Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitiveDUX1toDUX5homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.

How many homeobox genes and pseudogenes?

Using exhaustive database screening, followed by manual examination of sequences, we identified 300 homeobox loci in the human genome. Distinguishing which of these loci are functional genes and which are non-functional pseudogenes was difficult in some cases. Most loci classified as pseudogenes in this study are integrated reverse-transcribed transcripts, readily recognized by their dispersed genomic location, complete lack of intron sequences, and (in some cases) 3' homopolymeric run of adenine residues. A small minority are duplicated copies of genes, recognized by physical linkage to their functional counterparts and the same (or similar) exon-intron arrangement. In general, retrotransposed gene copies are non-functional (and therefore pseudogenes) from the moment of integration because they lack 5' promoter regions necessary for transcription. However, such sequences can occasionally acquire new promoters and become functional as 'retrogenes'. Duplicated gene copies often possess 5'promoter regions (as they are often encompassed by the duplication event); most degenerate to pseudogenes due to redundancy in a process known as non-functionalization, however some can be preserved as functional genes through sub- or neo-functionalization. Thus, in both instances, reliable indicators of non-functionality were sought in order to assign pseudogene status, notably frameshift mutations, premature stop codons and non-synonymous substitutions at otherwise conserved sites in the original coding region.

We currently estimate that the 300 human homeobox loci comprise 235 functional genes and 65 pseudogenes (Table1). These figures include three functional genes that possess partial homeobox sequences (PAX2,PAX5andPAX8) and retrotransposed pseudogenes that correspond to only part of the original transcript, whether or not it includes the homeobox region or indeed any of the original coding region. Consequently, 13 retrotransposed pseudogenes that lack homeobox sequences are included (NANOGP11,TPRX1P1,TPRX1P2,POU5F1P7,POU5F1P8,IRX4P1,TGIF2P2,TGIF2P3,TGIF2P4,CUX2P1,CUX2P2,SATB1P1,ZEB2P1). We do not includePAX1,PAX9andCERS1; these are functional genes without homeobox motifs, albeit closely related to true homeobox genes (the other PAX and CERS genes).

The total number of homeobox sequences in the human genome is higher than 300 for two reasons. First, several genes and pseudogenes possess more than one homeobox sequence, notably members of the Dux (double homeobox), Zfhx and Zhx/Homez gene families. Second, we have excluded a set of sequences related to humanDUX4(DUX1toDUX5), which have become part of 3.3 kb repetitive DNA elements present in multiple copies in the genome [12-14]. Few of these tandemly-repeated sequences are likely to be functional as expressed proteins, and all were probably derived by retrotransposition from functional DUX gene transcripts (see below). The fact that they are not included in the total count, therefore, is likely to have limited bearing on understanding the diversity and normal function of human homeobox genes. Hence, our figure of 300 homeobox loci is the most useful current estimate of the repertoire of human homeobox genes and pseudogenes.

Classification

We propose a simple classification scheme for homeobox genes, based on two principal ranks: gene class and gene family. A gene class contains one or more gene families, which in turn will contain one or more genes. In a few cases, it is useful to erect an intermediate rank between these levels, and for this we use the term subclass. For the entire set of homeobox genes, we use the term superclass.

For the rank of gene family, we use a specific evolutionary-based definition based on common practice in the field of comparative genomics and developmental biology. We define a gene family as a set of genes derived from a single gene in the most recent common ancestor of bilaterian animals (here defined as the latest common ancestor ofDrosophilaand human). This definition has been made explicitly in previous work [2,6] but is actually a principle that has been in widespread, but rather inconsistent, use for over a decade [15]. For example, amongst the homeobox genes, the En (engrailed) gene family was originally defined to include humanEN1andEN2, plusDrosophila enandinv[16]; these four genes arose by independent duplication from a single gene in the most recent common ancestor of insects and vertebrates. Moving outside the homeobox genes, this principle is also widespread; for example, the Hh (hedgehog) gene family was defined to include mouseShh,DhhandIhh, plusDrosophila hh[17]. To clarify boundaries between gene families, we conducted molecular phylogenetic analyses of human homeodomain sequences, using a range of protostome and occasionally cnidarian homeodomain sequences as outgroups (Additional files1 and2).

While the gene family definition described above is generally workable for homeobox genes, by necessity there are some exceptions. One type of exception relates to genes with an unknown ancestral number. For example, there is uncertainty as to whether there were one or two Dlx (distal-less) genes in the most recent common ancestor of bilaterians; however it is common practice to refer to a single Dlx gene family [18]. Thus, we stick with convention for this set of genes. There is similar uncertainty over the ancestral number of Irx (iroquois) genes [19], and again we treat these as a single gene family. The HOX genes are an interesting case as their precise number in the most recent common ancestor of bilaterians is unknown due to lack of phylogenetic resolution between 'central' genes [20]. Here we divide the HOX genes into seven gene families: the 'anterior' Hox1 and Hox2 gene families, the 'group 3' Hox3 gene family, the 'central' Hox4, Hox5 and Hox6-8 gene families, and the 'posterior' Hox9-13 gene family. Another type of exception relates to 'orphan' genes. These are genes that have been found in one species (for example human) but not in other species, or at least not in a wide diversity of Metazoa. Some of these will be ancient genes that have been secondarily lost from the genomes of some species, in which case these comply with our evolutionary definition of a gene family made above. Others, however, will be rapidly evolving genes that originated from another homeobox gene and then diverged to such an extent that their origins are unclear [21]. Whenever origins are unclear, we must define a new gene family to encompass those genes, even though they may not date back to the latest common ancestor of bilaterians. In these cases, the gene family is erected to recognize a set of distinct genes on the basis of DNA and protein sequence, rather than on evolutionary origins.

Using the aforementioned criteria, we recognize 102 homeobox gene families in the human genome (Table1). We are aware that other homeobox gene families exist in bilaterians but have been lost from humans (for example, Nk7, Ro, Hbn, Repo and Cmp; [7]), and we recognize that some gene family boundaries will alter as new information is obtained. Nonetheless, at the present time the 102 gene families provide a sound framework for the study of human homeobox genes.

It is much more difficult to propose a rigorous evolutionary definition for the rank of gene class. Every attempt to classify genes above the level of gene family involves a degree of arbitrariness. We define gene classes by taking two principal criteria into account. First, gene classes should ideally be monophyletic assemblages of gene families. To identify probable monophyletic groups of gene families, we conducted molecular phylogenetic analyses of homeodomain sequences, and looked for sets of gene families that group together stably, regardless of the precise composition of the dataset used (Figures1,2,3; Additional files3,4,5). Some gene families were difficult to place from sequence data alone, and were found in different gene classes (or subclasses) depending on the precise dataset analyzed or the phylogenetic method employed. This is perhaps not surprising as trees that encompass many homeobox genes can only be built with a short sequence alignment (the homeodomain); under these conditions, phylogenetic trees can only be used as a guide to possible classification, not the absolute truth. In ambiguous cases, we used the chromosomal location of genes to guide possible resolution between alternative hypotheses. Second, some homeobox gene classes can be characterized by the presence of additional protein domains outside of the homeodomain [2]. Recognized protein domains associated with homeodomains include the PRD domain, LIM domain, POU-specific domain, POU-like domain, SIX domain, various MEINOX-related domains, the CUT domain, PROS domain, and various ZF domains [2].

