*This article is dedicated to Dr John Paul with whom I had the privilege to work as a .... Fienberg et al., 1987; Holland & Hogan, 1988a). The transcripts are ...
Br. J. Cancer (1988), 58, Suppl. IX, 9-13
,'.
The Macmillan Press Ltd., 1988
Mammalian homeobox-containing genes: Genome organization, structure, expression and evolution* K. Schughart1, C. Kappen' & F.H. Ruddlel 2 Departments
of
'Biology and 2Human Genetics, Yale University, New Haven, Connecticut 06511, USA. Summary Mammalian homeobox-containing genes have been isolated by their sequence similarity to Drosophila homeotic selector genes. About 20 murine homeobox genes have been identified to date and their expression and structural organization has been described in detail. Most homeobox gene loci are organized in at least three major gene clusters in the mouse and human genome. The structure of homeobox genes within these clusters is very similar and in this paper the murine Hox-2.2 gene will be discussed as an example. Homeobox genes are expressed in region-specific patterns during different stages of vertebrate development and almost all mammalian homeobox genes are expressed in the central nervous system (CNS) of the developing embryo. Within the developing CNS of mouse embryos the anterior boundaries of expression are specific for each gene. Comparisons of nucleotide and amino acid sequences as well as the analysis of the structural organization of murine and human homeobox genes reveal strong paralogous relationships between genes in different clusters. These findings suggest that the homeobox gene clusters evolved in two steps. First, an ancestral gene cluster was created by duplications of individual genes along one linkage group and in a subsequent step duplications of the ancestral gene complex gave rise to the three (or possibly four) gene clusters observed in mouse and human to date. The possibility of the homeobox genes representing a functional array of genetic switches will he disCulssed.
The mammalian homeobox-containing genes were identified by their sequence similarities to Drosophila homeoboxcontaining genes (McGinnis et al., 1984a, b), such as Ultrabithorax (Ubx) and Antennapedia (Antp). Ubx and Antp are homeotic genes fundamentally involved in development (reviewed by Scott & O'Farrell, 1986; Gehring & Hiromi, 1986; Ingham, 1988; Akam, 1987). The function of the cognate mammalian genes is not yet clearly understood, but everything we do know strongly suggests that they play a crucial role in establishing developmental patterns in the vertebrate embryo (Fienberg et al., 1987). In insect and mammalian homeobox genes, the most highly conserved region is represented by the homeobox region. This region is specified by 183 nucleotide base pairs encoding 61 amino acids and is part of a larger coding region. The homeobox region averages similarities in nucleotide sequences of approximately 65-80% between insects and mammals. Amino acid sequences in some cases are 98% similar. Secondary structure prediction of the homeodomain reveals a helix turn helix motif in the most conserved subregion of the homeobox. Helix turn helix motifs have been shown to be involved in DNA binding of regulatory proteins in prokaryotes and yeast (Shepherd et al., 1984; Laughon & Scott, 1984). Direct experimental evidence exists that homeobox proteins are localized in nuclei, that they bind specifically to DNA, and that the homeodomain is necessary for DNA binding (Desplan et al., 1985, 1988; Fainsod et al., 1986; Kessel et al., 1987; Odenwald et al., 1987). Studies in Drosophila and mouse show that the homeobox protein may bind to its own regulatory region, and/or to regulatory sequences from other homeobox genes (Fainsod et al., 1986; Desplan et al., 1988). Recent analysis of the interaction of homeobox genes in Drosophila suggests that these interactions may serve a regulatory function (Ish-Horowicz & Pinchin, 1987). A more distantly related gene, Oct-2, has been recently isolated (Ko et al., 1988). The Oct-2 gene product represents a transcription factor involved in the regulation of expression of immunoglobulin genes. Thus, the concept is emerging that homeobox genes are controller genes which coordinate the activity of effector genes which in turn mediate developmental events. The importance of homeobox genes in the mediation of development is supCorrespondence: F.H. Ruddle. *This article is dedicated to Dr John Paul with whom I had the
privilege to work as a postdoctoral fellow, 1960-61. John taught me
that an essential part of creative work is a rich, but well-tested imagination- FHR.
