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, 5 (5), e10820

Improving Hox Protein Classification Across the Major Model Organisms

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Improving Hox Protein Classification Across the Major Model Organisms

Stefanie D Hueber et al. PLoS One.

Abstract

The family of Hox-proteins has been a major focus of research for over 30 years. Hox-proteins are crucial to the correct development of bilateral organisms, however, some uncertainty remains as to which Hox-proteins are functionally equivalent across different species. Initial classification of Hox-proteins was based on phylogenetic analysis of the 60 amino acid homeodomain. This approach was successful in classifying Hox-proteins with differing homeodomains, but the relationships of Hox-proteins with nearly identical homeodomains, yet distinct biological functions, could not be resolved. Correspondingly, these 'problematic' proteins were classified into one large unresolved group. Other classifications used the relative location of the Hox-protein coding genes on the chromosome (synteny) to further resolve this group. Although widely used, this synteny-based classification is inconsistent with experimental evidence from functional equivalence studies. These inconsistencies led us to re-examine and derive a new classification for the Hox-protein family using all Hox-protein sequences available in the GenBank non-redundant protein database (NCBI-nr). We compare the use of the homeodomain, the homeodomain with conserved flanking regions (the YPWM and linker region), and full length Hox-protein sequences as a basis for classification of Hox-proteins. In contrast to previous attempts, our approach is able to resolve the relationships for the 'problematic' as well as ABD-B-like Hox-proteins. We highlight differences to previous classifications and clarify the relationships of Hox-proteins across the five major model organisms, Caenorhabditis elegans, Drosophila melanogaster, Branchiostoma floridae, Mus musculus and Danio rerio. Comparative and functional analysis of Hox-proteins, two fields crucial to understanding the development of bilateral organisms, have been hampered by difficulties in predicting functionally equivalent Hox-proteins across species. Our classification scheme offers a higher-resolution classification that is in accordance with phylogenetic as well as experimental data and, thereby, provides a novel basis for experiments, such as comparative and functional analyses of Hox-proteins.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Classification schemes for Drosophila melanogaster and Mus musculus Hox-proteins.
The Hox-protein coding genes are depicted and classified according to the encoded proteins. A) Phylogeny-based classification of Hox-proteins according to their inferred ancestry based on their similarities across the homeodomains. Such classifications often include representation of a hypothesized common ancestor. B) Frequently depicted Hox-classification scheme in which synteny was used to further resolve the ANTP, UBX, ABD-A vs. Hox6, Hox7, Hox8 grouping. The difference between these classification schemes is best exemplified by the classification of the Drosophila ANTP, UBX and ABD-A proteins in relation to the vertebrate Hox6, Hox7 and Hox8 groups of proteins. In A) these proteins are grouped together and it remains unclear which of the proteins in this group are to be regarded as functionally most similar across the species. In B) these proteins are grouped according to the relative positions of their genes within the Hox-cluster.
Figure 2
Figure 2. Experiments supporting and conflicting with assignments of presumed functionally equivalent Hox-proteins.
The left side depicts the known expression patterns for the Hox-genes in four model organisms. The right side illustrates the corresponding chromosomal organization of the Hox-genes. Individual Hox-genes are represented by colored arrows. The color code is the same as used in the classification scheme in Figure1B (synteny-based). In C. elegans some genes have two colors, as a single equivalent protein in the other model organisms could not be determined. Experiments analyzing functional equivalence are shown as lines connecting genes in different Hox-clusters. Green and red lines indicate, respectively, supporting and conflicting experimental evidence regarding presumed functional equivalence of the proteins. The diagram is not to scale for either organism size, gene size or cluster size. Comparison of the Drosophila LAB and vertebrate HOXB1 was carried out in D. melanogaster using a Gallus gallus (chicken) HOXB1.
Figure 3
Figure 3. Homeodomain-only clustering.
