Triacylglycerol biosynthesis in shaded seeds of tung tree (Vernicia fordii) is regulated in part by Homeodomain Leucine Zipper 21.

Light quantity and quality affect many aspects of plant growth and development. However, few reports have addressed the molecular connections between seed oil accumulation and light conditions, especially dense shade. Shade-avoiding plants can redirect plant resources into extension growth at the expense of leaf and root expansion in an attempt to reach areas containing richer light. Here, we report that tung tree seed oil accumulation is suppressed by dense shade during the rapid oil accumulation phase. Transcriptome analysis confirmed that oil accumulation suppression due to dense shade was attributed to reduced expression of fatty acid and triacylglycerol biosynthesis-related genes. Through weighted gene co-expression network analysis, we identified 32 core transcription factors (TFs) specifically upregulated in densely shaded seeds during the rapid oil accumulation period. Among these, VfHB21, a class I homeodomain leucine zipper TF, was shown to suppress expression of FAD2 and FADX, two key genes related to α-eleostearic acid, by directly binding to HD-ZIP I/II motifs in their respective promoter regions. VfHB21 also binds to similar motifs in the promoters of VfWRI1 and VfDGAT2, two additional key seed lipid regulatory/biosynthetic genes. Functional conservation of HB21 during plant evolution was demonstrated by the fact that AtWRI1, AtSAD1, and AtFAD2 were downregulated in VfHB21-overexpressor lines of transgenic Arabidopsis, with concomitant seed oil reduction, and the fact that AtHB21 expression also was induced by shade. This study reveals some of the regulatory mechanisms that specifically control tung tree seed oil biosynthesis and more broadly regulate plant storage carbon partitioning in response to dense shade conditions.

Basic leucine zipper (bZIP) transcription factor genes and their responses to drought stress in ginseng, Panax ginseng C.A. Meyer.

BACKGROUND: Ginseng is an important medicinal herb in Asia and Northern America. The basic leucine zipper (bZIP) transcription factor genes play important roles in many biological processes and plant responses to abiotic and biotic stresses, such as drought stress. Nevertheless, the genes remain unknown in ginseng. RESULTS: Here, we report 91 bZIP genes identified from ginseng, designated PgbZIP genes. These PgbZIP genes were alternatively spliced into 273 transcripts. Phylogenetic analysis grouped the PgbZIP genes into ten groups, including A, B, C, D, E, F, G, H, I and S. Gene Ontology (GO) categorized the PgbZIP genes into five functional subcategories, suggesting that they have diversified in functionality, even though their putative proteins share a number of conserved motifs. These 273 PgbZIP transcripts expressed differentially across 14 tissues, the roots of different ages and the roots of different genotypes. However, the transcripts of the genes expressed coordinately and were more likely to form a co-expression network. Furthermore, we studied the responses of the PgbZIP genes to drought stress in ginseng using a random selection of five PgbZIP genes, including PgbZIP25, PgbZIP38, PgbZIP39, PgbZIP53 and PgbZIP54. The results showed that all five PgbZIP genes responded to drought stress in ginseng, indicating that the PgbZIP genes play important roles in ginseng responses to drought stress. CONCLUSIONS: These results provide knowledge and gene resources for deeper functional analysis of the PgbZIP genes and molecular tools for enhanced drought tolerance breeding in ginseng.

Ectopic Expressions of the GhLETM1 Gene Reveal Sensitive Dose Effects on Precise Stamen Development and Male Fertility in Cotton.

The homologous leucine zipper/EF-hand-containing transmembranes (LETMs) are highly conserved across a broad range of eukaryotic organisms. The LETM functional characteristics involved in biological process have been identified primarily in animals, but little is known about the LETM biological function mode in plants. Based on the results of the current investigation, the GhLETM1 gene crucially affects filament elongation and anther dehiscence of the stamen in cotton. Both excessive and lower expression of the GhLETM1 gene lead to defective stamen development, resulting in shortened filaments and indehiscent anthers with pollen abortion. The results also showed that the phenotype of the shortened filaments was negatively correlated with anther defects in the seesaw model under the ectopic expression of GhLETM1. Moreover, our results notably indicated that the gene requires accurate expression and exhibits a sensitive dose effect for its proper function. This report has important fundamental and practical significance in crop science, and has crucial prospects for genetic engineering of new cytoplasmic male sterility lines and breeding of crop hybrid varieties.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Protein-inorganic calcium-phosphate supraparticles as a robust platform for enzyme co-immobilization.