ported by mutations in Drosophila homeobox genes which dramatically affect epigenesis (Gehring & Hiromi, 1986). Unfortunately, no comparable mutations have yet been recorded for mammalian homeobox genes. Homeobox genes are clustered To date, about 20 homeobox genes in the mouse and human genome have been isolated and the total number of homeobox genes in the mouse might well exceed 50 gene loci. The mouse and human homeobox genes are organized in four major clusters (Figure 1). It was gratifying to see that Drosophila homeobox genes are organized similarly (Gehring & Hiromi, 1986). The mouse clusters contain as many as seven genes, and it is likely that this is a minimum number. The genes are spaced every 5 to 10 kb, and in all instances examined, they are transcribed in the same direction. To the extent examined, all homeobox genes in the mouse are expressed during embryonic development, and as yet no pseudogenes have been recognized. Moreover, the complexes show no evidence of highly repetitive sequences. These 4.2
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Figure 1 Chromosomal organization of murine homeobox gene clusters. Available mapping data on murine homeobox genes suggest the presence of at least four homeobox gene clusters on chromosome 2, 6, 11 and 15. Vertical bars indicate groups of related homeobox genes. (*): Personal communication from Pravtcheva et al. (Hox-4. 1). Question marks indicate that for some homeobox genes overlapping genomic clones have not been isolated which would allow these genes to be linked directly to other members of the respective cluster. Studies on the human Hox-3 cluster (Simeone et al., 1988) suggest the presence of a murine Hox-3.5 gene. Distances between homeobox loci are not drawn in scale. Instead, relative distances are given to illustrate the similar organization of parologous genes. *(4.2: suggested name for previously described Hox-5.1 gene).
10
K. SCHUGHART et al.
characteristics taken together with others to be reported below suggest a high level of conservation during the evolution of these clusters. The reasons for such a conservation are not known. One possibility might be that the expression pattern of a particular homeobox gene is dependent on its position within the cluster. Recently, reports have been published which suggest transcriptional read through over two or more adjacent genes within a cluster (Simeone et al., 1988).
Chromosomal organization of mouse and human homeobox gene clusters Homeobox gene clusters have been mapped to mouse chromosomes 2, 6, 11 and 15 (reviewed by Fienberg et al., 1987; Featherstone et al., 1988). Human cognate clusters have been mapped to human chromosomes 2, 7, 17 and 12, respectively (reviewed by Ruddle et al., 1987). Numerous genes syntenic with the homeobox gene clusters show orthologous homology between the two species (Figure 2). This is expected and already well documented. Unexpected is a strong paralogous homology between genes syntenic to the homeobox gene clusters (Figure 2). Thus, for example, in man, collagen loci are present on chromosomes 2, 7, 17 and 12. Cytokeratins are present on chromosomes 17 and 12, and another member of the intermediate filament family, desmin,
Paralogous homology Mouse chromosomes 2 6 11 Hox-4 Hox-1 Hox-2 Cola-1 Krt-2 Act-b Gcsf Gcg Erbb-2 Pkca Orthologous homology Human chromosomes .
