An autoregulatory element of the murine Hox-4.2 gene.

Hox-4.2 promoter activity was assayed by transient expression assays in P19 embryonal carcinoma (EC) cells. Cotransfection of a luciferase reporter gene construct driven by Hox-4.2 upstream sequences with an expression vector for the Hox-4.2 gene product resulted in a 20-fold increase in luciferase activity. This activity was specific in that the Hox-1.6 gene product had no effect in the same assay. Mutational analysis defined a cis-acting element with enhancer function which conferred most of this increase. Activation was largely dependent on two TAAT/ATTA motifs within this 217 bp fragment and HOX-4.2 bound specifically to both of these motifs. The 217 bp element maps within a highly conserved region of the human Hox-4.2 gene (HOX4B) which has been shown to display spatial enhancer activity in mice and flies. These findings suggest a conserved autoregulatory mechanism for the control of Hox-4.2 expression.     

Primary structure and embryonic expression pattern of the mouse Hox-4.3 homeobox gene

We report the cloning, genomic localization, primary structure and developmental expression pattern of the novel mouse Hox-4.3 gene. This gene is located within the HOX-4(5) complex, at a position which classifies it as a member of the Hox-3.1 and -2.4 subfamily, the DNA and predicted protein sequences further confirmed this classification. Hox-4.3 has a primary structure characteristic of a Hox gene but, in addition, contains several monotonic stretches of amino acids, one of the 'paired'-like type. As expected from its presence and position within the complex. Hox-4.3 is developmentally expressed in structures of either mesodermal or neurectodermal origin located or derived from below a precise craniocaudal level. However, a very important offset between anteroposterior boundaries within neuroectoderm versus mesoderm derivatives is observed. Like other genes of the HOX-4(5) complex, Hox-4.3 is expressed in developing limbs and gonads, suggesting that 'cluster specificity' could be a feature of the HOX network.     

The murine genes Hox-5.1 and Hox-4.1 belong to the same HOX complex on chromosome 2

Two different loci of Antennapedia-related homeobox-containing genes have been shown to map to mouse chromosome 2: the HOX-5 complex and the Hox-4.1 gene. These independently derived loci are likely to be parts of a single gene complex, although their close linkage has not yet been demonstrated. Since cosmid walks to extend the HOX-5 cluster and to potentially link the two loci were unsuccessful, we have used large restriction fragments separated by pulsed-field gel electrophoresis to demonstrate the linkage between probes from the HOX-5 region and sequences near Hox-4.1. To further define the distance between the two linked loci, we screened a NotI jumping library with sequences near the Hox-5.1 gene to obtain a marker within the region predicted to contain Hox-4.1. The jumping endpoint lies within genomic clones from a lambda phage walk extending from the 5' end of Hox-4.1, and thus provides clear evidence of linkage between the two Hox loci. Our results demonstrate that Hox-4.1 lies approximately 35 kb downstream of the Hox-5.1 gene and that the two loci do indeed thus constitute parts of the same HOX complex.     

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.     

The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes.

This paper reports the cloning of the fourth major murine homeogene complex, HOX-5. The partial characterization of this gene cluster revealed the presence of two novel genes (Hox-5.2, Hox-5.3) located at the 5' extremity of this complex. In situ hybridization experiments showed that these two genes are transcribed in very posterior domains during embryonic and foetal development. We also show that Hox-1.6, the gene located at the 3' most position in the HOX-1 complex, has a very anterior expression boundary during early development. These results clearly support the recently proposed hypothesis that the expression of murine Antp-like homeobox-containing genes along the antero-posterior developing body axis follows a positional hierarchy which reflects their respective physical positions within the HOX clusters, similar to that which is found for the Drosophila homeotic genes. Such a structural and functional organization is likely conserved in most vertebrates. Moreover, on the basis of sequence comparisons, we propose that the ordering of homeobox-containing genes within clusters has been conserved between Drosophila and the house mouse. Thus, very different body plans might be achieved, both in insects and vertebrates, by evolutionarily conserved gene networks possibly displaying similar regulatory interactions.     

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.     

