Human Homeobox Research Articles

Genomic organization and chromosome mapping of the human homeobox gene HHEX

In the present study, we report the genomic reconstruction of the human homeobox-containing gene HHEX by the use of the data available in public databases. This analysis allowed characterization of the gene organization showing that it is very similar to the mouse gene. Moreover the gene was mapped using FISH to 10q24.

Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects.

Inherited defects of skull ossification often manifest as symmetric parietal foramina (PFM; MIM 168500). We previously identified mutations of MSX2 in non-syndromic PFM and demonstrated genetic heterogeneity. Deletions of 11p11-p12 (proximal 11p deletion syndrome, P11pDS; MIM 601224) are characterized by multiple exostoses, attributable to haploinsufficiency of EXT2 and PFM. Here we identify ALX4, which encodes a paired-related homeodomain transcription factor, as the PFM disease gene in P11pDS.

A novel intronic polymorphism (intron 2 +130 (CT)n) in the human homeobox gene HOXB3.

Human MutationVolume 17, Issue 2 p. 156-157 Mutation and Polymorphism Report A novel intronic polymorphism (intron 2 +130 (CT)n) in the human homeobox gene HOXB3 † Susan A. Copeland-Yates, Susan A. Copeland-YatesSearch for more papers by this authorRon C. Michaelis, Ron C. MichaelisSearch for more papers by this author Susan A. Copeland-Yates, Susan A. Copeland-YatesSearch for more papers by this authorRon C. Michaelis, Ron C. MichaelisSearch for more papers by this author First published: 23 January 2001 https://doi.org/10.1002/1098-1004(200102)17:2<156::AID-HUMU20>3.0.CO;2-N † Communicated by Mark H. Paalman ‡ Online Citation: Human Mutation, Mutation and Polymorphism Report #210 (2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/mpr210.pdf § Acknowledgments:: We thank Dr. K.K. Kidd and Mr. William Speed for making available the genomic sequence of this region of HOXB3. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume17, Issue2February 2001Pages 156-157 RelatedInformation

Haploinsufficiency of ALX4 as a Potential Cause of Parietal Foramina in the 11p11.2 Contiguous Gene–Deletion Syndrome

Heterozygous mutations in MSX2 are responsible for an autosomal dominant form of parietal foramina (PFM). PFM are oval defects of the parietal bones that are also a characteristic feature of a contiguous gene-deletion syndrome caused by a proximal deletion in the short arm of chromosome 11 (Potocki-Shaffer syndrome). We have identified a human bacterial artificial chromosome (BAC) clone mapping to chromosome 11, containing a region homologous to the human homeobox gene MSX2. Further sequence analysis demonstrated that the human orthologue (ALX4) of the mouse Aristaless-like 4 gene (Alx4) is contained within this 11p clone. We used FISH to test for the presence-or for the heterozygous deletion-of this clone in two patients with the 11p11.2-deletion syndrome and showed that this clone is deleted in these patients. ALX4 and Alx4 were shown to be expressed in bone and to be absent from all other tissues tested. The involvement of Alx4 in murine skull development, its bone-specific expression pattern, the fact that Alx4 is a dosage-sensitive gene in mice, and the localization of a human genomic clone containing ALX4 to 11p11.2, with hemizygosity in patients with deletion of 11p11.2 who have biparietal foramina, support the contention that ALX4 is a candidate gene for the PFM in the 11p11.2-deletion syndrome.

Cloning and chromosomal localization of the human BARX2 homeobox protein gene

The human BARX2 gene encodes a homeodomain-containing protein of 254 amino acids, which binds optimally to the DNA consensus sequence YYTAATGRTTTTY. BARX2 is highly expressed in adult salivary gland and is expressed at lower levels in other tissues, including mammary gland, kidney, and placenta. The BARX2 gene consists of four exons, and is located on human chromosome 11q25. This chromosomal location is within the minimal deletion region for Jacobsen syndrome, a syndrome including craniosynostosis and other developmental abnormalities. This chromosomal location, along with the reported expression of murine barx2 in craniofacial development, suggests that BARX2 may be causally involved in the craniofacial abnormalities in Jacobsen syndrome.

Expression of human cardiac-specific homeobox protein in Escherichia coli.