2
HOX4 COL3,5,6 DES ACTL3 (GCG)
7 HOX1 COLIA2
ACTB IL6 EGFR-1 NPY
17 HOX2 COLIAl CYK13 ACTA GCSF EGFR-2 PKCA
15 Hox-3
Krt-1
12 HOX3 COL2A1 CYK4
PPY
Figure 2 Paralogous and orthologous relationships between genes syntenic to the homeobox gene clusters in mouse and human. Gene symbols: Hox-l/HOX-1, homeobox region 1; Hox2/HOX-2, homeobox region 2; Hox-3/HOX-3, homeobox region 3; Hox-4/HOX-4, homeobox region 4; Cola-l/COLIAl, collagen type I alpha 1; COL 3,5,6, collagen type III, type V, type VI, resp.; COLIA2, collagen type I alpha 2; COL2Al, collagen type 2 alpha 1; Krt-2, keratin type 2; Krt-l, keratin type 1; DES, desmin; CYK 13, cytokeratin type 13; CYK4, cytokeratin type 4; Act-b/ACTB, actin beta; ACTL3, actin-like sequence 3; ACTA, actin alpha skeletal muscle; Gcg/GCG, glucagon; IL6, interleukin 6; Gcsf/GCSF, granulocyte colony stimulating factor; Erbb-2, erythroblastic leukemia viral oncogene homolog 2; EGFR-1, epidermal growth factor receptor type 1; EGFR-2, epidermal growth factor receptor type 2; Pkca/PKCA, protein kinase C alpha peptide; NPY, neuropeptide Y; PPY, pancreatic polypeptide.
is
on chromosome 2. Interleukin 6 and granulocyte colony stimulating factor possess sequence similarities, and they map to chromosomes 7 and 17 (reviewed by Ruddle et al., 1987). The paralogies between human chromosomes 2, 7, 17
and 12 are consistent with the occurrence of at least two chromosome duplication events in mammals or their ancestors.
Structure of mammalian homeobox genes The typical mammalian homeobox gene is 5-10 kb in size. In comparison, Drosophila genes may be eight to ten times larger. The size difference in the insect genes can be largely accounted for in larger cis-flanking domains. The mouse Hox-2.2 gene may be taken as a prototype for mammalian homeobox genes (Figure 3; Schughart et al., 1988). Hox-2.2 genomic sequence extends over at least 10 kb and a transcribed region of about 2.6kb has been identified. The open reading frame deduced from cDNA sequences predicts a protein of 225 amino acids. Two exons have been identified, and it is probable that one or more additional exons exist. Comparisons of the amino acid sequence of Hox-2.2 with other homeobox genes showed that regions of conserved sequences are represented by a N-terminal octameric region, a hexameric region 5' of the homeodomain, the homeodomain, and an acidic C-terminal region. The hexameric region shows high sequence similarity to a sequence in f-like globins (Figure 3) and may be involved in protein-protein interactions. In haemoglobins, the region similar to the homeobox gene hexamer is involved in a1 -,B2 subunit interactions. This is consistent with findings which suggest that homeodomain proteins may function as protein dimers (Desplan et al., 1988). Repeats of acidic amino acid residues are frequently seen in the Cterminal coding portions of homeodomain proteins, such as strings of glutamic acid residues. The Hox-2.2 protein contains five glutamic acid residues downstream of the homeodomain and other homeodomain proteins contain up to 15 glutamic acid residues. Their function is not established, but they may be involved in the transcriptional activation of target genes (Ma & Ptashne, 1987). An AU-rich region and a consensus polyadenylation signal (AAUAAA) is seen in the 3' non-coding flanking region of the Hox-2.2 transcription unit.
