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Molecular Cloning, Chromosomal Mapping, and Characterization of the Human Cardiac-Specific Homeobox Gene hCsx
Molecular Medicinevolume 2, pages86–96 (1996)
Csx/Nkx2.5, a murine nonclustered homeobox gene expressed primarily in the heart, has significant sequence similarity to the Drosophila tinman gene. Tinman is essential for heart and gut formation in Drosophila. Targeted mutation in the mouse gene, Csx/Nkx25, arrests cardiac development during early embryonic stages, suggesting an evolutionary conservation in cardiogenesis.
Materials and Methods
We have isolated and characterized a human homolog, hCsx, from an adult cardiac cDNA library. Northern blotting and ribonuclease protection was used to define the pattern of expression during normal development and in disease states. Chromosomal localization of the gene was determined by somatic cell hybrid analysis and fluorescent in situ hybridization.
The predicted amino acid sequence of hCsx has 87% overall homology to the murine gene with 100% identity in the homeodomain. The homeodomain sequence of hCsx is 95% identical to its Xenopus homolog, and 65% to tinman. hCsx mRNA was detected exclusively in the heart. hCsx transcript was detected at 12 weeks in human embryonic heart, the earliest time point examined, and was up-regulated 5-fold between 12 and 19 weeks. There was no significant alteration of hCsx message level in the myocardium of 14 patients with end stage heart failure compared to a normal control. The human gene mapped to the distal portion of chromosome 5, the 5q34–q35 region. This defines a new synteny region between human chromosome 5q and the t-locus of mouse chromosome 17, where the mouse Csx gene is located.
hCsx, the human homolog of Drosophila tinman, is expressed in heart in a tissue restricted manner. Distal 5q trisomies produce several phenotypic abnormalities, including a high incidence of congenital heart disease. Isolation of the hCsx gene will allow further studies of mutations in this gene and their potential associations with some forms of congenital heart disease in humans.
The coordinated activation of regulatory genes plays a determinant role in the developmental process. The identity of any given cell lineage is finally determined by the complement of genes activated in the correct spatiotemporal fashion. Genes encoding transcription factors are particularly important because they control the expression of downstream target genes in this process. Activation of transcription factors can initiate a genetic cascade determining the final developmental destinies of groups of cells that will become a specific organ or tissue. The discovery of the MyoD family of transcription factors was a major breakthrough for the understanding of the genetic cascade leading to skeletal muscle development (1). These myogenic regulatory factors (MyoD, myogenin, myf-5 and MRF4) share a basic helix-loop-helix motif that binds to a cis-regulatory element called the E-box in the enhancers of different muscle specific genes (2). Skeletal and cardiac myocytes share several contractile proteins. Nevertheless, mice homozygous for null mutations in MyoD and myf-5 genes show a complete absence of skeletal muscle but no gross abnormality of the heart pointing to a divergent regulation between these two striated muscle cell types (3).
Homeobox genes are master regulatory genes that specify the body plan of metazoans (4). They share a common motif of 180 bp, the homeobox, which encodes the 60 amino acid homeodomain, the DNA-binding domain of this family of proteins (5). Some homeobox genes are organized in complexes (clustered), while others are randomly dispersed (non-clustered) throughout the genome (6). The clustered superfamily of homeobox genes seems to arise from a single cluster in the primitive metazoans (7). It is likely that as different organisms became genetically and anatomically more complex, duplications of the primordial genes arose. There are two clusters (Antennapedia and Bithorax) in Drosophila and four clusters (HoxA to HoxD) in mouse and human. These genes are organized in the same order along the chromosome as they are expressed along the antero-posterior body axis in what is known as the colinearity rule (8). Over- and mis-expression of some members of the Hox gene family, as well as null mutation analyses suggest that vertebrate Hox-type homeobox genes function in a similar way as their invertebrate homologs (8). In the non-clustered homeobox superfamily there are several mammalian homologs of each Drosophila gene which are named after the insect gene. They include the POU-family, paired-family, LIM-family, and NK-family, among others (6). These randomly inserted genes do not follow the colinearity rule, often contain additional conserved motifs, and may play a critical role in tissue specification during development (8).
