Chromosomal mapping of ANTP class homeobox genes in amphioxus: piecing together ancestral genomes

Homeobox genes encode DNA-binding proteins, many of which are implicated in the control of embryonic development. Evolutionarily, most homeobox genes fall into two related clades: the ANTP and the PRD classes. Some genes in ANTP class, notably Hox, ParaHox, and NK genes, have an intriguing arrangement into physical clusters. To investigate the evolutionary history of these gene clusters, we examined homeobox gene chromosomal locations in the cephalochordate amphioxus, Branchiostoma floridae. We deduce that 22 amphioxus ANTP class homeobox genes localize in just three chromosomes. One contains the Hox cluster plus AmphiEn, AmphiMnx, and AmphiDll. The ParaHox cluster resides in another chromosome, whereas a third chromosome contains the NK type homeobox genes, including AmphiMsx and ArnphiTlx. By comparative analysis we infer that clustering of ANTP class homeobox genes evolved just once, during a series of extensive cis-duplication events of genes early in animal evolution. A trans-duplication event occurred later to yield the Hox and ParaHox gene clusters on different chromosomes. The results obtained have implications for understanding the origin of homeobox gene clustering, the diversification of the ANTP class of homeobox genes, and the evolution of animal genomes.

Ol-Prx 3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes)

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization of Ol-Prx 3 (Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996) Proc. Natl. Acad. Sci. USA 93, 10691–10696] and with the human SHOX gene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., et al. (1997) Nat. Genet. 16, 54–62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for the PRX 3 family of homeobox genes.

Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp), Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost. We make further inferences concerning the evolution of heads and Hox genes in arthropods.

Cloning and characterization of four murine homeobox genes.

Four novel murine homeobox genes, Uncx-4.1, OG-2, OG-9, and OG-12, were cloned and partially sequenced. The amino acid sequence of the mouse Uncx-4.1 homeodomain is closely related to the sequence of the unc-4 homeodomain of Caenorhabditis elegans. However, the OG-2, OG-9, and OG-12 homeodomains are relatively diverged and are not closely related to any previously described homeodomain. Northern blot analyses revealed multiple bands of Uncx-4.1, OG-2, OG-9, and OG-12 poly(A)+ RNA in RNA from mouse embryos and adults that change during development and showed that each gene is expressed in a tissue-specific manner. OG-12 cDNAs were cloned that correspond to two alternatively spliced species of OG-12 mRNA. Three major bands of Uncx-4.1 poly(A)+ RNA were found only in RNA from adult mouse brain, but an additional band was observed in RNA from all of the other tissues tested. Major bands of OG-9 and OG-2 poly(A)+ RNA were found only in RNA from striated muscle; however, trace bands were detected in RNA from other tissues.

Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells.

Smooth muscle cell plasticity is considered a prerequisite for atherosclerosis and restenosis following angioplasty and bypass surgery. Identification of transcription factors that specify one smooth muscle cell phenotype over another therefore may be of major importance in understanding the molecular basis of these vascular disorders. Homeobox genes exemplify one class of transcription factors that could govern smooth muscle cell phenotypic diversity. Accordingly, we screened adult and fetal human smooth muscle cell cDNA libraries with a degenerate oligonucleotide corresponding to a highly conserved region of the homeodomain with the idea that homeobox genes, if present, would display a smooth muscle cell phenotype-dependent pattern of expression. No homeobox genes were detected in the adult human smooth muscle cell library; however, five nonparalogous homeobox genes were uncovered from the fetal library (HoxA5, HoxA11, HoxB1, HoxB7, and HoxC9). Northern blotting of adult and fetal tissues revealed low and restricted expression of all five homeobox genes. No significant differences in transcripts of HoxA5, HoxA11, and HoxB1 were detected between adult or fetal human smooth muscle cells in culture. HoxB7 and HoxC9, however, showed preferential mRNA expression in fetal human smooth muscle cells that appeared to correlate with the age of the donor. This phenotype-dependent expression of homeobox genes was also noted in rat pup versus adult smooth muscle cells. While similar differences in gene expression have been reported between subsets of smooth muscle cells from rat vessels of different-aged animals or clones of rat smooth muscle, our findings represent a demonstration of a transcription factor distinguishing two human smooth muscle cell phenotypes.