Expression of homeobox gene transcripts within the central nervous system The homeobox genes are expressed both prenatally and in the adult organism, predominately in ectodermal and mesodermal tissues and their derivatives (reviewed by Fienberg et al., 1987; Holland & Hogan, 1988a). The transcripts are expressed specifically in respect to time and place, and do not necessarily follow recognized organ or tissue boundaries. The central nervous system (CNS) provides an opportunity to compare expression patterns of individual homeobox gene transcripts, since almost all of the homeobox genes described to date are expressed in the CNS. It should be emphasized that homeobox gene expression is a complex subject that cannot be adequately dealt with here. Most genes encode a variety of transcripts and these may differ in expression, depending on the tissue and time of development. A particular transcript may also appear in different tissues at a particular time, or be expressed, extinguished, then reappear. The patterns are also involved and complex in terms of dorsal/ventral, anterior/posterior, and lateral expression. Here, only one aspect of expression will be addressed, namely the anterior boundaries of expression in the CNS. A number of homeobox genes have been studied in regard to their anterior level of expression in the CNS, using in situ hybridization techniques to detect mRNAs in tissue sections obtained from mouse embryos. For example, the Hox-1.5, 3.1, 2.2, 2.1 and 2.5 homeobox genes are all expressed in the CNS of 13.5 day p.c. mouse embryos, but exhibit specific
MAMMALIAN HOMEOBOX-CONTAINING GENES
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Hox-2.2: V Y P W M Q R B-globin: V Y P W T 0 R Figure 3 Structure of the Hox-2.2 gene. The restriction map of the Hox-2.2 gene and structure of the isolated cDNA clone are illustrated in the upper part of the figure. Exons are illustrated by thick bars. The lower part of the figure shows the predicted structure of the Hox-2.2 mRNA. The open reading frame is boxed, the three stippled regions indicate the positions of the conserved N-terminus, the hexapeptide, and the glutamic acid-rich region. The homeobox is shown as a striped box. Amino acid sequences of the three conserved regions are given below. The alignment of the Hox-2.2 hexapeptide with the corresponding region of f-like globins is shown. The underlined amino acid residues in the 3-globin sequence are involved in al-,B2 subunit interactions. An AU-rich region and a consensus transcriptional stop signal is present in the predicted 3' untranslated region of the Hox-2.2 mRNA.
anterior boundaries of expression in the developing spinal cord (Fainsod et al., 1987; Awgulewitsch et al., 1986; Utset et al., 1987; Bogarad et al., submitted). The Hox-1.5 homeobox gene is expressed in the extreme anterior position of the medulla, and Hox-3.1 transcripts can be detected in the spinal cord with an anterior boundary at the level of the third cervical vertebra. An interesting relationship emerges when one compares the anterior position of expression of the Hox-2 homeobox genes in the spinal cord in relationship to their location in the homeobox gene clusters. The results are shown schematically in Figure 4. Hox-2.2 specific transcripts can be detected throughout the developing spinal cord of 13.5 day p.c. mouse embryos showing an anterior boundary of expression within the hindbrain (Schughart et al., 1988). This pattern of expression is highly reminiscent of the pattern reported for the Hox-2.1 gene (Utset et al., 1987; Holland & Hogan, 1 988b). A direct comparison in crosssections shows that Hox-3. 1 expression extends more rostrally into the hindbrain than Hox-2.2 expression (Schughart et al., 1988). The anterior boundaries of Hox-2.1 and -2.2 expression are located behind Hox-1.5 in the medulla with Hox-2.1 anterior to Hox-2.2. Hox-2.5 RNA can be detected in the developing spinal cord, but the anterior boundary of expression appears to be at the level of the first cervical vertebra (Bogarad et al., submitted). By Northern analysis, Deschamps et al. (1987) detected Hox-2.3 transcripts in the spinal cord with an anterior boundary in the developing hindbrain. Thus, there is an overlapping pattern of the transcript expression throughout the medulla and spinal cord with defined anterior boundaries for each homeobox gene. Furthermore, these findings document an unexpected but very interesting observation, namely that the order of anterior limits of homeobox gene expression parallels the order of genes in the cluster (Figure 1). The same correlation
e
.a
MB
*Hox-2.Z
Hox-2.1
Hox-2.5
Figure 4 Region-specific expression of murine Hox-2 genes within the developing CNS of 13.5-day p.c. embryos. (a) Pattern of Hox-2.2 expression. Transcripts are detected within the medulla and the spinal cord. FB, forebrain; MB, midbrain; SC, spinal cord. (b) and (c) Schematized developing CNS indicating Hox-2.1 and Hox-2.5 (Bogarad et al., submitted) expression patterns, respectively. (b) Hox-2.1 transcripts accumulate within the medulla and spinal cord. (c) Hox-2.5 transcripts accumulate within the dorsal half of the spinal cord posterior to the first cervical vertebra.
holds true for other homeobox genes which have been studied (reviewed by Holland & Hogan, 1 988a). The functional significance, if any, of this structural arrangement is not apparent. One might speculate that the relative position of homeobox genes on the chromosome may be important for their region-specific expression. It is intriguing
12
K. SCHUGHART et al.
that in Drosophila a similar relationship of expression patterns and organization of homeobox genes has been described (Harding et al., 1985).