A homeobox gene whose expression pattern is restricted to specific cell lineages during development would be of significant interest as a candidate tissue-specification gene. There has been a recent interest in the Drosophila NK homeobox gene family (9), as a group of genes which may specify cell fates of various tissues due to the restricted tissue expression of many of these genes. For example, the NK-1/S59 gene is expressed in the somatic mesoderm, in anterior portions of the central nervous system, and in the midgut of Drosophila (10). Rodent NK-2 homologs are expressed in a tissue restricted fashion: mNKx2.1/TTF-1 is expressed in the thyroid anlage, the lung bud, and the developing brain; mNKx2.2 and mNKx2A are expressed in certain areas of the central nervous system; and mNKx23 is expressed in the developing gut (11, 12). Expression of the NK-3/bagpipe gene is restricted to the gut and a subset of heart progenitors in Drosophila (10). In the bagpipe mutant embryos, visceral mesoderm formation is disrupted, suggesting the requirement of bagpipe for gut development (13).
The NK-4/tinman gene is expressed in the heart precursor cells of the dorsal mesoderm (14). Tinman mutants do not form the dorsal vessel, the Drosophila equivalent of the vertebrate heart (13,15). Tinman has several different vertebrate relatives (16). The mouse gene, Csx/Nkx2.5 is expressed from Day 7.5 postcoitum (p.c.) in the cadiac progenitor cells and its cardiac expression continues through the adult stage (17,18). Targeted disruption of Csx/Nkx2.5 results in abnormal heart morphogenesis and embryonic lethality around 9–10 days p.c. (19). In null mutant embryos, the beating linear heart tube forms normally but cardiac myogenesis is arrested at 8.5 days p.c. when the looping process occurs, with subsequent death within 1–2 days (19).
It is important to point out that in addition to the homeobox genes, there are two other transcription factors, GATA-4 and MEF2C, expressed in the murine precardiac mesoderm that are also candidate regulatory genes for cardiac morphogenesis (20,21). To date, the exact roles of these transcription factors in mammalian cardiac development have not been elucidated.
Due to the significant functional conservation of the Csx-like genes among species, it may be hypothesized that a human homolog of tinman exists and may be related to some forms of congenital heart disease and other cardiac pathologic states. In the present study, we report the cloning of the human homolog of the Csx/Nkx2.5 gene, hCsx. Its sequence, chromosomal location, expression pattern in normal tissues and disease states are presented.
Materials and Methods
Cardiac cDNA Library Screening and DNA Sequencing
A lambda gt10 library (a gift from Dr. Seth Alper) prepared from a single adult human left ventricle (22) was screened with a PstI fragment of mouse Csx containing the conserved homeobox and NK2 box (17). Low stringency hybridizations were performed with 35% formamide, 1% SDS, 2×SSC, 10% Dextran sulfate and salmon sperm DNA at 42°C. DNA sequencing reactions were carried out using the Taq DyeDeoxy Terminator or DyePrimer cycle sequencing kit (Applied Bio-systems, Foster City, CA, U.S.A.). Both strands were sequenced on an automated DNA Sequenator (Applied Biosystems 370A) by the DNA Sequencing Core Facility (Ann Arbor, MI, U.S.A.). Sequence analysis was performed with MacVector and AssemblyLIGN software (Eastman Kodak, Rochester, NY, U.S.A.). Database searches were performed using the BLAST program (Whitehead Institute for Biomedical Research, Cambridge, MA, U.S.A.).
Ribonuclease Protection Analysis
We analyzed left ventricle RNA previously isolated from adult patients with end-stage heart failure of different etiologies, undergoing cardiac transplant surgery at Brigham and Women’s Hospital (Boston, MA, U.S.A.) (23). The subjects included six patients with idiopathic dilated cardiomyopathy, six with ischemic cardiomyopathy, and two adults with congenital heart disease (one with L-transposition of great vessels and one with an atrial septal defect and congenital mitral stenosis, both of whom had undergone prior cardiac surgery). RNA samples of organ donors with no known cardiac pathology were used as controls. To examine developmental changes, the previously described ventricular RNA samples (23) from artificially aborted fetuses (gestation 12–19 weeks) were used. The original protocol of human cardiac tissue procurement (Dr. Paul D. Allen, Brigham and Women’s Hospital) was approved by the institutional review board as previously described (23).
Ribonuclease (RNase) protection assays were performed essentially as described (24). The 32P-labeled hCsx probe was synthesized by runoff transcription with T3 RNA polymerase from the pBluescript plasmid (Stratagene, La Jolla, CA, U.S.A.) containing the full length hCsx cDNA linearized with AvaII. The γ-actin probe was similarly synthesized with SP6 RNA polymerase from a HinfI-linearized plasmid, pSP64 (a gift from Dr. Ellis J. Neufeld) containing a portion of the 3′-untranslated region of the human γ-actin gene. Five micrograms of total human ventricular RNA was used for each protection with 10 µg of yeast tRNA as a negative control in each experiment. All samples were simultaneously hybridized with hCsx and γ-actin probes. All hybrids were digested with RNase A and RNase Tl at 37°C for 30 min. The digested products were run on 6% denaturing gels, followed by autoradiography. Relative amounts of mRNA were determined by densitometry in the linear response range of the X-ray films for each message.