Using the aforementioned criteria, we recognize eleven homeobox gene classes in the human genome: ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS, ZF and CERS (Table1). There is no expectation that the eleven gene classes will be of similar size, simply because some classes will have undergone more expansion by gene duplication than others. In the human genome, the ANTP and PRD classes are much larger than the other classes. Although gene classes should ideally be monophyletic, it is possible that the ZF homeobox gene class, characterized by the presence of zinc finger motifs in most of its members, is polyphyletic (Figure3; Additional file5). In other words, domain shuffling may have brought together a homeobox sequence and a zinc finger sequence on more than one occasion. The same may also be true for the LIM class; alternatively the apparent polyphyly of LIM-class homeodomains could be a consequence of LIM domain loss or artefactual placement of some ZF-class homeodomains in phylogenetic analyses (Figure3; Additional file5).

In theory, it is possible to recognize higher level associations above the level of the gene class, because the diversification of homeobox genes will have taken place by a continual series of gene duplication events. We do not propose names for hierarchical levels above the rank of class, and consider that gene name, gene family and gene class (and occasionally subclass) convey sufficient information for most purposes.

We use a consistent convention for writing gene classes and gene families. We present the names of all gene classes in abbreviated non-italicized upper case – for example, the ANTP and PRD classes – to avoid confusion with gene symbols (Antpandprd) or indeed gene names (Antennapediaandpaired). In contrast, we present the names of all gene families in non-italicized title case; for example, the Cdx, En and Gsc gene families. We have used this style consistently in recent work [6,21-23] and note that several other authors have done likewise [4,7,24]. We suggest that this style, and most of these gene family names, can be used in other bilaterian genomes. Extending the scheme to non-bilaterians is more difficult, however, and awaits clarification of the relationship between the homeobox genes of sponges, placozoans, cnidarians and bilaterians [7,25].

The ANTP homeobox class

The ANTP class derives its name from theAntennapedia(Antp) gene, one of the HOX genes within the ANT-C homeotic complex ofDrosophila melanogaster. The human genome has 39 HOX genes, arranged into four Hox clusters. Here we divide the HOX genes into seven gene families: Hox1, Hox2, Hox3, Hox4, Hox5, Hox6-8 and Hox9-13. The HOX genes are not the only ANTP-class genes, and we recognize a total of 37 gene families in this class (Table1). We divide these 37 gene families between two subclasses that are relatively well-supported in phylogenetic analyses: the HOXL and the NKL subclasses (Figure1; Additional file3). As previously discussed, the subclasses are largely consistent with the chromosomal positions of genes [26,27]. The HOXL (HOX-Like or HOX-Linked) genes primarily map to two fourfold paralogous regions: the Hox paralogon (2q, 7p/q, 12q and 17q) and the ParaHox paralogon (4q, 5q, 13q and Xq) (Figure4). The NKL (NK-Like or NK-Linked) genes are more dispersed, but there is a concentration on the NKL or MetaHox paralogon (2p/8p, 4p, 5q and 10q) (Figure4). Somewhat aberrantly, the Dlx and En gene families group with the NKL subclass in phylogenetic analyses (Figure1; Additional file3), but with the HOXL subclass on the basis of chromosomal positions (Figure4).

Most of the 37 gene families in the ANTP class have been clearly defined before. We draw attention here to several cases that could cause confusion. Other details can be found in Table2.

∘ Cdx, Gsx and Pdx gene families. Some authors refer to the Pdx gene family as the Xlox gene family [28]. One gene from each of these families (CDX2,GSX1andPDX1) forms the ParaHox cluster at 13q12.2 (Figure4), and clustering of Cdx, Gsx and Pdx genes is ancestral for chordates [28].

∘ Mnx gene family. This gene family name derives from a previous study [29]. The family includes one gene in the human genome:MNX1(formerlyHLXB9), and two genes in the chicken genome:Mnx1(formerlyHB9) andMnx2(formerlyMNR2). Some authors refer to the Mnx gene family as the Exex gene family due to theDrosophilaorthologexex[7].

∘ Dlx gene family. It is currently unclear if this gene family is derived from one or more genes in the common ancestor of bilaterians [18]. Phylogenetic analyses place this gene family firmly within the NKL subclass (Figure1; Additional file3), but chromosomal positions (on the Hox chromosomes 2, 7 and 17) place it within the HOXL subclass (Figure4). Here we favor placement of the Dlx gene family within the NKL subclass due to strong phylogenetic support.

∘ En gene family. Phylogenetic analyses place this gene family either within the NKL subclass (maximum likelihood; Figure1) or close to the division between the NKL and HOXL subclasses (neighbor-joining; Additional file3). Here we place the En gene family within the NKL subclass, although we note that humanEN2maps close to the clear HOXL-subclass genesGBX1andMNX1on chromosome 7 (Figure4).

∘ Nk2.1 and Nk2.2 gene families. The genesNKX2-1(formerlyTITF1),NKX2-4,NKX2-2andNKX2-8divide into two distinct gene families each with an invertebrate ortholog, not a single Nk2 gene family.NKX2-1andNKX2-4are collectively orthologous toDrosophila scroand amphioxusAmphiNk2-1[30,31]; these comprise one gene family: Nk2.1.NKX2-2andNKX2-8are collectively orthologous toDrosophila vndand amphioxusAmphiNk2-2[31,32]; these comprise a second gene family: Nk2.2.

∘ Nk4 gene family. The genesNKX2-3,NKX2-5andNKX2-6form a gene family, quite distinct from other human genes that confusingly share the prefixNKX2. These three genes are actually orthologs ofDrosophila tin(formerlyNK4); they are not orthologs ofDrosophila vnd(formerlyNK2) orscro[33]. Therefore, they do not belong to the Nk2.1 or Nk2.2 gene families, but belong to a separate Nk4 gene family. As the three gene names have very extensive current usage, it may be difficult for revised names to be used consistently. In this situation, we don't alter the current names, but raise for discussion the possibility of these genes being renamed to the more logicalNKX4-1(NKX2-5),NKX4-2(NKX2-6) andNKX4-3(NKX2-3), or toCSX1(NKX2-5),CSX2(NKX2-6) andCSX3(NKX2-3), based on the alternative nameCSX1forNKX2-5[34].

∘ Noto gene family. This gene family falls close to the division between the ANTP and PRD classes in phylogenetic analyses (Additional files1 and2). We favor placement within the ANTP class as the humanNOTOgene is chromosomally linked to the clear ANTP-class (NKL-subclass) genesEMX1,LBX2,TLX2andVAX2on chromosome 2 (Figure4), suggesting ancestry by ancient tandem duplication.

Most of the 100 genes in the ANTP class have been adequately named previously. However, several genes were unnamed or misnamed prior to this study. We have updated these as follows.

∘GSX2[Entrez Gene ID: 170825] is the second of two human members of the Gsx gene family. This previously unnamed gene has clear orthology to mouseGsh2, inferred from sequence identity and synteny. We designate the geneGSX2and revise the nomenclature of the other human member of the family fromGSH1toGSX1[Entrez Gene ID: 219409], in accordance with homeobox gene nomenclature convention.

∘MNX1[Entrez Gene ID: 3110] is the only member of the Mnx gene family in the human genome. This gene was previously known asHLXB9; we rename itMNX1because it is not part of a series of at least nine related genes.

∘PDX1[Entrez Gene ID: 3651] is the only member of the Pdx gene family in the human genome. This gene was previously known asIPF1; we rename itPDX1because the majority of published studies use this as the gene symbol.

∘BSX[Entrez Gene ID: 390259] is the only member of the Bsx gene family in the human genome. We designate this previously unnamed geneBSXon the basis of clear orthology to the mouseBsxgene, inferred from sequence identity and synteny.

∘DBX1[Entrez Gene ID: 120237] andDBX2[Entrez Gene ID: 440097] are the only two members of the Dbx gene family in the human genome. We designate these previously unnamed genesDBX1andDBX2on the basis of clear orthology to mouseDbx1andDbx2, inferred from sequence identity and synteny.

