corresponding homeodomains in all loci can share the maximal peptide sequence identity. This correspondence of individual homeoboxes in different chromosomal loci suggests the hypothesis of large-scale duplications of a single complex locus. The existence of an ancestral complex locus might have predated the divergence of vertebrates and invertebrates.     

Gene organization of murine homeobox-containing gene clusters

A chromosomal walk which links a previously described and a new homeobox to the Hox-2 murine homeobox gene cluster is described, and the nucleotide sequence of the new homeobox is presented. With these new data the Hox-2 gene cluster contains seven loci on an approximately 100-kb-long physical map. Homology comparisons reveal that a significant number of vertebrate homeoboxes are in fact analogous. We also find that the linear order of homologous homeoboxes is similar in the two murine gene complexes, Hox-1 and Hox-2, and among the human homeobox loci on chromosome 17. Conservation of the homeo-domain and the linear gene order of homeobox-containing genes in vertebrates is discussed in light of the interactions and the anteroposterior linear order of homeotic loci in insects.     

Molecular cloning, chromosomal assignment, and nucleotide sequence of the feline homeobox HOX3A.

The feline homolog to the mammalian homeobox locus, HOX3A, was isolated by screening a domestic cat genomic library with the murine Hox-3.1 probe. The nucleotide sequence similarity of the feline homeobox was 96% to human HOX3A, 94% to mouse Hox-3.1, and 94% to rat R4. The deduced amino acid sequence (homeodomain) of this feline homeobox was identical to all homeodomains of these cognate genes. Using a panel of feline x rodent somatic cell hybrids, the HOX3A locus was assigned to feline chromosome B4. Human HOX3A and mouse Hox-3.1 have been mapped previously to human chromosome 12 and mouse chromosome 15, respectively, both of which share syntenic homology to feline chromosome B4. These data demonstrate evolutionary conservation of both HOX3A gene sequences and chromosomal location during mammalian evolution.     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

Assignment of the HOX2 and HOX3 gene clusters to the bovine chromosome regions 19q17-qter and 5q14-23

The homeobox 2 (HOX2) and homeobox 3 (HOX3) clusters have been chromosomally assigned in cattle by in situ hybridization. The probes employed were a murine probe for the mapping of HOX2 to 19q17-qter and human probes for the mapping of HOX3 to 5q14-q23. These assignments confirm the chromosomal assignment of two syntenic groups, consisting of loci located on human chromosome 12 (bovine chromosome 5) and the long arm of human chromosome 17 (bovine chromosome 19).     

Classification and nomenclature of all human homeobox genes

Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.     

Structure and sequence of the human homeobox gene HOX7.

A cosmid containing the human sequence HOX7, homologous to the murine Hox-7 gene, was isolated from a genomic library, and the positions of the coding sequences were determined by hybridization. DNA sequence analysis demonstrated two exons that code for a homeodomain-containing protein of 297 amino acids. The open reading frame is interrupted by a single intron of approximately 1.6 kb, the splice donor and acceptor sites of which conform to known consensus sequences. The human HOX7 coding sequence has a very high degree of identity with the murine Hox-7 cDNA. Within the homeobox, the two sequences share 94% identity at the DNA level, all substitutions being silent. This high level of sequence similarity is not confined to the homeodomain; overall the human and murine HOX7 gene products show 80% identity at the amino acid level. Both the 5' and 3' untranslated regions also show significant similarity to the murine gene, with 79 and 70% sequence identity, respectively. The sequence upstream of the coding sequence of exon 1 contains a GC-rich putative promoter region. There is no TATA box, but a CCAAT and numerous GC boxes are present. The region encompassing the promoter region, exon 1, and the 5' region of exon 2 have a higher than expected frequency of CpG dinucleotides; numerous sites for rare-cutter restriction enzymes are present, a characteristic of HTF islands.     

HOX11L1, a gene involved in peripheral nervous system development, maps to human chromosome 2p13.1-->p12 and mouse chromosome 6C3-D1.

HOX11L1 is a homeobox gene involved in peripheral nervous system development as confirmed by knockout mice exhibiting megacolon with enteric ganglia, a phenotype associated in human with Intestinal Neuronal Dysplasia (IND). Using FISH and radiation hybrids we have localized HOX11L1 to human chromosome 2p13.1-->p12, in a 14-cR interval between WI-5987 (D2S2088) and GCT1B4 (D2S2497), and confirmed the synteny between mouse 6C3-D1 and human 2p13.1-->p12 chromosomes by mapping an EST cDNA clone corresponding to mouse HOX11L1 (Tlx2).     