Human cardiac-specific homeobox protein cDNA (hCsx) was cloned into expression plasmid pET32a and fused with Escherichia coli thioredoxin (Trx). The Trx-Csx fusion protein was under the control of bacteriophage T7 promoter. When expressed in E. coli BL21(DE3), about half of the recombinant Trx-Csx products existed in the form of insoluble inclusion bodies. When coexpressed with human protein disulfide isomerase, more than 90% of Trx-Csx products accumulated in the soluble form in the cell lysate. The recombinant Csx fusion protein was purified by one-step metal-chelating affinity chromatography.

Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification.

The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non-syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34-q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation.

Isolation and characterization of the human homeobox gene HOX D1.

Homeobox genes, first identified in Drosophila, encode transcription factors that regulate embryonic development along the anteroposterior axis of an organism. Vertebrate homeobox genes are described on the basis of their homology to the genes found within the Drosophila Antennapedia and Bithorax homeotic gene complexes. Mammals possess four paralogous homeobox (HOX) gene clusters, HOX A, HOX B, HOX C and HOX D, each located on different chromosomes, consisting of 9 to 11 genes arranged in tandem. We report the characterization of the human HOX D1 gene. This gene consists of two exons, encoding a 328 amino acid protein, separated by an intron of 354 bp. The human HOX D1 protein is one amino acid longer (328 amino acids) than the mouse protein (327 amino acids) and is 82% identical to the mouse HOX D1 homolog. The DNA binding homeodomain region of the human protein exhibits a 97% and 80% identity between mouse Hoxd1 and Drosophila labial homeodomains, respectively. The exon/intron and intron/exon splice junctions are conserved in position between human and mouse genes. Determination of the human HOX D1 gene structure permits the use of PCR based analysis of this gene for the assessment of mutations, for diseases that link to the HOXD cluster (such as Duanes Retraction Syndrome (DRS)), or polymorphisms associated with human variation. Molecular characterization of the HOXD1 gene may also permit analysis of the functional role of this gene in human neurogenisis.

Cardiac Expression of the Ventricle-Specific Homeobox Gene Irx4 Is Modulated by Nkx2-5 and dHand

We report the isolation and characterization of the cDNAs encoded by the murine and human homeobox genes, Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0–8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5–11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression was also assessed in embryos with aberrant cardiac development: mice lacking RXRα or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development, which is downstream of Nkx2-5 and dHand.

Genomic Cloning and Characterization of the Human Homeobox Gene SIX6 Reveals a Cluster of SIX Genes in Chromosome 14 and Associates SIX6 Hemizygosity with Bilateral Anophthalmia and Pituitary Anomalies

The Drosophila gene sine oculis (so), a nuclear homeoprotein that is required for eye development, has several homologues in vertebrates (the SIX gene family). Among them, SIX3 is considered to be the functional orthologue of so because it is strongly expressed in the developing eye. However, embryonic SIX3 expression is not limited to the eye field, and SIX3 has been found to be mutated in some patients with holoprosencephaly type 2 (HPE2), suggesting that SIX3 has wide implications in head development. We report here the cloning and characterization of SIX6, a novel human SIX gene that is the homologue of the chick Six6(Optx2) gene. SIX6 is closely related to SIX3 and is expressed in the developing and adult human retina. Data from chick and mouse suggest that the human SIX6 gene is also expressed in the hypothalamic and the pituitary regions. SIX6 spans 2567 bp of genomic DNA and is split in two exons that are transcribed into a 1393-nucleotide-long mRNA. Chromosomal mapping of SIX6 revealed that it is closely linked to SIX1 and SIX4 in human chromosome 14q22.3–q23, which provides clues about the origin and evolution of the vertebrate SIX family. Recently three independent reports have associated interstitial deletions at 14q22.3–q23 with bilateral anophthalmia and pituitary anomalies. Genomic analyses of one of these cases demonstrated SIX6 hemizygosity, strongly suggesting that SIX6 haploinsufficiency is responsible for these developmental disorders.