∘NKX1-1[Entrez Gene ID: 54729] andNKX1-2[Entrez Gene ID: 390010] are the only two members of the Nk1 gene family in the human genome. These genes were previously known asHSPX153andC10orf121respectively; we rename themNKX1-1andNKX1-2on the basis of clear orthology to mouseNkx1-1andNkx1-2, inferred from sequence identity and synteny.

∘NKX2-1[Entrez Gene ID: 7080] is the first of two human members of the Nk2.1 gene family. This gene was previously known asTITF1; we rename itNKX2-1to show that it is a member of the Nk2.1 gene family.

∘NKX2-6[Entrez Gene ID: 137814] is the third of three human members of the Nk4 gene family. We designate this previously unnamed geneNKX2-6on the basis of clear orthology to mouseNkx2-6, inferred from sequence identity and synteny, although nomenclature revision for the entire Nk4 gene family should be discussed (see above).

∘NKX3-2[Entrez Gene ID: 579] is the second of two human members of the Nk3 gene family. This gene was previously known asBAPX1; we rename itNKX3-2to show that it is a member of the Nk3 gene family.

∘NKX6-3[Entrez Gene ID: 157848] is the third of three human members of the Nk6 gene family. We designate this previously unnamed geneNKX6-3on the basis of clear orthology to mouseNkx6-3, inferred from sequence identity and synteny.

∘VENTX[Entrez Gene ID: 27287] is the only functional member of the Ventx gene family in the human genome. This gene was previously known asVENTX2. We remove the numerical suffix from this gene symbol because we discovered that the sequence formerly known asVENTX1is actually a retrotransposed pseudogene derived from this gene. Accordingly, we also replace theVENTX1symbol withVENTXP7(see below).

In contrast to the previous descriptions of probable functional genes, there has been much less research on pseudogenes within the ANTP class. Eleven pseudogenes derived from the humanNANOGgene have been described previously [22], while four pseudogenes in the Ventx gene family have been reported following routine annotation of the human genome. We have identified two additional Ventx-family pseudogenes (VENTXP5andVENTXP6), and also found two cases of pseudogenes that were originally mistaken for functional genes (MSX2P1andVENTXP7). In all cases, we have clarified the origins and organization of these pseudogenes. This research brings the total number of ANTP-class pseudogenes in the human genome to 19.

∘MSX2P1[Entrez Gene ID: 55545]. A short cDNA sequence [EMBL:X74862] related to the Msx gene family was reported previously [35]; the former Entrez Gene record labeledHSHPX5was based on this sequence. This locus was later provisionally calledMSX4, as it was distinct from humanMSX1andMSX2, and by synteny it was clearly not the ortholog of mouseMsx3[27]. It is now clear that this locus was formed by retrotransposition of mRNA fromMSX2and hence we name itMSX2P1. The genomic sequence ofMSX2P1can now be accessed via the Reference Sequence collection [RefSeq:NR_002307]. The pseudogene shares 91% sequence identity withMSX2mRNA, lacks intronic sequence, and has remnants of a 3' poly(A) tail. It is intriguing, but probably coincidental, that theMSX2P1pseudogene has integrated at 17q23.2, close to several ANTP-class genes (HOXB cluster,MEOX1,DLX3andDLX4).

∘NANOGP1[Entrez Gene ID: 404635]. We follow Booth and Holland [22] and classifyNANOGP1as a pseudogene that arose by tandem duplication ofNANOG. The alternative view, argued by Hart et al [36], is that this locus is a functional gene, and should be namedNANOG2. There is evidence for transcription of this locus in human embryonic stem cells [36], and for selection-driven conservation of the open reading frame [37], but as yet no clear evidence for function.

∘NANOGP8[Entrez Gene ID: 388112]. We follow Booth and Holland [22] and classifyNANOGP8as a retrotransposed pseudogene. The alternative view, argued by Zhang et al [38], is that this locus is a functional retrogene. There is evidence for transcription and translation of this locus in cancer cell lines and tumors [38], but no evidence yet for a role in normal tissues.

∘VENTXP1[Entrez Gene ID: 139538],VENTXP2[Entrez Gene ID: 347975],VENTXP3[Entrez Gene ID: 349814] andVENTXP4[Entrez Gene ID: 152101]. These fourVENTXretrotransposed pseudogenes have been reported previously, and were originally known asVENTX2P1toVENTX2P4. The correction of theVENTX2gene symbol to simplyVENTX(see above) means that each of the pseudogene names should also change; we rename themVENTXP1toVENTXP4.VENTXP1is transcribed but due to mutations it can no longer encode a homeodomain protein; it can however encode an antigenic peptide (NA88A) responsible for T-cell stimulation in response to melanoma [39].

∘VENTXP5[Entrez Gene ID: 442384]. We designate this previously unnamed sequenceVENTXP5because it is clearly a retrotransposed pseudogene ofVENTX. The genomic sequence ofVENTXP5can now be accessed via the Reference Sequence collection [RefSeq:NG_005091]. The pseudogene shares 83% identity withVENTXmRNA (after masking of an Alu element in the parental mRNA sequence), lacks intronic sequence, and has remnants of a 3' poly(A) tail.

∘VENTXP6[Entrez Gene ID: 552879]. We designate this previously unannotated sequenceVENTXP6because it is clearly a retrotransposed pseudogene ofVENTX. Its lack of annotation may reflect the fact that it is located within an intron of an unrelated and well characterized gene,STAU2. The genomic sequence ofVENTXP6can now be accessed via the Reference Sequence collection [RefSeq:NG_005090]. The pseudogene shares 87% identity withVENTXmRNA (after masking of an Alu element in the parental mRNA sequence) and lacks intronic sequence.

∘VENTXP7[Entrez Gene ID: 391518]. A short cDNA sequence [EMBL:X74864] was reported previously and namedHPX42[35]. This was later renamed theVENTX1gene, after it was found to be related toXenopusVentx-family genes. Our analysis of the genomic sequence at this locus reveals that it is actually a retrotransposed pseudogene of theVENTXgene (formerlyVENTX2); thus we designate itVENTXP7. The genomic sequence ofVENTXP7can now be accessed via the Reference Sequence collection [RefSeq:NR_002311]. The pseudogene shares 86% identity withVENTXmRNA (after masking of an Alu element in the parental mRNA sequence), lacks intronic sequence, and has remnants of a 3' poly(A) tail.

One other gene could conceivably be included in the ANTP class, but is excluded from our survey. This gene [Entrez Gene ID: 360030; GenBank:AY151139], has been annotated as a homeobox gene and is located just 20 kb fromNANOG. However, no homeodomain was detected when the deduced protein was analyzed for conserved domains. Also, secondary structure prediction did not predict the expected organisation of alpha helices. Alignment with the NANOG homeodomain reveals identity of the KQ and WF motifs, either side of the same intron position (44/45), but few other shared residues. It is possible, but unproven, that the locus arose by tandem duplication of part, or all, of theNANOGhomeobox gene. This gene has generated two retrotransposed pseudogenes: one at 2q11.2 and another at 12q24.33.