Isolation and analysis of embryonic expression of Hox-4.9, a member of the murine labial-like gene family.

Two members of the murine labial (lab) subfamily of Antennapedia-like homeobox-containing genes, Hox-1.6 and Hox-2.9, have been identified previously. Here we describe a third member genetically linked to the Hox-4 cluster on chromosome 2. This gene, designated Hox-4.9, is similar in structure to the other lab subfamily members. However, little coding sequence other than the homeobox and sequences immediately upstream of it have been conserved. By in situ hybridization analysis, Hox-4.9 mRNA is first detected at the end of the late streak stage (E7.75) in presumptive lateral and extraembryonic mesoderm. During early neurogenesis (E8.0-8.5), Hox-4.9 is detected solely in lateral mesoderm; its lack of expression in somitic mesoderm and the neural tube makes it unique among the Hox genes. By late neurogenesis and through mid-gestation (E9.0-E11.5), Hox-4.9 is no longer detected in lateral mesoderm but is found instead in a restricted region of presumed trunk neural crest and in the dermatome. These data are discussed in comparison with what is known about expression of the other members of the lab subfamily.     

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 regional localization of the murine homeobox-containing gene Hox-3.3 to mouse chromosome region 15E

A murine homeobox-containing cDNA clone has been isolated from an adult spinal cord library. Using in situ hybridization and somatic cell genetics techniques, the newly isolated homeobox gene has been mapped to mouse chromosome region 15E. Because of its chromosomal location, we called this gene locus Hox-3.3. Nucleotide sequence analysis revealed that the Hox-3.3 gene represents the murine cognate of the human homeobox gene c8. The presumptive organization of the murine Hox-3 homeobox gene cluster is discussed.     

Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.     

Murine genes related to the Drosophila AbdB homeotic genes are sequentially expressed during development of the posterior part of the body.

The cloning, characterization and developmental expression patterns of two novel murine Hox genes, Hox-4.6 and Hox-4.7, are reported. Structural data allow us to classify the four Hox-4 genes located in the most upstream (5') position in the HOX-4 complex as members of a large family of homeogenes related to the Drosophila homeotic gene Abdominal B (AbdB). It therefore appears that these vertebrate genes are derived from a selective amplification of an ancestral gene which gave rise, during evolution, to the most posterior of the insect homeotic genes so far described. In agreement with the structural colinearity, these genes have very posteriorly restricted expression profiles. In addition, their developmental expression is temporally regulated according to a cranio-caudal sequence which parallels the physical ordering of these genes along the chromosome. We discuss the phylogenetic alternative in the evolution of genetic complexity by amplifying either genes or regulatory sequences, as exemplified by this system in the mouse and Drosophila. Furthermore, the possible role of 'temporal colinearity' in the ontogeny of all coelomic (metamerized) metazoans showing a temporal anteroposterior morphogenetic progression is addressed.     

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.     

Homeobox-containing genes in the newt are organized in clusters similar to other vertebrates.

In vertebrates, the majority of homeobox (HBox) genes are found in four clusters and this structural organization is believed to be of functional importance. Many HBox genes sustain their expression in the appendages of the adult newt. To further understand their regulation, the genomic loci of four newt HBox genes (two from the human HBox (HOX)-2 complex and two from the HOX-3 complex) were analysed and compared with homologous loci in other vertebrates. Notophthalmus viridescens HBox (NvHBox) genes were selected from a lambda EMBL3 library and analysed by restriction mapping and nucleotide (nt) sequencing. The nt sequences of the NvHBox genes have a very high degree of homology (more than 90%) with the human and mouse HBox genes, HOX-3.3, HOX-3.4, HOX-2.7 and HOX-2.8. The sequences flanking the HBox are also very homologous to their human and mouse counterparts. Moreover, the size of the DNA spacer separating NvHBox-3.3 from NvHBox-3.4, and NvHBox-2.7 from NvHBox-2.8 in the newt is similar in the homologous regions of the mouse and human, despite there being a C value ten times greater in the newt genome. Finally, three of these NvHBox genes are expressed in the limbs of the adult newt.