Southern and Northern Blot Analysis
Human genomic DNA was extracted from mononuclear blood cells as described previously (24), and used for Southern blotting. Ten micrograms genomic DNA samples were digested with BamH1, EcoRI, or HindIII then electrophoresed in a 0.8% agarose gel and transferred to a Hybond-N Nylon membrane (Amersham Life Science, Arlington, IL, U.S.A.). The membranes were hybridized as previously described (25) and washed under high stringency to a final wash in 0.1×SSC, 0.5% SDS at 65°C. After this, the blots were subjected to autoradiography.
A human multiple tissue Northern blot containing approximately 2 µg of poly A+ RNA per lane from different human tissues was obtained (Clontech, Palo Alto, CA, U.S.A.). The blot was hybridized and washed following the manufacturer’s directions.
Chromosomal Location and Intron-Exon Mapping
Chromosomal mapping was first determined by somatic cell hybrid analysis. The Human/Rodent Somatic Cell Hybrid Panel #2 was obtained from NIGMS (Coriell Institute for Medical Research, Camden, NJ, U.S.A.). The presence of the hCsx gene was determined by polymerase chain reaction (PCR) performed with 200 ng hybrid genomic DNA, 500 µM each primer, 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 200 µM dNTPs, and 1.5 units of Ampli-Taq DNA polymerase (Perkin Elmer-Cetus, Norwalk, CT, U.S.A.) in a final volume of 25 µl. The primers used were, sense (from the 3′ coding region, P1S): 5′-AACTTCGTGAACTTCGGCGTC-3′, and antisense (from the 3′ untranslated region, P1AS): 5′-GTCTCCGCAGGAGTGAATG-3′ (see Figure 2B below). After an initial denaturation step for 5 min at 95°C, the samples were amplified for 30 additional cycles at 94°C for 1 min, 60°C for 30 sec and 72°C for 30 sec. The 229-bp human specific product was visualized on a 2% agarose gel stained with ethidium bromide, subcloned into the pCR II vector (Invitrogen, San Diego, CA, U.S.A.) and sequenced.
A human genomic P1 clone containing the hCsx gene was obtained by screening a human foreskin fibroblast P1 library by PCR (26,27) with the primers described above. The primers used to amplify the intronic sequence of the hCsx P1 clone were, sense (from the 5′ coding region, P4S): 5′-TGTGCGTCTGCCTTTCC-3′ and anti-sense (from homeodomain, P4AS): 5′-TGCGTG GACGTGAGTTTCAG-3′ (see Fig. 2B below) following the same PCR conditions described above. The P1 plasmid containing the hCsx genomic clone was used as a probe and fluorescence in situ hybridization (FISH) was performed as described previously (28) by the Cancer Center Cytogenetic Core Facility (Ann Arbor, MI, U.S.A.).
Cloning of the Human Cardiac-Specific Homeobox (hCsx) cDNA
Screening of a human ventricular cDNA library using the murine Csx cDNA probe resulted in the isolation of five positive clones from approximately 2 × 106 recombinants. Partial sequencing and restriction mapping indicated that these clones represented a single transcript, hCsx. Sequence analysis (Fig. 1) revealed a single open reading frame with a putative initiation codon preceded by a strong Kozak consensus initiation sequence (29). The longest hCsx cDNA clone is 1585 nucleotides in length that predicts a protein of 323 amino acids with no poly(A) tail at the 3′ end. Overall, the predicted hCsx protein sequence has 87% identity with its mouse homolog and about 60% with the Xenopus homolog, XCsx2/XNkx2.5 (Ref. 30 and Issei Komuro, Yu Chang Fu, D.T., and S.I., unpublished data). The homeodomain sequence of hCsx is 100% identical to its mouse homolog and 95% to its Xenopus homolog (Fig. 2A, a). There are two other conserved domains in the hCsx sequence, the TN-domain and the NK2 box, located amino- and carboxy-terminal to the homeobox, respectively. Interestingly, in the regions corresponding to the conserved TN-domain and the NK2 box, the identity is 100% among the human, mouse, and Xenopus genes (Fig. 2A, b and c). The hCsx homeodomain shows very low homology (less than 50%) to most known homeodomains with the exception of those in the NK2 gene family of Drosophila, Xenopus, and mouse.