The PRD homeobox class

The PRD class derives its name from thepaired(prd) gene ofDrosophila melanogaster. In previous studies, the PRD class has been subdivided in several different ways, often based on identify of the amino acid at residue 50 in the homeodomain, for example S50, K50 and Q50. These categories are not monophyletic groupings of genes and so can be misleading if we aim for a classification scheme that reflects evolution [5]. Here we divide the PRD class into two subclasses of unequal size: the PAX subclass (containing seven PAX genes, excludingPAX1andPAX9), and the PAXL subclass (containing 43 non-PAX genes and many pseudogenes) (Table1). PAX genes are defined by possession of a conserved paired-box motif, distinct from the homeobox, coding for the 128-amino-acid PRD domain. Of the nine human genes possessing a paired-box (PAX1toPAX9), only four also contain a complete homeobox (PAX3,PAX7,PAX4andPAX6). Three genes have a partial homeobox (PAX2,PAX5andPAX8), while two lack a homeobox entirely (PAX1andPAX9). Phylogenetic analyses using PAX genes from a range of species suggest that these are secondary conditions, and that the ancestral PAX gene probably possessed both motifs [40]. The PAX genes do not constitute a single gene family, because it is clear that the latest common ancestor of the Bilateria contained four PAX genes. Three of these are ancestors of the PRD-class homeobox gene families Pax2/5/8, Pax3/7 and Pax4/6; the fourth is the ancestor ofPAX1andPAX9. Thus the PAX subclass contains three gene families. We divide the PAXL subclass into 28 gene families, although as explained below not all of these date to the base of the Bilateria. Thus, we recognize a total of 31 gene families in the PRD class (Table1).

Many of the 31 gene families in the PRD class have been clearly defined before. We draw attention here to newly defined gene families and cases that could cause confusion. Other details can be found in Table3.

∘ Argfx, Dprx and Tprx gene families. There are no known invertebrate members of these three gene families. Therefore, these are exceptions to the rule defining gene families as dating to the base of the Bilateria. The Dprx and Tprx gene families may have arisen by duplication and very extensive divergence fromCRX, a member of the Otx gene family, during mammalian evolution; origins ofARGFXare obscure [21].

∘ Dux gene family. Members of this gene family are characterized by the presence of two closely-linked homeobox motifs. Most members are intronless sequences present in multiple polymorphic copies within the 3.3 kb family of tandemly repeated elements associated with heterochromatin. These comprise the sequences known asDUX1toDUX5reported in previous studies [12-14] and numerousDUX4copies detected in this study (see below). The absence of introns suggests that these sequences may have originated by retrotransposition from an mRNA transcript, thus they are probably non-functional. There are two noticeable exceptions; these members known asDUXAandDUXBpossess introns, thus either one could be the progenitor for the large number of intronless Dux-family sequences found in the human genome.DUXAhas spawned 10 retrotransposed pseudogenes and has been described previously [21].DUXBis described here (see below).

∘ Hopx gene family. Phylogenetic analyses places this gene family, containing a single very divergent homeobox geneHOPX(formerlyHOP), either within the PRD class (maximum likelihood; Additional file1) or close to Zhx/Homez-family genes (neighbor-joining; Additional file2). We favor placement in the PRD class for three reasons. First, the HOPX homeodomain has highest sequence identity with PRD-class homeodomains (GSC: 38% and PAX6: 36%). Second, the HOPX homeodomain possesses the same combination of residues that are invariably conserved across human PRD-class homeodomains (Additional file6). Third, the HOPX homeodomain shares the 46/47 intron position seen in many PRD-class homeodomains.HOPXdoes not map particularly near any other homeobox genes, although the closest isGSX2in the ANTP class at 4q12 (Figure4).HOPXis not a typical PRD-class homeobox gene; the homeodomain has a single amino acid insertion between helix I and helix II (Additional file6), and lacks the ability to bind DNA [41,42].

∘ Leutx gene family. This gene family contains a single gene in the human genome,LEUTX, and no known invertebrate members. We placeLEUTXin the PRD class for four reasons. First, there is weak phylogenetic support for this placement (Additional files1 and2). Second, the LEUTX homeodomain possesses the same combination of residues that are invariably conserved across human PRD-class homeodomains (except for a leucine at position 20; Additional file6). Third, the LEUTX homeodomain shares the 46/47 intron position seen in many PRD-class homeodomains. Fourth, theLEUTXgene is located close to the PRD-class genesTPRX1,CRX,DPRXandDUXAon the distal end of the long arm of chromosome 19 (Figure4). This fourth observation leads us to hypothesize that this gene family arose by tandem duplication and extensive divergence during mammalian evolution.

∘ Nobox gene family. This gene family falls close to the division between the ANTP and PRD classes in both maximum likelihood and neighbor-joining phylogenetic analyses (Additional files1 and2). We favor placement within the PRD class because the NOBOX homeodomain has higher sequence identity with PRD-class homeodomains (up to 55%) than with ANTP-class homeodomains (up to 46%). Chromosomal position does not shed light on the issue, as its location at 7q35 is close to both ANTP- and PRD-class genes (Figure4).

∘ Otx gene family. This very well known gene family was originally considered to contain humanOTX1andOTX2(and their mouse orthologs) and theDrosophila otdgene [43]. Later, it was shown that theCRXgene is a member of the same gene family, deriving from the same ancestral gene. Thus,CRXcould be considered the trueOTX3gene [44]. Unfortunately, theOTX3symbol was formerly used erroneously for a gene in a different family, now calledDMBX1, thus complicating its future use. The gene family name Otx is derived by majority rule from the constituent genes.

∘ Pax2/5/8 gene family. This gene family is also known as Pax group II; it containsPAX2,PAX5andPAX8, clearly derived from a single ancestral gene [45]. These genes have partial homeoboxes.

∘ Pax3/7 gene family. This gene family is also known as Pax group III; it containsPAX3andPAX7, clearly derived from a single ancestral gene [46].

∘ Pax4/6 gene family. This gene family is also known as Pax group IV; it containsPAX4andPAX6. There is confusion as to whether this should be split into two gene families, because invertebrate homologs generally group withPAX6in phylogenetic analyses and not as an outgroup to the two genes as might be expected. We follow the generally accepted view and groupPAX4andPAX6into a single gene family, proposing thatPAX4is a divergent member, not an ancient gene [40].

∘ Rhox gene family. The mouse Rhox cluster was first described as comprising twelve X-linked homeobox genes, all selectively expressed in reproductive tissues [47]. Subsequent studies reported a total of 32 genes in the cluster, with the additional genes attributed to recent tandem duplications [48-51]. The human genome contains three homeobox genes at Xq24 that are clearly members of the Rhox gene family based on sequence identity, molecular phylogenetics, intron positions and chromosomal location. These areRHOXF1(formerlyOTEX/PEPP1),RHOXF2(formerlyPEPP2) andRHOXF2B(formerlyPEPP2b/PEPP3).

Most of the 50 genes in the PRD class have been adequately named previously. However, several genes were unnamed or misnamed prior to this study. We have updated these as follows.

∘ALX1[Entrez Gene ID: 8092] is the first of three human members of the Alx gene family. This gene was previously known asCART1; we rename itALX1because it is related toALX3andALX4; all three genes were formed by duplication from a single ancestral invertebrate gene [52].

∘DRGX[Entrez Gene ID: 117065] is the only member of the newly defined Drgx gene family in the human genome. This gene was previously known asPRRXL1andDRG11, and there is a clear mouse ortholog (Prrxl1). The symbolPRRXL1is misleading because it infers membership of the Prrx gene family, containingPRRX1andPRRX2in the human genome. Several lines of evidence suggest it belongs to a different gene family. First, this gene (at 10q11.23) is not located in the same paralogon asPRRX1(1q24.3) andPRRX2(9q34.11) so they are not three paralogs generated during genome duplication in early vertebrate evolution. Second, it has a completely different exon-intron structure from the Prrx-family genes, and it does not contain a Prrx domain or an OAR domain (present inPRRX1andPRRX2; [53]). Third, the homeodomain is only 73% identical to PRRX1 and PRRX2 homeodomains, much lower than the 80-100% usually encountered for members of the same gene family in humans. Finally, we have identified theDrosophilaortholog,IP09201. The homeodomains ofDrosophilaIP09201 and human DRGX form a highly supported monophyletic group in our maximum likelihood (90%; Additional file1) and neighbor-joining (97%; Additional file2) phylogenetic analyses. The new symbolDRGX(dorsal root ganglia homeobox) incorporates the root of the former symbolDRG11, referring to expression of the rodent ortholog in dorsal root ganglia neurons [54].