A) CLANS overview of the pairwise sequence similarities for the set of 15,788 sequences identified as potentially Hox-related. P-cutoff  =  10−15; coloring: red  =  Paired, yellow  =  Irx, turquoise  =  NK-cluster. B) Detailed view of the Hox/ParaHox/Nk-cluster identified in A). P-cutoff  = 10−18; coloring as in A). C) Detailed view of the Hox and Hox-like sequences identified in B) (including the non-Hox-protein ‘Cdx/Cad’, Gsx/Ind and Mox clusters). P-cutoff  = 10−18; coloring as in Figure 1B. 5-pointed stars represent Drosophila melanogaster, 4-pointed stars represent Caenorhabditis elegans, circles represent Branchiostoma floridae, rectangles represent Mus musculus and rhomboids represent Danio rerio sequences.
Figure 4
Figure 4. Overview of Hox-clusters generated by CLANS.
A) 60 amino acid homeodomain sequences, B) Full-length protein sequences and C) Extended homeodomain sequences. Irrespective of the type of sequence used, the CLANS analyses generate very similar cluster maps in which the seven major groups identified in Figure 3C can be found in comparable positions.
Figure 5
Figure 5. Detailed clustering of sequences from groups 1 and 2 identified in Figure 3C .
P-cutoff  = 10−18. Coloring as in Figure 1B: pink  =  LAB+Hox1, purple  =  PB+Hox2, beige  =  Hox3. 5-pointed stars represent Drosophila melanogaster, 4-pointed stars represent Caenorhabditis elegans, circles represent Branchiostoma floridae, rectangles represent Mus musculus and rhomboids represent Danio rerio sequences. Group 2 sequences can further be subdivided into groups 2A (Hox2-like) and 2B (Hox3-like).
Figure 6
Figure 6. 2D clustering of group 3.
P-value 10−25; coloring as in Figure 1B: red  =  DFD+Hox4, orange  =  SCR+Hox5, yellow  =  ANTP+Hox6, green  =  UBX+Hox7, light-blue  =  ABD-A+Hox8. 5-pointed stars represent Drosophila melanogaster, 4-pointed stars represent Caenorhabditis elegans, circles represent Branchiostoma floridae, rectangles represent Mus musculus and rhomboids represent Danio rerio sequences. Separate clusters are formed by the protein sequences for 3A  =  DFD/Hox4, 3B  =  SCR/Hox5, 3C  =  ANTP/Hox7, 3D  =  Hox6, 3E  =  Hox8, 3F  =  UBX and 3G  =  ABD-A.
Figure 7
Figure 7. 2D-representations of a 3D homeodomain clustering of group 4.
This group was clustered in 3D, as a 2D clustering was unable to provide sufficient resolution to show the sub-structure present within this group. The two figures differ by a 90° rotation around the X-axis; P-value cutoff  = 10−15. Three major groups are visible: 4A  =  Drosophila ABD-B, 4B  =  vertebrate Hox9 and 4C  =  vertebrate Hox10 sequences.
Figure 8
Figure 8. 2D homeodomain clustering of groups 5 to 7.
P-value cutoff  = 10−18. Group 5 combines vertebrate Hox11, group 6 vertebrate Hox12 and group 7 vertebrate Hox13 sequences. Amphioxus Hox13 and Hox14 sequences can be seen grouping in close proximity, but not as part of group 5 (vHox11).
Figure 9
Figure 9. Comparative 2D clustering of the Drosophila ANTP, UBX, ABD-A and the vertebrate Hox6, Hox7 and Hox8 sequence groups.
The sequence groups were identified based on Figure 6. Depicted are the clusters for homeodomain, full-length as well as extended homeodomain sequences. Coloring as in Figure 1B: yellow  =  ANTP+Hox6, green  =  UBX+Hox7, light-blue  =  ABD-A+Hox8. 5-pointed stars represent Drosophila melanogaster, circles represent Branchiostoma floridae, rectangles represent Mus musculus and rhomboids represent Danio rerio sequences. A) 60 amino acid homeodomain sequences. Separate clusters are apparent for ANTP+Hox7, ABD-A, UBX and Hox8 sequences. Vertebrate Hox6 sequences are dispersed across multiple clusters. B) Full-length protein sequences. Separate clusters are formed for arthropod and vertebrate sequences. ANTP lies in close proximity to Hox6 and Hox7 sequences. C) Extended homeodomain sequences. ANTP and Hox7 appear in one cluster while, ABD-A, UBX, Hox8 and vertebrate Hox6 sequences form separate groups. These figures were generated using the same CLANS P-value cut-off of 10−26.
Figure 10
Figure 10. Proposed classification.
The organisms are ordered to show the clearest representation of the Hox-proteins most similar in sequence and thus expected to be most similar in function. The figure is not supposed to indicate that Drosophila is descended from Caenorhabditis or that the chordates descended from Drosophila. Vertical gray lines delineate sequence similarity groups. Colored lines linking Hox-genes indicate which Hox-proteins are most sequence similar to one another. Links within a species indicate a presumed multiplication, or loss, of the corresponding proteins in a lineage, while links between species indicate the most sequence similar pairs of Hox-proteins in these species. The colors are used to represent groups of similar sequences, except for the ‘non-colors’ white and gray. These ‘non-colors’ indicate proteins with considerable sequence divergence to any other sequence in the model organisms we compare. Zebrafish is not depicted as the assignment between tetrapods and zebrafish is clear.

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