Expression of hCsx in Human Tissues
To examine the tissue distribution of hCsx transcripts, a Northern blot containing poly A+ RNA from several adult human tissues was examined. A band corresponding to a transcript size of approximately 1.6 kb was detected exclusively in the heart (Fig. 3). There was another lighter band in the heart lane of approximately 3 kb as well. It may correspond to unprocessed RNA of the same transcript or the product of a related gene. No signal was detected in the other adult tissues examined including brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
To examine the level of expression of hCsx in human fetal heart and in different pathologic states, RNase protection analysis was performed using a probe corresponding to the 3′ end of hCsx cDNA (Fig. 2B, Probe D). A human γ-actin probe corresponding to the 3′ untranslated region was used as an internal control (Fig. 4A). Densitometric analysis of hCsx transcript normalized by γ-actin message is shown in Fig. 4B. The hCsx transcript was detected at 12 weeks of embryonic development, the earliest time point examined. There was 5-fold increase in the hCsx message occurring between 12 and 19 weeks (Fig. 4B). To determine whether expression of hCsx mRNA is altered in the failing human myocardium, we analyzed left ventricular RNA samples from patients with ischemic cardiomyopathy (n = 6) and idiopathic dilated cardiomyopathy (n = 6) and two patients with congenital heart disease. No significant difference in abundance of hCsx message in the different pathologic states was detected. (Fig. 4 A and B).
Isolation of Genomic Clone of hCsx
In order to begin to characterize the genomic organization of hCsx gene, a human genomic P1 clone containing the hCsx gene was isolated by PCR as described in Materials and Methods. PCR analysis of the P1 genomic clone confirmed that it contained sequences of the cDNA from the 5′ untranslated and coding region using primer pair P2S/P2AS (Fig. 2B), and the homeodomain region using primer pair P3S/P3AS (Fig. 2B). The primers used were, P2S: 5′-CGTGGGCAGCG CCGCTTT-3′, P2AS: 5′-GTCGGGGTCGCTGTA GGCAC G-3′, P3S: 5′-CCCCGGGCGCGACGGCG GAGG-3′, P3AS: 5′-CAGAGTCTGGTCCTGCCG CTG-3′. An intronic sequence upstream of the homeobox was also amplified using primer pair P4S/P4AS (Fig. 2B). PCR reactions using primer pair P4S/P4AS with the hCsx cDNA template gave an expected size band (288 bp), while an approximately 1.8-kb product was detected when the hCsx P1 genomic clone was used as a template (data not shown).
Hybridization of a human genomic Southern blot with Probe A (containing the 5′ end of the cDNA, Probe A, Fig. 2B) and subsequently with Probe B (containing the homeobox and the NK2 box. Probe B, Fig. 2B), detected a predominant band in each lane as well as other faint bands (data not shown). These results suggest that this gene is present in a single locus in the human genome, but that there are likely other related genes (perhaps containing an hCsx-like homeodomain and NK2 box) that remain to be isolated in the human genome.
Chromosomal Location of hCsx Gene
The mouse Csx/Nkx2.5 gene maps within the t-locus of the mouse chromosome 17 (Ref. 31 and Issei Komuro, Neal Copeland, D.T., and S.I., unpublished data). This region of mouse chromosome 17 has been shown to have synteny homology to human chromosome 6 (32). To determine chromosomal location of the hCsx gene, somatic cell hybrid PCR analysis was performed using the Human/Rodent Somatic Cell Hybrid Panel #2. A band of the expected size (229 bp) was reproducibly detected in human chromosome 5 (Fig. 5). This PCR product was subcloned and sequenced, which showed 100% homology with the hCsx cDNA sequence.
In order to determine the chromosomal location of hCsx more specifically, FISH analysis was performed using the entire P1 clone as a probe. A pair of signals from both chromatids was present in the long arm of a B-group chromosome. After banding, the signal was localized in the distal portion of chromosome 5 on band q35, close to the junction with band 5q34 (Fig. 6 A-C). No signal was detected by FISH on chromosome 6, confirming the results of somatic cell hybrid PCR analysis.
Homeobox genes play critical roles in tissue organization by regulating downstream target genes (8). As with the widely studied Hox genes, the NK family seems to be evolutionarily conserved and duplicates may have arisen from a single gene (6). In this report we describe the first human gene isolated to date that belongs to the NK family of homeobox genes. The homeodomain regions of the NK family members are more closely related to each other than to other homeodomains (9). While the functional significance of the additional conserved domains, the NK-2 domain and the TN domain, are not known, these domains represents another area of close relationship among these genes in several species. In adult human tissues, hCsx expression appears to be restricted to the heart. This tissue restricted pattern of expression is evolutionarily conserved among diverse species including Drosophila (15), Xenopus (30), mouse (17,18), and humans.