∘DUXB[Entrez Gene ID: 100033411] is a human member of the Dux (double homeobox) gene family. As previously discussed, most members of this gene family are intronless and are probably derived by retrotransposition of an mRNA transcript from a functional intron-containing Dux gene (or duplication of such an integrant). Booth and Holland [21] described theDUXAgene containing five introns (including one within each homeobox), and noted the existence of a second intron-containing human Dux-family gene provisionally designatedDUXB. TheDUXBnomenclature is endorsed here. No cDNA or EST sequences have been reported forDUXB.

∘GSC2[Entrez Gene ID: 2928] is the second of two human members of the Gsc gene family. This gene was previously known asGSCL; we rename itGSC2to remove the inadvertent implication that it is not a true gene, and also to reflect the clear orthology to chickGsc2as inferred by phylogenetic analysis and synteny.

∘HOPX[Entrez Gene ID: 84525] is the only member of the newly defined Hopx gene family in the human genome. The mouse version of the gene was first identified first and namedHop(homeodomain only protein) because the encoded protein is just 73 amino acids long, with 61 of these making up the homeodomain [41,42]. TheHOPgene symbol is not ideal as it is also used for unrelated genes, includinghopscotchinDrosophilaandhop-sterilein mouse. Therefore, we revise the gene symbol fromHOPtoHOPX(HOP homeobox) in accordance with homeobox gene nomenclature convention.

∘LEUTX[Entrez Gene ID: 342900] is the only member of the newly defined Leutx gene family in the human genome. We designate this previously unnamed geneLEUTX(leucine twenty homeobox) to reflect the presence of a leucine residue at the otherwise highly conserved homeodomain position 20; other PRD-class homeodomains have a phenylalanine at this position (Additional file6). Studies of mutations in other homeobox genes suggest that mutation to leucine alters transcriptional activity of a homeodomain protein [55].

∘RAX2[Entrez Gene ID: 84839] is the second of two human members of the Rax gene family. This gene was previously known asRAXL1; we rename itRAX2to standardize nomenclature.

∘RHOXF1[Entrez Gene ID: 158800] andRHOXF2[Entrez Gene ID: 84528] are two of three human members of the Rhox gene family. These genes were previously known asOTEX/PEPP1andPEPP2respectively. The prefixPEPPis not suitable as it is used for numerous aminopeptidase P-encoding genes. Thus, we replace the gene symbolsOTEX/PEPP1andPEPP2withRHOXF1andRHOXF2respectively, to reflect their orthologous relationship with the mouse Rhox cluster (containing 32 genes, see above) whilst avoiding inadvertent equivalence to specific genes within the cluster.

∘RHOXF2B[Entrez Gene ID: 727940] is the third human member of the Rhox gene family. This locus was referred to in previous studies asPEPP2b[56] andPEPP3[51]. The prefixPEPPcannot be approved for reasons noted above.RHOXF2Bis located very close toRHOXF1andRHOXF2at Xq24 and is clearly a very recent duplicate ofRHOXF2. The genomic sequences at these two loci share 99% identity over exonic, intronic and approximately 20 kb flanking regions. Over the coding region, there are just two nucleotide substitutions (both nonsynonymous); one of these results in an unusual change within the homeodomain (arginine to cysteine at position 18). We currently listRHOXF2Bas a functional gene, although it is possible that it is a duplicated pseudogene.

∘SEBOX[Entrez Gene ID: 645832] is the only member of the Sebox gene family in the human genome. The human gene is the ortholog of mouseSeboxbased on their locations in syntenic chromosomal regions (17q11.2 and 11B5 respectively) and presence of the same intron positions. However, sequence identity is lower than normal for orthologous genes in mouse and human (78% amino acid identity over the homeodomain) and there is evidence that the human gene has undergone divergence. Most surprisingly, the human sequence has two unusual substitutions in the homeodomain [57]. At homeodomain position 51, the human sequence codes for lysine whereas mouse has asparagine; an earlier analysis of 346 homeodomain sequences found asparagine to be invariant at this position [1,2]. Similarly, at homeodomain position 53, human has tryptophan whereas mouse has arginine; this position is almost invariably arginine [1,2]. These sequence changes in the important third helix raise the possibility that humanSEBOXcould have accumulated mutations as a non-functional pseudogene. Until this is shown more clearly we consider it to be a functional, but divergent, gene. This gene was previously known asOG9XwithSEBOXas the alternative symbol; we favorSEBOXbecause theOGprefix was originally used for several unrelated homeobox genes.

∘UNCX[Entrez Gene ID: 340260] is the only member of the Uncx gene family in the human genome. This gene was previously known asUNCX4.1; we remove the numerals to giveUNCXas these do not denote a series within a gene family.

∘VSX2[Entrez Gene ID: 338917] is the second of two human members of the Vsx gene family. This gene was previously known asCHX10; we rename itVSX2to better reflect its paralogous relationship toVSX1.VSX2has been used as an alias for this gene in other vertebrate species and the gene symbolCHX10has the disadvantage of implicitly suggesting presence of at least nine paralogs in human (CHX1toCHX9), which do not exist.

Unlike the situation with the ANTP class, many of the pseudogenes within the PRD class have been well characterized. A previous study has described and named two pseudogenes in the Argfx gene family, seven pseudogenes in the Dprx gene family, four pseudogenes in the Tprx gene family, and 10 pseudogenes derived from theDUXAgene [21]. There is also a possibility that theSEBOXandRHOXF2Bloci are non-functional pseudogenes, as described above. We have identified a previously undescribed pseudogene from the Otx gene family (OTX2P1), and argue that the majority of Dux-family sequences are pseudogenes.

∘OTX2P1[Entrez Gene ID: 100033409]. We designate this previously undescribed sequenceOTX2P1because it is clearly a retrotransposed pseudogene ofOTX2. The genomic DNA sequence ofOTX2P1shares significant homology withOTX2transcript variant 2 [RefSeq:NM_172337]. There is an Alu element (AluSx subfamily) insertion, a Made1 (Mariner derived element 1) insertion, and a 1182-nucleotide deletion inOTX2P1compared toOTX2. TheOTX2P1sequence lacks introns, ends with a poly(A) tail, and harbors critical sequence alterations (including a three-nucleotide insertion introducing a stop codon into the deduced homeodomain).

∘DUX1[EMBL:AJ001481],DUX2[GenBank:AF068744],DUX3[GenBank:AF133130] andDUX5[GenBank:AF133131]. These sequences have been cloned in previous studies [12,13]. We detected no matches with 100% identity toDUX1,DUX2,DUX3orDUX5in build 35.1 of the human genome sequence, which covers the euchromatic regions of each chromosome. This concurs with previous studies indicating thatDUX1,DUX2,DUX3andDUX5are found in heterochromatin on human acrocentric chromosomes; each is apparently present in multiple copies within members of the 3.3 kb family of tandemly repeated DNA elements [12,13]. Because the majority of human heterochromatin has not been sequenced, and may be variable between individuals, the exact number of copies ofDUX1,DUX2,DUX3andDUX5is unknown. It is also debatable whether these loci encode functional proteins. These sequences lack introns and, as discussed above, are most likely derived from intron-containing genes in the Dux family, such asDUXAorDUXB.