Immobilization of enzymes provides many benefits, including facile separation and recovery of enzymes from reaction mixtures, enhanced stability, and co-localization of multiple enzymes. Calcium-phosphate-protein supraparticles imbued with a leucine zipper binding domain (ZR ) serve as a modular immobilization platform for enzymes fused to the complementary leucine zipper domain (ZE ). The zippers provide high-affinity, specific binding, separating enzymatic activity from the binding event. Using fluorescent model proteins (mCherryZE and eGFPZE ), an amine dehydrogenase (AmDHZE ), and a formate dehydrogenase (FDHZE ), the efficacy of supraparticles as a biocatalytic solid support was assessed. Supraparticles demonstrated several benefits as an immobilization support, including predictable loading of multiple proteins, structural integrity in a panel of solvents, and the ability to elute and reload proteins without damaging the support. The dual-enzyme reaction successfully converted ketone to amine on supraparticles, highlighting the efficacy of this system.

Genome-wide identification and characterization of HD-ZIP genes in potato.

HD-ZIP (Homeodomain leucine zipper) transcription factors play an important regulatory role in stress resistance in plants. The purpose of this study was to analyze the characteristics of the HD-ZIP genes/proteins and to study their expression profiles under high and low temperature conditions in potato (Solanum tuberosum L.). A strict homology search was used to find 43 HD-ZIP genes located on potato chromosomes 1-12. Exons/introns, protein features and conserved motifs were analyzed, and six segment duplications were identified from 43 HD-ZIP genes. Then, we analyzed the data from the PGSC (Potato Genome Sequencing Consortium) database regarding the expression of 43 HD-ZIP genes that were induced by biotic and abiotic stresses and phytohormone treatments and conducted an expression analysis for these genes across all potato life stages. Additionally, the expression levels of 13 HD-ZIP genes were analyzed under high temperature (37 °C) and low temperature (4 °C) conditions. The results showed that the transcript levels of all 13 genes changed, which indicated that these genes respond to heat and cold in plants. Especially for StHOX20, the expression significantly upregulated in roots at 37 °C and 4 °C. Our findings laid the foundation and provided clues for understanding the biological functions of HD-ZIP family genes.

Genome-Wide Identification and Characterization of the AREB/ABF/ABI5 Subfamily Members from Solanum tuberosum.

Abscisic acid (ABA) plays crucial roles in plant development and adaption to environmental stresses. The ABA-responsive element binding protein/ABRE-binding factor and ABA INSENSITIVE 5 (AREB/ABF/ABI5) gene subfamily members, which belong to the basic domain/leucine zipper (bZIP) transcription factors family, participate in the ABA-mediated signaling pathway by regulating the expression of their target genes. However, information about potato (Solanum tuberosum) AREB/ABF/ABI5 subfamily members remains scarce. Here, seven putative AREB/ABF/ABI5 members were identified in the potato genome. Sequences alignment revealed that these members shared high protein sequence similarity, especially in the bZIP region, indicating that they might possess overlapping roles in regulating gene expression. Subcellular localization analysis illustrated that all seven AREB/ABF/ABI5 members were localized in the nucleus. Transactivation activity assays in yeast demonstrated that these AREB/ABF/ABI5 members possessed distinct transcriptional activity. Electrophoretic mobility shift assays (EMSA) confirmed that all of these AREB/ABF/ABI5 members could have an affinity to ABRE in vitro. The expression patterns of these AREB/ABF/ABI5 genes showed that they were in response to ABA or osmotic stresses in varying degrees. Moreover, most AREB/ABF/ABI5 genes were induced during stolon swelling. Overall, these results provide the first comprehensive identification of the potato AREB/ABF/ABI5 subfamily and would facilitate further functional characterization of these subfamily members in future work.