PCR survey of Hox genes in the goldfish Carassius auratus auratus

A tetraploidization event took place in the cyprinid lineage leading to goldfishes about 15 million years ago. A PCR survey for Hox genes in the goldfish Carassius auratus auratus (Actinopterygii: Cyprinidae) was performed to assess the consequences of this genome duplication. Not surprisingly, the genomic organization of the Hox gene clusters of goldfish is similar to that of the closely related zebrafish (Danio rerio). However, the goldfish exhibits a much larger number of recent pseudogenes, which are characterized by indels. These findings are consistent with the hypothesis that dosage effects cause selection pressure to rapidly silence crucial developmental regulators after a tetraploidization event.

The pathophysiology of HOX genes and their role in cancer

The HOM-C clustered prototype homeobox genes of Drosophila, and their counterparts, the HOX genes in humans, are highly conserved at the genomic level. These master regulators of development continue to be expressed throughout adulthood in various tissues and organs. The physiological and patho-physiological functions of this network of genes are being avidly pursued within the scientific community, but defined roles for them remain elusive. The order of expression of HOX genes within a cluster is co-ordinated during development, so that the 3' genes are expressed more anteriorly and earlier than the 5' genes. Mutations in HOXA13 and HOXD13 are associated with disorders of limb formation such as hand-foot-genital syndrome (HFGS), synpolydactyly (SPD), and brachydactyly. Haematopoietic progenitors express HOX genes in a pattern characteristic of the lineage and stage of differentiation of the cells. In leukaemia, dysregulated HOX gene expression can occur due to chromosomal translocations involving upstream regulators such as the MLL gene, or the fusion of a HOX gene to another gene such as the nucleoporin, NUP98. Recent investigations of HOX gene expression in leukaemia are providing important insights into disease classification and prediction of clinical outcome. Whereas the oncogenic potential of certain HOX genes in leukaemia has already been defined, their role in other neoplasms is currently being studied. Progress has been hampered by the experimental approach used in many studies in which the expression of small subsets of HOX genes was analysed, and complicated by the functional redundancy implicit in the HOX gene system. Attempts to elucidate the function of HOX genes in malignant transformation will be enhanced by a better understanding of their upstream regulators and downstream target genes.

Dispersal of NK homeobox gene clusters in amphioxus and humans

The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx-Lbx and NK3-NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

Isolation and characterization of a novel human Mix -like homeobox gene MIXL

Molecular events involved in specification of early hematopoietic system are not well known. In Xenopus, a paired-box homeodomain family (Mix.1–4) has been implicated in this process. Although Mix-like homeobox genes have been isolated from zebrafish (bon), chicken (CMIX) and mice (MmI/MIXL1), isolation of a human Mix-like gene has remained elusive. ^ We have recently isolated and characterized a novel human Mix-like homeobox gene with a predicted open reading frame of 232 amino acids designated the Mix.1 homeobox (Xenopus laevis)-like gene (MIXL). The overall identity of this novel protein to CMIX and MmI/MIXL1 is 41% and 69%, respectively. However, the identity in the homeodomain is 66% to that of Xenopus Mix.1, 79% to that of CMIX, and 94% to that of MmI/MIXL1. In normal hematopoiesis, MIXL expression appears to be restricted immature B and T lymphoid cells. Several acute leukemic cell lines of B, T and myeloid lineages express MIXL suggesting a survival/block in differentiation advantage. Furthermore, Xenopus animal cap assay revealed that MIXL could induce expression of the α-globin gene, suggesting a functional conservation of the homeodomain. ^ Biochemical analysis revealed that MIXL proteins are phosphorylated at multiple sites. Immunoprecipitation and immunoblotting confirmed that MIXL is tyrosine phosphorylated. Mutational analysis determined that Tyr20 appears to be the site for phosphorylation. However, deletion analysis preliminarily showed that the proline-rich domain appears not to be necessary for tyrosine phosphorylation. The novel finding will help us make a deeper understanding of the regulation on homeodomain proteins by rarely reported tyrosine phosphorylation. ^ Taken together, isolation of the MIXL gene is the first step toward understanding novel regulatory circuits in early hematopoietic differentiation and malignant transformation. ^