∘DUX4[GenBank:AF117653]. This sequence has been extensively studied as some of its multiple copies exist within the 3.3 kb repetitive elements of the D4Z4 locus at 4q35 [14]. The polymorphic D4Z4 locus is linked to facioscapulohumeral muscular dystrophy (FSHD); between 12 and 96 tandem copies of 3.3 kb elements are present in unaffected individuals and deletions leaving a maximum of eight such elements have been associated with FSHD [58]. In build 35.1 of the human genome sequence, we identified 35 loci at 10 chromosomal locations containing a total of 58DUX4(and highly similar) homeobox sequences. This should not be taken as a precise figure due to copy number polymorphism and the possibility of additional copies existing in currently unsequenced heterochromatic regions. Some of the copies are 100% identical to the previously reportedDUX4sequence over the homeobox regions, others have single nucleotide polymorphisms, some have critical sequence mutations, and others have just a single homeobox. Most of the copies are located in tandemly repeated arrays (for example, on chromosomes 4, 10 and 16) and others are alone in the genome (for example, a single copy resides at 3p12.3). The majority ofDUX4copies are unlikely to encode functional proteins as suggested by their intronless, mutated and tandemly repeated nature. The lack of introns indicates they are most likely derived from intron-containing genes in the Dux family, such asDUXAorDUXB.

The LIM homeobox class

The LIM class encodes proteins with two LIM domains (named from the nematodelin-11, mammalianIsl1and nematodemec-3genes) N-terminal to a typical (i.e. 60-amino-acid) homeodomain. The LIM domain is a protein-protein interaction domain of approximately 55 amino acids comprising two specialised cysteine-rich zinc fingers in tandem [59]. Importantly, human genes also exist that encode LIM domains but not homeodomains. These LIM domains are divergent from the LIM domains encoded by LIM homeobox genes, and hence these genes are unlikely to be derived by loss of the homeobox. There is one exception: the human Lmo gene family encodes LIM domains that have been grouped by sequence similarity and domain arrangement with the LIM domains of the LIM homeobox gene class [59]. Thus, this gene family may have secondarily lost the homeobox, although this remains untested. Only genes encoding both LIM domains and homeodomains are included in our LIM homeobox gene count.

We have identified a total of twelve LIM-class homeobox genes in the human genome (Tables1 and4), consistent with previous work [60]. Phylogenetic analyses of homeodomains do not always recover the LIM class as a monophyletic group, depending on the dataset and method used (Figure3; Additional files1,2 and5), but it is likely that the class evolved from a single fusion event that brought together LIM domains and a homeodomain. Phylogenetic analyses of homeodomains divide the LIM class into six gene families (Figure3; Additional files1,2 and5), consistent with previous studies [60]. Each gene family has two human members and dates to a single ancestral gene in the most recent common ancestor of bilaterians [60]. We have not found any human LIM-class pseudogenes.

The POU homeobox class

The POU class generally encodes proteins with a POU-specific domain (named from the mammalian genesPit1(nowPou1f1),OCT1andOCT2(nowPOU2F1andPOU2F2), andnematodeunc-86) N-terminal to a typical homeodomain. The POU-specific domain is a DNA-binding domain of approximately 75 amino acids; the POU-specific domain and the homeodomain are collectively known as the bipartite POU domain [61].

We have identified a total of 16 POU-class homeobox genes in the human genome (Tables1 and4). The genes form a distinct grouping even if the POU-specific domain is disregarded – phylogenetic analyses of homeodomains recover the POU class as a monophyletic group (Figure3; Additional files1,2 and5). There are six widely recognized gene families within the POU class (Pou1 to Pou6), and nomenclature revisions approximately 10 years ago clarified which genes belong to which gene family [62]. We have placed two additional genes (HDXandPOU5F2) in the POU class on the basis of their deduced homeodomain sequences, even though one of these genes (HDX) does not encode a POU-specific domain. We have erected a new gene family for this gene, bringing the total number of gene families in the POU class to seven. We have also identified a total of eight POU-class pseudogenes in the human genome (Tables1 and4); we have named six of these (POU5F1P2,POU5F1P4toPOU5F1P8), and revised the nomenclature of one other (POU5F1P3).

∘HDX[Entrez Gene ID: 139324]. This gene was previously known asCXorf43. The gene encodes a highly divergent atypical (68-amino-acid) homeodomain but not a POU-specific domain, and thus it is debatable whether it should be placed within the POU class. Phylogenetic analyses of homeodomains place it basally in a clade with the POU class (Figure3; Additional files1 and5), or within the POU class (Additional file2), suggesting that the HDX protein either diverged before the POU-specific domain became associated with the homeodomain or lost the POU-specific domain during evolution. Further information on this gene may allow this tentative classification to be revisited.

∘POU5F2[Entrez Gene ID: 134187]. We designate this previously unnamed genePOU5F2on the basis of clear orthology to the mouseSprm1gene, which has been assigned the second member of the Pou5 gene family [63]. The symbolPOU5F2ensures the gene conforms with standardized nomenclature for the POU class.

∘POU5F1P2[GeneID: 100009665],POU5F1P3(formerlyPOUF51L) [GeneID: 5461],POU5F1P4[GeneID: 100009666],POU5F1P5[GeneID: 100009667],POU5F1P6[GeneID: 100009668],POU5F1P7[GeneID: 100009669] andPOU5F1P8[GeneID: 100009670]. Prior to this study, a single retrotransposed pseudogene of thePOU5F1gene had been annotated and designatedPOU5F1P1[Entrez Gene ID: 5462]. AnotherPOU5F1-related sequence of unknown status had been annotated and designatedPOUF5F1L[GeneID: 5461]. We replace the gene symbolPOUF5F1LwithPOU5F1P3as this sequence is a retrotransposed pseudogene ofPOUF51. Our analyses of the human genome sequence identified a further six pseudogenes ofPOU5F1, which we name sequentiallyPOU5F1P2,POU5F1P4through toPOU5F1P8. Each clearly aligns to the mRNA sequence ofPOU5F1but with sequence alterations, indicating origin by retrotransposition.POU5F1P2andPOU5F1P6have frameshift mutations in the homeobox.POU5F1P5andPOU5F1P6have stop codons in the homeobox.POU5F1P7andPOU5F1P8are partial integrants ofPOU5F1mRNA excluding the homeobox –POU5F1P7covers part of the 3' untranslated region andPOU5F1P8a short region around the start codon.

The HNF homeobox class

The HNF class (named after the rat geneHnf1) encodes proteins with a POU-like domain N-terminal to a highly atypical homeodomain. The POU-like domain, as its name indicates, is weakly similar in sequence to the POU-specific domain [64]; more importantly, it has nearly the same three-dimensional structure and mode of DNA binding as the POU-specific domain [65].

We have identified a total of three HNF-class homeobox genes in the human genome (Tables1 and4), consistent with previous work [66,67]. The homeodomains encoded by the humanHNF1AandHNF1Bgenes are atypical in possessing 21 extra amino acid residues between the second and third alpha helices (Additional file6). We place these two genes in a single gene family (Hnf1) within the HNF class, implying derivation from a single invertebrate gene. Examination of their chromosomal locations concurs with this view.HNF1AandHNF1Bmap to parts of the genome known to have duplicated in early vertebrate evolution, namely 12q24.31 (HNF1A, nearLHX5and on the same arm as the HOXC cluster) and 17q12 (HNF1B, betweenLHX1and the HOXB cluster) (Figure4). The use of the A and B suffixes is unfortunate, as numerals are generally used to distinguish paralogs of this age, but is retained at present due to widespread and stable use. The homeodomain encoded by the humanHMBOX1gene is atypical in possessing 15 extra amino acid residues between the second and third alpha helices (Additional file6). Phylogenetic analyses confirm previous suggestions [67] thatHMBOX1is more distantly related toHNF1AandHNF1B(Figure3; Additional files1,2 and5). We place this gene in a separate gene family (Hmbox) within the same class. We have not found any human HNF-class pseudogenes.