Human ZHX1: Cloning, Chromosomal Location, and Interaction with Transcription Factor NF-Y

NF-YA, B, and C comprise the heterotrimeric transcription factor known as nuclear factor Y (NF-Y) or CCAAT-binding protein (CBF). NF-Y binds many CCAAT and Y box (an inverted CCAAT box, ATTGG) elements. Mutations of these elements that disrupt the binding of NF-Y result in decreased transcription from various tissue-specific and inducible promoters. We employed a yeast two-hybrid system to screen a human liver cDNA library in an effort to isolate proteins that interact with NF-Y and that may play a role in tissue-specific or hormone-inducible promoter activity. Using a fragment of the NF-YA subunit as bait we isolated a cDNA that encodes most of the open reading frame of the human zinc fingers and homeobox 1 (ZHX1) protein. The complete open reading frame was subsequently isolated and found to encode a protein of 873 amino acids that contains two zinc fingers and five homeodomain motifs. Northern blot analysis of poly(A)+ RNA isolated from various tissues revealed two major ZHX1 transcripts of about 4.5 and 5 kilobases. Both transcripts were expressed ubiquitously, although the 5-kilobase transcript is of greater abundance in most tissues examined. The human ZHX1 gene is located on chromosome 8q, between markers CHCL.GATA50B06 and CHLC.GATA7G07.

Volume 35 Contents and Index

A novel human homeobox gene related to the mouse Esx1 homeobox gene, which we have designated ESXR1, has been identified. ESXR1 and Esx1 share 65% identity within their homeodomains and have glutamic acid-rich and proline-rich N- and C-terminal regions, respectively. Unlike Esx1, ESXR1 contains 12 repeats of a unique nine amino acid motif, PPMAP(V/L)PPG, located C-terminal to the homeodomain. The general exon–intron structures of ESXR1 and Esx1 appear to be conserved. ESXR1 has been localized to human Xq22.1–q22.3, the same region of synteny shared by the map position of Esx1. ESXR1 expression appears to be restricted to the placenta and testis, the tissues in which Esx1 is also expressed. These data suggest that ESXR1 may be the orthologue of Esx1. The findings that there are similarities between ESXR1 and Esx1, yet differences between their encoded products, are consistent with the idea that placental genes evolve rapidly between mammalian species.

Modulation of the Human Homeobox Genes PRX-2 and HOXB13 in Scarless Fetal Wounds

Scarless healing of cutaneous wounds occurs in humans during the first two trimesters of development, but by birth all wounds are repaired with scar formation. To search for transcriptional regulatory genes that might mediate fetal tissue regeneration, we surveyed homeobox gene expression in proliferating fetal fibroblasts and in wounded and unwounded skin. Two novel human homeobox genes, PRX-2 and HOXB13, were identified that were differentially expressed during fetal versus adult wound healing. Both genes were predominantly expressed in proliferating fetal fibroblasts and developing dermis, and PRX-2 was downregulated in adult skin. In a model of scarless fetal skin regeneration, PRX-2 expression was strongly increased compared with unwounded skin and the signal was localized to the wounded dermis, the site of scarless repair. Conversely, in adult skin weak epidermal PRX-2 expression was observed, mRNA levels were not increased by wounding, and no dermal expression was detected. HOXB13 expression was decreased in wounded fetal tissue relative to unwounded fetal controls or wounded adult skin. Thus both HOXB13 and PRX-2 are expressed in patterns consistent with roles in fetal skin development and cutaneous regeneration.

A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD.

We report here the identification of a new human homeobox gene, PITX3, and its involvement in anterior segment mesenchymal dysgenesis (ASMD) and congenital cataracts in humans. The PITX3 gene is the human homologue of the mouse Pitx3 gene and is a member of the RIEG/PITX homeobox gene family. The protein encoded by PITX3 shows 99% amino-acid identity to the mouse protein, with 100% identity in the homeodomain and approximately 70% overall identity to other members of this family. We mapped the human PITX3 gene to 10q25 using a radiation-hybrid panel. A collection of 80 DNA samples from individuals with various eye anomalies was screened for mutations in the PITX3 gene. We identified two mutations in independent patients. A 17-bp insertion in the 3'-end of the coding sequence, resulting in a frame shift, occurred in a patient with ASMD and cataracts, and a G-->A substitution, changing a codon for serine into a codon for asparagine, in the 5'-end of the gene occurred in a patient with congenital cataracts. Both mutations cosegregate with the disease phenotype in families, and neither were found in up to 300 control individuals studied. Further expression analysis of Pitx3 in the mouse supports a unique role in early ocular development, with later expression extending to the midbrain, tongue, incisors, sternum, vertebrae and limbs. These data strongly suggest a role for PITX3 in ASMD and cataracts and provide new evidence of the contribution of the RIEG/PITX gene family to the developmental program underpinning normal eye formation.

Isolation and androgen regulation of the human homeobox cDNA, NKX3.1.