In mammals, cardiac muscle cells are derived from the anterior lateral plate mesoderm. In humans, commitment of mesodermal cells to the cardiac lineage occurs very early, when cells migrate to form the cardiogenic area at the beginning of the third week of human embryonic development (33). By the end of the 3rd week the primitive heart is formed and soon becomes functional, perfusing the different embryonic tissues and allowing normal development of the human embryo (33). We detected the hCsx transcript at 12 weeks, the earliest time point available. Since in the mouse, Csx/Nkx2.5 transcript is present as early as 7.5 days p.c. (17,18), it is likely that hCsx transcript may have been present much earlier, perhaps as early as at the time of commitment of the cardiogenic area (around 16 to 18 days of human development) (33,34). There was a significant increase in hCsx transcript level between 17 and 19 weeks gestational age (Fig. 4B). At this time there is rapid growth of the heart and an associated increase in the ventricular stroke volume (35–37). In the mouse, Csx/Nkx2.5 expression is relatively constant from late embryonic to adult stages (17). Similarly, levels of expression of hCsx appear relatively stable from the mid-embryonic stage to the adult; however, a more detailed study is necessary to make this a final conclusion.
Because Drosophila tinman and mouse Csx/Nkx2.5 are essential for heart development, we hypothesized that expression of hCsx may be altered in disease states. However, there was no significant change in the level of expression of the hCsx transcript in human idiopathic dilated and ischemic cardiomyopathy as well as the two congenital heart disease samples examined. It should be noted that these samples are from the end stage of heart failure and we can not rule out the possibility that levels of expression of hCsx may be altered in the earlier stages of the disease processes. Furthermore, our results neither rule out the possibility of an altered expression of the hCsx protein nor possible existence of point mutations in the hCsx gene in other forms of congenital cardiopathies (38).
By FISH, hCsx maps to the 5q34–q35 region. Synteny homology of mouse chromosome 17, where the Csx/Nkx2.5 gene maps, suggested that the human homolog would likely reside on chromosome 6 (Fig. 7). The localization of hCsx to the distal portion of chromosome 5q is unexpected since adjacent human genes surrounding the mouse Csx/Nkx2.5 locus have been assigned to human chromosome 6. However, no synteny has been studied in the specific region where the mouse Csx/Nkx2.5 gene is located, the th20 region (31). The unambiguous assignment of hCsx to human chromosome 5q (Figs. 5 and 6 A-C), suggests the introduction of a novel synteny group between human chromosome 5q and mouse chromosome 17. There is another gene, Fer, located on human chromosome 5, which is also present on mouse chromosome 17, outside of the t-locus (Figure 7) (32). Alternatively, this disrupted region of homology may be due to a chromosomal rearrangement.
In the genetic cascade for heart morphogenesis, a number of genes are activated sequentially to produce proteins which are necessary for normal development. When any of these genes are altered, a phenotypic abnormality of the heart may occur, depending on where the gene is located in this epigenetic cascade (39–41). A strictly anatomical classification of congenital heart disease is evolving into a newer classification schema according to the affected developmental stage of cardiac morphogenesis (42). In general, duplications of any given chromosome portion produce a less severe phenotype than deletions (43). In the case of chromosome 5q, there are at least 25 cases reported in the literature with distal 5q trisomies. The predominant phenotype includes growth and mental retardation as well as facial abnormalities and a high incidence (at least 67%) of congenital heart disease. Among the cardiac malformations, ventricular septal defect is the most common in these patients (44). To date, no homozygous deletions have been reported in the distal portion of the chromosome 5q (43). This raises the possibility of an extremely critical role of this segment during human embryogenesis, with its loss resulting in fetal demise during the early embryonic period (45,46). In fact, a lethal spontaneous deletion of the th20 locus has been reported in the mouse; however, whether the lethality of this deletion is directly related to the loss of Csx/Nkx2.5 or to other closely linked genes is not known (47).
In summary, we have isolated the first human member of the NK family of homeobox genes. This gene, hCsx, appears to be the homolog of murine Csx/Nkx25 and Drosophila tinman. Since null mutations of Csx/Nkx2.5 and tinman genes cause a severe early cardiac defect, it is possible that mutations in hCsx may be discovered in some forms of congenital heart disease. Future studies will test this hypothesis.
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We thank Ann Burgess, Madhu Prasad, and Mary Richmond for excellent technical assistance; T. Takahashi and P. D. Allen for RNA samples; and N. G. Copeland, T. W. Glover, S. A. Camper, and D. Law for helpful comments on the manuscript. This work was supported in part by grants from the National Institutes of Health and the American Heart Association to SI.
Contributed by T. Yamada on October 11, 1995.