Genomic and transcriptomic analyses of HD-Zip family transcription factors and their responses to abiotic stress in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis (L.) O. Kuntze) is a perennial evergreen woody plant, and its leaves contain various beneficial ingredients and have healthy efficacy. HD-Zip (homeodomain-leucine zipper) transcription factors (TFs) are widely distributed in plants and play an important role in plant growth and environmental response. To date, knowledge on HD-Zip gene family in tea plant is still limited. In this study, 33 HD-Zip TFs were selected based on the genomic and transcriptomic databases of tea plant. The conserved domains and common motifs of these TFs were predicted and analyzed. These 33 Cshdz TFs were divided into four groups (HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV). The interaction network of the HD-Zip proteins of tea plant was established based on the data of Arabidopsis. In addition, the expression levels of these Cshdz genes in tea plant cv. 'Longjing43' were detected and analyzed under five abiotic stress treatments. Results showed that the different expression profiles of Cshdz genes were associated with different abiotic stress treatments. Our findings suggested a potential relationship between the resistance of tea plant and its Cshdz genes.

Assembly properties of human immunodeficiency virus type 1 Gag-leucine zipper chimeras: implications for retrovirus assembly.

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant "Gag-Zipper" proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the "dimerizing" Gag-Zipper protein misassembles into very small particles, while the "trimerizing" protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the "dimerizing" Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.

Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper-like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper-like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants.

A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer.

Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

Identification of a chromosomal breakpoint and detection of a novel form of an MLL-AF17 fusion transcript in acute monocytic leukemia with t(11;17)(q23;q21).

More than 40 genes have been reported as translocation partners of the mixed lineage leukemia gene (MLL) in hematologic malignancies. AF17 was identified earlier than most other MLL translocation partners. On the other hand, there is only 1 report of an MLL-AF17 fusion transcript in acute myeloid leukemia (AML). Here we describe a 40-year-old man with a diagnosis of AML involving t(11;17)(q23;q21). We identified a chromosomal breakpoint for t(11;17)(q23;q21) at MLL intron 6 and AF17 intron 8. Although the previously reported form of the MLL-AF17 fusion transcript was not detected by reverse transcriptase-polymerase chain reaction (PCR) analysis, a novel form of an MLL-AF17 fusion transcript joining MLL exon 6 to AF17 exon 9 was detected by complementary DNA panhandle PCR. The fact that 2 forms of MLL-AF17 retain the leucine zipper domain of AF17 suggests that the dimerization domain of AF17 is critical for leukemogenesis by the MLL-AF17 fusion gene.

Cloning and analysis of a novel cDNA from Trichinella spiralis encoding a protein with an FYVE zinc finger domain.

A cDNA library from Trichinella spiralis adults 3 days post-infection was screened with a cDNA probe, designated T 54, derived from a newborn larvae subtracted cDNA library. Sequence analysis showed that the positive clone contained a cDNA insert of 1464 bp in length with a single open reading frame of 1290 bp, which encoded a protein of 429 amino acids with a putative molecular mass of 49.9 k Da. Database analysis predicted the deduced protein had a leucine zipper motif and an FYVE zinc finger domain. The recombinant fusion protein was expressed and rabbit anti-recombinant protein sera reacted with a single peptide migrating at approximately 55 k Da in crude worm extract from muscle larvae, adults and newborn larvae stages.

A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors.

We have developed a high-throughput fluorescence anisotropy screen, using a 384-well format, to identify small molecules that disrupt the DNA binding of B-ZIP proteins. Binding of a B-ZIP dimer to fluorescently labeled DNA can be monitored by fluorescence anisotropy. We screened the National Cancer Institute diversity set of 1990 compounds to identify small molecules that disrupt the B-ZIP|DNA complex of CREB, C/EBPbeta, VBP, and AP-1 (FOS|JUND) bound to their cognate DNA sequence. We identified 21 compounds that inhibited the DNA binding of at least one B-ZIP protein, and 12 representative compounds were grouped depending on whether they displaced ethidium bromide from DNA. Of the 6 compounds that did not displace ethidium bromide, 2 also inhibited B-ZIP binding to DNA in a secondary electrophoretic mobility shift assay screen with some specificity. Thermal stability monitored by circular dichroism spectroscopy demonstrated that both compounds bound the basic region of the B-ZIP motif. NSC13778 preferentially binds C/EBPalpha 1000-fold better than it binds C/EBPbeta. Chimeric proteins combining C/EBPalpha and C/EBPbeta mapped the binding of NSC13778 to three amino acids immediately N terminal of the leucine zipper of C/EBPalpha. These experiments suggest that the DNA binding of B-ZIP transcription factors is a potential target for clinical intervention.