The SINE homeobox class

The SINE class (named after theDrosophilageneso:sine oculis) encodes proteins with a SIX domain N-terminal to a typical homeodomain. The SIX domain is a DNA-binding domain of approximately 115 amino acids; both the SIX domain and the homeodomain are required for DNA binding [68].

We have identified a total of six SINE-class homeobox genes in the human genome (Tables1 and4), consistent with previous work [68,69]. The genes form a distinct grouping even if the SIX domain is disregarded – phylogenetic analyses of homeodomains recover the SIX class as a monophyletic group (Figure3; Additional files1,2 and5). Phylogenetic analyses of homeodomains divide the SIX class into three gene families (Figure3; Additional files1,2 and5), consistent with previous studies [68,69]. Each gene family has two human members and dates to a single ancestral gene in the most recent common ancestor of bilaterians [68,69]. We have not found any human SINE-class pseudogenes.

The TALE homeobox class

TALE (three amino acid loop extension) class genes are distinguished by the presence of three extra amino acids between the first and second alpha helices of the encoded homeodomain [1,2,70]. Genes belonging to the TALE class encode proteins with various domains outside of the atypical homeodomain.

We have identified a total of 20 TALE-class homeobox genes in the human genome (Tables1 and4). The genes form a distinct grouping in phylogenetic analyses even when the three extra homeodomain residues are excluded from the sequence alignment (Figure3; Additional file5). Bürglin [2] has given the TALE group the rank of 'superclass' and distinguished between several 'classes' by the presence of distinct domains outside of the homeodomain. These are the IRX domain, MKX domains, the MEIS domain, the PBC domain and TGIF domains [2,71-73]. Along with some others [4,7,24], we have given the TALE group the rank of 'class' containing several 'gene families'; this maintains consistent terminology throughout the present paper. Phylogenetic analyses of homeodomains divide the TALE class into six gene families (Figure3; Additional files1,2 and5), including an Mkx family containing the recently describedMKXgene, which is distinguished from Irx-family genes phylogenetically and by absence of an IRX domain [73,74]. It should be noted that the established name of the Pknox gene family does not indicate orthology with Knox-family genes of plants. We have also identified a total of 10 TALE-class pseudogenes in the human genome (Tables1 and4); we have named six of these (IRX4P1,TGIF1P1andTGIF2P1toTGIF2P4), and revised the nomenclature of two others (IRX1P1andPBX2P1).

∘IRX1P1[Entrez Gene ID: 646390]. This sequence was previously known asIRXA1; we rename itIRX1P1because it is clearly a retrotransposed pseudogene ofIRX1and not a functional gene. TheIRX1P1sequence aligns to the mRNA ofIRX1but has a frameshift mutation and two stop codons in the homeobox.

∘IRX4P1[Entrez Gene ID: 100009671]. We designate this previously unannotated sequenceIRX4P1because it is clearly a retrotransposed pseudogene ofIRX4. TheIRX4P1sequence is a partial integrant derived from a region of theIRX4mRNA around the stop codon; it lacks the homeobox.

∘PBX2P1[Entrez Gene ID: 5088]. This sequence was previously known asPBXP1; we rename itPBX2P1because it is clearly a retrotransposed pseudogene ofPBX2. The former name ofPBXP1did not indicate its transcript of origin. ThePBX2P1sequence aligns to the mRNA ofPBX2but has a frameshift mutation in the coding region.

∘TGIF1P1[Entrez Gene ID: 126052]. We designate this previously unannotated sequenceTGIF1P1because it is clearly a retrotransposed pseudogene ofTGIF1. The locus has many sequence alterations when compared toTGIF1mRNA, including a 48 nucleotide insertion within the homeobox.

∘TGIF2P1[GeneID: 126826],TGIF2P2[GeneID: 100009674],TGIF2P3[GeneID: 100009672] andTGIF2P4[GeneID: 100009673]. These four sequences were unannotated prior to this study. We designate themTGIF2P1toTGIF2P4because they are clearly pseudogenes ofTGIF2. Each aligns to the mRNA sequence ofTGIF2but with sequence alterations, indicating origin by retrotransposition.TGIF2P1has many sequence alterations, including a frameshift mutation in the homeobox.TGIF2P2andTGIF2P3are very similar neighboring loci that must have originated by tandem duplication of a retrotransposedTGIF2mRNA; neither includes the homeobox.TGIF2P4is a short partial integrant derived from part of the 3' untranslated region ofTGIF2mRNA.

The CUT homeobox class

The CUT class (named after theDrosophilagenecut) generally encodes proteins with one or more CUT domains N-terminal to a typical homeodomain. The CUT domain is a DNA-binding domain of approximately 75 amino acids [75]. There are three widely recognized gene families within the CUT class in humans (Onecut, Cux, Satb; [76]). A fourth gene family (Cmp), lacking a CUT domain but sharing a CMP domain with the Satb gene family, is absent from vertebrates. Bürglin and Cassata [76] have proposed that the vertebrate Satb gene family evolved from the invertebrate Cmp gene family.

We have identified a total of seven CUT-class homeobox genes in the human genome (Tables1 and4). Although grouped together by presence of CUT domains, the homeodomains of the Onecut, Cux and Satb gene families are quite divergent and do not always form a monophyletic group in phylogenetic analyses (Additional files2 and5). Topologies that separate the gene families are also only weakly supported, so it is most parsimonious to assume that the class is actually monophyletic but the constituent genes underwent rapid sequence divergence following their initial duplications. We have revised the nomenclature of two CUT-class genes (CUX1andCUX2). We have also identified a total of three CUT-class pseudogenes in the human genome (Tables1 and4); we have named all of these (CUX2P1,CUX2P2andSATB1P1).

∘CUX1[Entrez Gene ID: 1523] andCUX2[Entrez Gene ID: 23316]. These genes were previously known asCUTL1andCUTL2respectively. We rename themCUX1andCUX2in accordance with homeobox gene nomenclature convention.

∘CUX2P1andCUX2P2. These sequences were unannotated prior to this study. We designate themCUX2P1andCUX2P2because they are clearly retrotransposed pseudogenes ofCUX2. Both are short partial integrants derived fromCUX2mRNA, excluding the homeobox –CUX2P1covers part of the coding region at the 5' end andCUX2P2part of the 3' untranslated region.

∘SATB1P1[Entrez Gene ID: 100033410]. We designate this previously unannotated sequenceSATB1P1because it is clearly a retrotransposed pseudogene ofSATB1.SATB1P1is a short partial integrant derived from part of the 3' untranslated region ofSATB1mRNA; it does not encompass the homeobox.

The PROS homeobox class

The PROS class (named after theDrosophilagenepros) encodes proteins with a PROS domain C-terminal to an atypical homeodomain. The PROS domain is a DNA-binding domain of approximately 100 amino acids [77]. PROS-class genes encode a highly divergent homeodomain with three extra amino acids. These additional residues are inserted at a different position compared to the TALE class, being between the second and third alpha helices (Additional file6).

We have identified a total of two PROS-class homeobox genes in the human genome (Tables1 and4), which we have placed in a single gene family (Prox). The highly divergent homeodomain sequence and unusual structural features provide justification for PROS being a separate gene class, despite the small number of genes. In phylogenetic analyses, PROS-class homeodomains are situated on a long branch, very distant from other classes (Figure3; Additional files1,2 and5). The humanPROX1gene is well characterized; we have identified and named its paralog,PROX2. We have not found any human PROS-class pseudogenes.