Paralogous relationships between homeobox gene clusters Comparisons of the murine homeobox gene clusters provide evidence for strong paralogous homologies (Schughart et al., in preparation). The comparison of nucleotide and deduced amino acid sequences of homeobox domains, as well as comparisons of flanking regions (dot matrix comparisons of whole cDNA sequences, position of splice sites, spacing between the homeodomain and the conserved hexapeptide), shows that individual homeobox genes can be grouped together (Figure 5). These relationships have been confirmed using parsimony algorithms provided in the PAUP program (D. Swoffold, Illinois Natural History Survey, IL). It is striking that genes most highly related are arranged in identical order between Hox clusters 1, 2 and 3. Only fragmentary data is available for the Hox-4 cluster, but it also appears to fit this pattern. In addition to their sequence similarity, the spacing of paralogous loci within the clusters is remarkably similar. These relationships are schematically illustrated in Figure 1. Because of the high conservation of both the sequence and the position of a particular homeobox gene, homologous genes can be easily identified. This makes it possible to draw conclusions about the evolution of mammalian homeobox genes and to use these genes to study molecular evolution of gene families in general. Evolution of the murine homeobox genes The findings reviewed above prompted us to speculate about the evolution of mammalian homeobox genes. The results obtained so far are consistent with a process of gene duplications resulting in the expansion of homeobox genes within an ancestoral gene cluster, followed by two chromosomal duplications giving rise to four gene clusters. In earlier discussions of this hypothesis, we favored one chromosome duplication event, followed by translocations to other chromosomes (Ruddle, 1988). This concept is now excluded by recent findings showing the presence of cognate homeobox genes in three or more clusters. 10
20
Chromosome duplication most probably occurred by genome duplication, since polyploidy would obviate problems associated with genetic imbalance. Sequence similarities, patterns of gene and gene complex organization, and the parallelism between anterior/posterior position of gene expression in the CNS and position within the gene clusters strongly suggest that several of the genes discussed here are cognates between mammals and insects. If this assumption is correct, then it follows that the lateral expansion of the genes within a primordial homeobox gene cluster occurred before the divergence of protostomes and deuterostomes, an event estimated to have taken place about 500 million years ago. Consistent with this interpretation is the fact that in lower insects, such as Tribolium, the Bithorax and Antennapedia complexes appear to be contiguous (Beeman, 1987). At some later time, this cluster separated in the insect lineage into two separate gene clusters, but it is still located on the same chromosome arm, 3R, in Drosophilia. In the insects, there is no evidence for chromosome duplication to produce more gene clusters. However, the genes as pointed out earlier are significantly larger in terms of their cis-flanking domains, and this most probably can be considered as a more modern, advanced feature. The chromosome duplication events in the deuterostomes may have occurred after the divergence of the echinoderms and ancestral protochordates, because preliminary evidence suggests that present-day echinoderms have only a modest number of Antennapedia-like genes. It will be of considerable interest to survey the gene number and patterns of gene organization in the extant echinoderms and primitive chordates in order to obtain more information on this question. The evidence obtained today for the mouse and to a lesser degree in man argues convincingly for genome duplication events in the history of the chordata. This idea has been advanced previously but based primarily on the existence of sequence related gene families, such as the immunoglobulins. The advantage of the homeobox system is the structural concordance of the homeobox clusters and the paralogous relationships of their linked genes which allows the identification of homologous genes. These patterns can be 30