The prostate is dependent on androgens for development and maintenance of its differentiated phenotype. We have applied the technique of differential display PCR to the androgen-sensitive prostate cancer cell line LNCaP to isolate androgen-responsive genes. The technique of DD-PCR was applied to androgen-stimulated LNCaP cell RNA to detect and isolate androgen-responsive genes. The human homeobox gene NKX3.1, the homologue to mouse Nkx3.1, recently isolated by Beiberich et al. [J Biol Chem 1996;271:31779-31782], was detected and cloned. NKX3.1 is induced by androgens in a time- and concentration-dependent manner. NKX3.1 is induced 6- to 7-fold in 12 hr, with a significant induction seen in 2 hr. This regulation is at the level of transcription, as androgens increase the number of new NKX3.1 transcripts, and de novo protein synthesis is not required. In humans, NKX3.1 is expressed most highly in the prostate, with a much lower level of expression seen in the testis. No other tissue examined showed detectable levels of NKX3.1 expression. NKX3.1 is an androgen-regulated, prostate-specific homeobox gene. We hypothesize that it may play a role in the development and differentiation of the prostate.

SHOT, a SHOX-related homeobox gene, is implicated in craniofacial, brain, heart, and limb development.

Deletion of the SHOX region on the human sex chromosomes has been shown to result in idiopathic short stature and proposed to play a role in the short stature associated with Turner syndrome. We have identified a human paired-related homeobox gene, SHOT, by virtue of its homology to the human SHOX and mouse OG-12 genes. Two different isoforms were isolated, SHOTa and SHOTb, which have identical homeodomains and share a C-terminal 14-amino acid residue motif characteristic for craniofacially expressed homeodomain proteins. Differences between SHOTa and b reside within the N termini and an alternatively spliced exon in the C termini. In situ hybridization of the mouse equivalent, OG-12, on sections from staged mouse embryos detected highly restricted transcripts in the developing sinus venosus (aorta), female genitalia, diencephalon, mes- and myelencephalon, nasal capsula, palate, eyelid, and in the limbs. SHOT was mapped to human chromosome 3q25-q26 and OG-12 within a syntenic region on chromosome 3. Based on the localization and expression pattern of its mouse homologue during embryonic development, SHOT represents a candidate for the Cornelia de Lange syndrome.

A new human homeobox gene OGI2X is a member of the most conserved homeobox gene family and is expressed during heart development in mouse.

Homeodomain (HD) proteins are transcription regulators controlling a variety of cell fates. The HD region characterizing this protein family is a domain of 60 amino acid residues that recognizes and binds a site in the regulatory region of the target gene. It has been suggested that regions outside the HD may determine the specific functions of the various HD proteins by forming additional contacts with DNA sequences or by interactions with other proteins. We have identified a 14 amino acid motif within the C-terminal region of the protein encoded by the RIEG1 gene that is conserved among several HD proteins. Overlapping expression of the genes encoding these proteins during craniofacial development suggested that they might interact with a common factor. In order to identify additional genes possessing this motif we screened a human craniofacial cDNA library with oligoprobes. A novel gene was identified, exhibiting the most homology to murine Og12x (formerly OG12) and the recently reported human SHOX gene. Human OG12X and murine Og12x are highly homologous and the OG12X and Og12x proteins are 100% identical. In situ hybridization on mouse embryos ranging from 9 to 16 days post-coitum localized murine Og12x mRNA in the heart, otic region, maxillary and mandibular components of the first branchial arch, nasal processes, eyelid, midbrain, medulla oblongata, limbs, dorsal root ganglia and genital tubercle. OG12X was mapped to human chromosome 3q22-26 and murine Og12x to the syntenic region on mouse chromosome 3. Based upon the expression pattern of its mouse cognate, OG12X represents a candidate for the blepharophimosis (BPES) and Cornelia de Lange syndromes previously mapped to this region.