∘PROX2[Entrez Gene ID: 283571]. We designate this previously unannotated genePROX2on the basis of clear orthology to the mouseProx2gene, inferred from sequence identity and synteny. The homeobox of humanPROX2has two introns and unusually the splice sites of the first (5') intron (AT-AA) do not follow the GT-AG donor-acceptor rule. This has also been noted for mouseProx2[78].

The ZF homeobox class

The ZF (zinc finger) class generally encodes proteins with zinc finger motifs, in addition to one or more homeodomains. As noted earlier, phylogenetic analyses of homeodomains does not recover the ZF class as a monophyletic group (Figure3; Additional files1,2 and5). We recognize that this suggests that zinc finger motifs and homeodomains may have been brought together on three separate occasions in evolution; nonetheless, it is convenient and informative to group these into a single class. Inclusion of theHOMEZgene in the ZF class may be surprising, as this gene does not encode zinc fingers. However, as previously noted [79] and reproduced in our phylogenetic analyses (Figure3; Additional files1,2 and5), the multiple homeodomain sequences of this gene are clearly related to those encoded by theZHX1,ZHX2andZHX3genes.

We have identified a total of 14 ZF-class homeobox genes in the human genome (Tables1 and4), which we have placed in five gene families (Adnp, Tshz, Zeb, Zfhx and Zhx/Homez). We have also identified one ZF-class pseudogenes in the human genome (Tables1 and4). We have revised the nomenclature of five of these loci (ADNP2,ZEB1,ZEB2,ZEB2P1andZFHX3).

∘ADNP2[Entrez Gene ID 22850]. This gene was previously known asZNF508; we rename itADNP2to reflect its paralogous relationship toADNP.

∘ZEB1[Entrez Gene ID: 6935] andZEB2[Entrez Gene ID: 9839]. These genes were previously known asZFHX1AandZFHX1Brespectively. We rename themZEB1andZEB2to distinguish them from genes belonging to the distantly related Zfhx gene family.

∘ZEB2P1[Entrez Gene ID: 100033412]. This retrotransposed pseudogene ofZEB2has been described previously [80]. Our new nomenclature (ZEB2P1) reflects the origin of this locus.

∘ZFHX3[Entrez Gene ID: 463]. This gene was previously known asATBF1; we rename itZFHX3to reflect its close relationship toZFHX2andZFHX4; indeedZFHX3was a synonym for this gene.

The CERS homeobox class

The highly unusual CERS (ceramide synthase) class, also known as the LASS (longevity assurance) class, comprises a single gene family that is highly conserved amongst eukaryotes and includes the yeast gene and original memberLAG1. There are six CERS-class genes in the human genome (CERS1toCERS6) and five of these (CERS2toCERS6) encode proteins with a homeodomain sequence [81,82]. These are, however, extremely divergent from the homeodomains of other gene classes. Secondary structure prediction analyses suggest these sequences have the potential to encode three alpha helices in the appropriate positions (data not shown). The most surprising characteristic of these genes is that biochemical studies predict them to encode transmembrane proteins, with the homeodomain on the cytosolic side of the endoplasmic reticulum membrane, and hence they could not act as DNA-binding proteins or transcription factors [81,82]. It is possible that an ancestor of these genes gained a homeobox through exon shuffling, or alternatively this could represent convergent evolution. We include onlyCERS2toCERS6in our comprehensive compilation of human homeobox genes, asCERS1lacks a homeobox motif.

Chromosomal distribution of human homeobox genes

The chromosomal locations of genes can give clues to evolutionary ancestry, including patterns of gene duplication, and the possible existence of gene clusters. In Figure4, we show the chromosomal locations of all human homeobox genes. We do not include probable pseudogenes on these ideograms, because most of these have originated by reverse transcription of mRNA and secondary integration into the genome, and hence give no insight into ancestral locations of genes. The highly repetitiveDUX1toDUX5sequences are also not shown, as these have undergone secondary amplification and are also most likely non-functional (see above).

The first observation is that there are homeobox genes on every human chromosome. Even the two sex chromosomes harbor homeobox genes, withSHOX(short stature homeobox) in the PAR1 pseudoautosomal region at the tip of the short arms of X and Y being the best known. Haploinsufficiency ofSHOXis implicated in the short stature phenotype of Turner syndrome patients who lack one copy of the X chromosome [83]. There are also nine other homeobox genes in non-pseudoautosomal regions of the X chromosome, including three tandemly-arranged members of the Rhox gene family, collectively homologous to the multiple Rhox (reproductive homeobox) genes of mouse. Only one of the homeobox genes on the X chromosome, the TALE-class geneTGIF2LX, has a distinct homolog on the Y chromosome, calledTGIF2LY. These genes map to the largest homology block shared by the unique regions of the X and Y chromosomes, spanning 3.5 Mb. It has been proposed that the ancestor of these two genes arose by retrotransposition ofTGIF2mRNA [84].

The autosomes with the lowest number of homeobox genes are chromosomes 21 (with justPKNOX1) and 22 (withGSC2andISX). Examination of the remaining autosomes reveals that homeobox genes are quite dispersed with some interesting regional accumulations. The best known examples of close linkage between homeobox genes are the four Hox clusters on human chromosomes 2, 7, 12 and 17, comprising 9, 11, 9 and 10 genes respectively; each of these is shown as just a single line on each ideogram for simplicity (Figure4). These should not be considered in isolation, however, because many other ANTP-class genes map in the vicinity of the Hox clusters [26,27]. These include genes very tightly linked to the Hox clusters, notably the Evx-family genes (on chromosomes 2 and 7), Dlx-family genes (on chromosomes 2 and 17), and Meox-family genes (on chromosomes 2 and 17).

There are other concentrations of ANTP-class genes away from the Hox clusters. These are the ParaHox cluster (GSX1,PDX1,CDX2) on chromosome 13, and four sets of NKL-subclass genes on 2p/8p (split), 4p, 5q and 10q, hypothesized to be derived from an ancestral array by duplication [26,33]. The accumulation on the distal half of the long arm of chromosome 10 is particularly striking, comprising eleven ANTP-class genes from 10 gene families. This is not a tight gene cluster, but it is compatible with ancestry by extensive tandem gene duplication followed by dispersal. Discounting the rather aberrant case of the Hox clusters, this region of the long arm of chromosome 10 is the most homeobox-rich region of the human genome.

There are additional groupings of homeobox genes outside the ANTP class. These include two TALE-class Irx clusters on chromosomes 5 and 16 homologous to the described mouse Irx clusters [19], and a set of PRD-class genes on chromosome 19 proposed to be derived from theCRXhomeobox gene by duplication and rapid divergence [21]. Perhaps the most interesting case, however, is found on the tip of the long arm of chromosome 9, where there is a concentration of homeobox genes from disparate gene classes. Four LIM-class genes, one ANTP-class gene, one PRD-class gene and one TALE-class gene are found in this location. Although dispersed over a large region, and not forming a tight gene cluster, the linkages are nonetheless intriguing. It is possible that these linkages reflect ancestry from the very ancient gene duplications that must have generated the distinctive homeobox gene classes found within animal genomes.

Acknowledgements

We thank Rebecca Furlong, Tokiharu Takahashi, Hidetoshi Saiga, Naohito Takatori, David Ferrier, Mario Pestarino, Thomas Bürglin and reviewers for helpful advice. Research undertaken by PWHH and HAFB was supported by the BBSRC and the Wellcome Trust. The work of EAB and the HUGO Gene Nomenclature Committee is supported by NHGRI grant P41 HG003345 and the Wellcome Trust.

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