Cloning and analysis of three new homeobox genes from the nematode Caenorhabditis elegans

Three homeobox-containing genes from the nematode Caenorhabditis elegans are described. Two of them (ceh-11 and ceh-12) were isolated from a genomic library by hybridization at low stringency with the Ascaris lumbricoides homeobox AHB-1. The first clone contains a homeobox defining a new class of homeoboxes (ceh-11). This gene maps on the third chromosome of C. elegans, at the same locus as egl-5, a gene already known to be essential for the determination of specific neurons. In the second clone, sequence analysis revealed the existence of the third helix of a putative homeobox (ceh-12) which is interrupted by an intron located upstream of the codon for the amino acid 45 of the homeodomain. Using the ceh-11 homeobox as a probe, a third homeobox (ceh-13) was isolated from a cDNA library. As ceh-13 belongs to the labial class of homeoboxes, we conclude that, at the time when the nematode lineage diverged from the myriapod-insect and the vertebrate lineages, the duplication which led to the Antp and the labial families of homeoboxes had already taken place.     

Sequence analysis of the homeobox-containing exon of the murine Hox-4.3 homeogene.

A homeobox-containing gene * was detected by Southern analysis of a cosmid spanning a region of the murine HOX-4 complex between Hox-4.4 (Hox-5.2) and Hox-4.2 (Hox-5.1) with a probe derived from the Hox-4.2 homeobox. The sequence of a cross-hybridizing region revealed an open reading frame encoding an Antennapedia (Antp) class homeodomain highly homologous to the products of human HOX4C (Hox-5.4/HOX4E), mouse Hox-3.1 and Hox-2.4. This, together with strong conservation of sequences 3' to the homoebox, indicates that we have cloned the murine Hox-4.3 gene. No other homeobox sequences were detected in this screen suggesting that the HOX-4 complex lacks paralogous genes represented in the equivalent regions of other HOX loci.     

Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells.

We studied the expression of 38 human homeobox genes belonging to the four HOX complex loci in embryonal carcinoma (EC) cells induced to differentiate by culturing them in a medium containing retinoic acid (RA). Genes located at the 3' end of each one of the four HOX loci are activated by RA in a sequential order colinear with their 3' to 5' arrangement in the cluster: 3' HOX genes respond early to the drug while upstream genes respond progressively later. Among the genes located at the 5' end of HOX loci RNase protection analysis reveals that one HOX3 gene and four HOX4 genes are weakly expressed in EC stem cells and downregulated upon treatment with 10(-5) M RA. While activation of early responding genes does not require continuous protein synthesis, the observed timing and polarity of gene activation is disrupted in the absence of protein synthesis.     

Gene fishing: the use of a simple protocol to isolate multiple homeodomain classes from diverse invertebrate taxa

Abstract Comparison of relevant gene sequence and functional data is central to understanding the evolution of metazoan development. The conservation of portions of regulatory genes, such as homeoboxes, allows for the design of PCR-based sequence isolation and amplification strategies. Here we describe a simple protocol that uses a degenerate primer pair to isolate a variety of homeobox-containing genes from diverse metazoan taxa. In a nonexhaustive survey, we have isolated 28 gene sequence fragments from 15 taxa, representing eight invertebrate phyla (Mollusca, Echiura, Annelida, Platyhelminth, Acoela, Ctenophora, Cnidaria, and Porifera). Based on BLAST and parsimony analyses, these gene fragments affiliate with several gene groups (PAIRED-like, HOX, and ParaHOX) and several single genes, including pancreas/duodenum homeoboxes (Pdx), empty spiracles (ems/Emx), gastrulation brain homeoboxes (Gbx), hematopoietically expressed homeoboxes (HEX), brain specific homeobox (bsh/BarH1/BarH2), NK-1 (NK-1/s59/slouch), and ladybird (Lbl/Lbe/Lbx). In several cases, these fragments represent the first reported orthologue for the phylum or superphyletic group (i.e., Lophotrochozoa).     

Structural analysis of the human HOX4A homeobox gene.

The HOX4A gene, one of a cluster of homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with the HOX4B cDNA probe. The amino acid sequence was predicted according to the conceptual translation of 13 homology groups of human HOX genes (1). The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. The HOX4A protein predicted from the nucleotide sequence of the HOX4A gene is comprised of 416 amino acid residues. Comparison of the predicted HOX4A protein with the HOX2G protein revealed three regions of sequence similarity: an N-terminal octapeptide, a hexapeptide (pre-box) upstream of the homeodomain, and the homeodomain at the C-terminus.