A regulatory element of the empty spiracles homeobox gene is composed of three distinct conserved regions that bind regulatory proteins

Homeobox genes encode a class of highly evolutionarily conserved transcription factors that control embryonic development. The Drosophila melanogaster empty spiracles gene is the homolog of the two human homeobox genes EMX1 and EMX2. These genes are necessary for central nervous system development. We used a regulatory element of the empty spiracles gene to study the control of homeobox gene expression. The 1.2-kilobase (kb) cis-regulatory element located 3 kb 5' of the transcription start site of the empty spiracles gene was analyzed by evolutionary sequence comparisons, gel mobility shift assays, DNase footprinting, and the generation of transgenic flies. The corresponding element from a related species, Drosophila hydei, was cloned. Three discrete, approximately 100 base pair (bp) regions of sequence homology were identified. Each had two blocks of 10 to 40 bp of near perfect sequence identity. Fusion proteins were produced containing the Abdominal-B homeodomain or the empty spiracles homeodomain, known regulators of empty spiracles gene expression. Gel mobility shift assays showed that each of the three regions is bound by both proteins. DNase footprinting revealed closely linked empty spiracles and Abdominal-B binding sites. We then generated transgenic flies containing a reporter linked to individual conserved regions of the enhancer. Reporter expression was evident only outside of the usual empty spiracles expression domain. These elements are not sufficient alone; a combinatorial model is proposed. Conserved discrete areas within a homeobox gene regulatory element, which function as homeodomain protein transcription factor binding sites, are used in a combinatorial fashion to regulate these developmentally important genes.

The human homeobox genes MSX-1, MSX-2, and MOX-1 are differentially expressed in the dermis and epidermis in fetal and adult skin

In order to identify homeobox genes which may regulate skin development and possibly mediate scarless fetal wound healing we have screened amplified human fetal skin cDNAs by polymerase chain reaction (PCR) using degenerate oligonucleotide primers designed against highly conserved regions within the homeobox. We identified three non- HOX homeobox genes, MSX-1, MSX-2, and MOX-1, which were differentially expressed in fetal and adult human skin. MSX-1 and MSX-2 were detected in the epidermis, hair follicles, and fibroblasts of the developing fetal skin by in situ hybridization. In contrast, MSX-1 and MSX-2 expression in adult skin was confined to epithelially derived structures. Immunohistochemical analysis of these two genes suggested that their respective homeoproteins may be differentially regulated. While Msx-1 was detected in the cell nucleus of both fetal and adult skin; Msx-2 was detected as a diffuse cytoplasmic signal in fetal epidermis and portions of the hair follicle and dermis, but was localized to the nucleus in adult epidermis. MOX-1 was expressed in a pattern similar to MSX early in gestation but then was restricted exclusively to follicular cells in the innermost layer of the outer root sheath by 21 weeks of development. Furthermore, MOX-1 expression was completely absent in adult cutaneous tissue. These data imply that each of these homeobox genes plays a specific role in skin development.

Human genes for dental anomalies.

The development of the tooth at gene level is beginning to be understood. This paper reviews current knowledge and the advances in research on human genes whose defect leads to dental anomalies. Amelogenesis imperfecta (AI) is a diverse group of hereditary disorders characterized by a variety of developmental enamel defects including hypoplasia and hypomineralization, some of which have been revealed to be associated with defective amelogenin genes. The human amelogenin genes on X and Y chromosomes have been cloned and investigated extensively. Although autosomally inherited forms of AI are more common than the X-linked forms, most studies on the genes causing AI have been performed on the genes of X-linked forms. Recently, the gene for the human tuftelin protein (an enamelin) has been cloned as a candidate gene for the autosomal forms of AI with another gene on chromosome 4 involved in some families. Dentinogenesis imperfecta (DI) may be associated with osteogenesis imperfecta (OI), which is an autosomal dominant bone disease. Most patients with OI have mutations in either the COLIA1 or COLIA2 genes, which encode the alpha 1(I) or alpha 2(I) subunits of type I collagen, the major organic component of bone and dentin. Gene defects causing isolated DI have not been identified. Recently, it was demonstrated that a missense mutation of MSXI, a human homeobox gene, causes autosomal dominant agenesis of second premolars and third molars. Data indicating an important function for MSXI, the mouse counterpart of the human MSXI gene, in mouse tooth development have been accumulating since 1991. Knockout mice lacking this gene exhibited multiple craniofacial anomalies including complete tooth agenesis. X-linked anhidrotic ectodermal dysplasia (EDA), characterized by abnormal hair, teeth, and sweat glands, was demonstrated to be caused by a mutation in a novel transmembrane protein gene that is expressed in epithelial cells and in other adult and fetal tissues. The predicted EDA protein may belong to a novel class of proteins with a role in epithelial-mesenchymal signaling. Several mutations have been reported in genes causing hypophosphatasia, which is characterized by defective mineralization of the skeletal and dental structures.