40
50
60
Antp
RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKENK
Hoxl7 Hox32
TRKK-CP--KH---------L--M----D--Y-V-RL-N-----V----------M--I-TRKK-CP--K----------L--M----D--Y-V-RV-N-----V----------M--M--
Hox24 -R------S-----------L--P----K----VS---G-----V---------------N
Hox3l -RT-----S-----------L--P----K----VS---G-----V---------------N Hoxll ___________________-----------------------------------------HHox23 -Y-----------------Y--------------T---------------Hoxl2 GR---------------------------------N------------------------Hox22 GR-------------------Y-------------------------------------SHOX33 -R--- -I-S--------------------------N-----------------------SN
Hoxl3 G--A-TA---------------------------------S-----------------D-Hox2l G--A-TA---------------------------------S-----------------D-Hoxl4 P--S-TA---Q-V----------------------- T---S---V-------------DHHox26 P--S-TA---Q-V--------Y---------V--------S-----------------DHHOX51 P--S-TA---Q-V-----------------------T---P-----------------DHHoxl5 S----TA---P-LV------------M-P--V-M-NL-N----------------Y--DQHox27 S--A-TA--SA-LV------------C-P--V-M-NL-N-S--------------Y--DQHOX41 S-SV-T---SA-LV------------C-P--V-M-NL-N----------------Y--DQ-
Hoxl6 PNAV-TNF-TK-LT---------K----A--V---AS-Q-N-T-V----------Q--REFigure 5 Amino acid sequences of the homeodomain of murine homeobox genes. Sequences are arranged in groups of related homeobox genes. Dashes in the murine homeobox sequences indicate amino acid residues identical to the sequence of the Drosophila Antp homeodomain.
MAMMALIAN HOMEOBOX-CONTAINING GENES
convincingly attributed to whole chromosome or genome duplications. At the moment, only rough estimates can be made about the timing of these events, because of the lack of extensive sequence data and paralogous relationships in vertebrates other than mouse and man. However, it is of interest to point out that there are an average of 13 nucleotide substitutions in homeobox regions of four mouse-human orthologous pairs. This represents the changes that have occurred since the man/mouse divergence. There is an average of 30 substitutions in 15 mouse-mouse paralogous pairs, and this corresponds to changes accumulated, since the presumed chromosome duplication event. This means that the duplication event occurred before the divergence of mouse and man. Under the assumption that mutation rates have been linear over time, these figures would suggest that the duplication occurred prior to 150-200 million years ago. Further sequence information from lower vertebrates will be necessary to test this hypothesis. Is the homeobox gene system an informational array? The homeobox genes as we know them today have the properties of switching mechanisms, because of the 'selfregulating' properties of the protein products. That is to say, there are several well-documented instances where the homeodomain protein can bind to specific operator sites in
13
its own cis-flanking region. We presume these circuits may be feedback loops, and may serve a regulatory function. Thus, single homeobox genes have the potential to operate as genetic switches. If a gene undergoes duplication, a more complex switching apparatus is created, since now the two genes not only have the capacity to self regulate, but also to cross regulate. As the gene family expands and evolves with respect to the nature of these interactions, the potential exists for the development of a highly sophisticated switching device. If such an information array possessed 50 genes, each capable of at least two expression states, then 250 (approximately 1 x 1015) combinations of expression could be achieved. It should be kept in mind that a large mammalian organism is composed of only 1014-15 cells. Thus, it is interesting to consider the possible existence of informational (environmental) cues feeding into such a system, cybernetic cues informationally linking the system, and effector cues emanating from the system, and in turn coordinating the patterns of expression of effector genes.
This work was supported by NIH GM09966. CK and KS are supported by Postdoctoral Fellowships from the Deutsche Forschungsgemeinschaft (FRG). We thank Mrs Marie Siniscalchi for her help in the preparation of the manuscript.
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