Homeodomain-leucine zipper proteins interact with a plant homologue of the transcriptional co-activator multiprotein bridging factor 1.

StMBF1 (Solanum tuberosum multiprotein bridging factor 1) is a plant member of the MBF1 family of transcriptional co-activators. In an attempt to understand the role of StMBF1, we analyzed its interaction with plant transcription factors of the homeodomain-leucine zipper (Hd-Zip) family, a group of proteins with a typical leucine zipper motif adjacent to a homeodomain. StMBF1 is able to interact in vitro with the Hd-Zip protein Hahb-4 both in the presence and absence of DNA. Upon binding, StMBF1 increases the DNA binding affinity of Hahb-4, and of another plant homeodomain containing protein from the GL2/Hd-Zip IV family, HAHR-1. The biological role of interactions is discussed in this paper.

Basic leucine zipper transcription factors C/EBP and MafL in the hydrozoan jellyfish Podocoryne carnea.

Members of the CCAAT/enhancer binding protein (C/EBP) and the Maf protein subfamilies have been characterized in a variety of bilaterian organisms. This is the first report of C/EBP and MafL genes in a basal organism, the hydrozoan jellyfish Podocoryne carnea. Transcripts of both genes are present in all life cycle stages: egg, embryo, larva, polyp, and medusa. During early development, both factors appear to regulate metamorphosis of the larva to the primary polyp. Both genes are also expressed in the striated muscle of the developing and adult medusa. During in vitro transdifferentiation of striated muscle cells to smooth muscle and nerve cells, C/EBP is continuously expressed, whereas MafL expression is turned off during transdifferentiation and reactivated when nerve cells differentiate. Thus, both factors may be involved in muscle and nerve cell differentiation. In the mature medusa both genes are also implicated in gametogenesis. Developmental and evolutionary aspects of the gene structures and expression patterns are discussed.

Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1.

The role of the NH(2)-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH(2)-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 +/- 1.3 min compared with 3.2 +/- 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH(2) terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 degrees C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH(2) terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.

Contrasting patterns of expression of transcription factors in pancreatic alpha and beta cells.

Pancreatic alpha and beta cells are derived from the same progenitors but play opposing roles in the control of glucose homeostasis. Disturbances in their function are associated with diabetes mellitus. To identify many of the proteins that define their unique pathways of differentiation and functional features, we have analyzed patterns of gene expression in alphaTC1.6 vs. MIN6 cell lines by using oligonucleotide microarrays. Approximately 9-10% of >11,000 transcripts examined showed significant differences between the two cell types. Of >700 known transcripts enriched in either cell type, transcription factors and their regulators (TFR) was one of the most significantly different categories. Ninety-six members of the basic zipper, basic helix-loop-helix, homeodomain, zinc finger, high mobility group, and other transcription factor families were enriched in alpha cells; in contrast, homeodomain proteins accounted for 51% of a total of 45 TFRs enriched in beta cells. Our analysis thus highlights fundamental differences in expression of TFR subtypes within these functionally distinct islet cell types. Interestingly, the alpha cells appear to express a large proportion of factors associated with progenitor or stem-type cells, perhaps reflecting their earlier appearance during pancreatic development. The implications of these findings for a better understanding of alpha and beta cell dysfunction in diabetes mellitus are also considered.

The Aspergillus nidulans metR gene encodes a bZIP protein which activates transcription of sulphur metabolism genes.

The identification, isolation and characterization of a new Aspergillus nidulans positive-acting gene metR, which encodes a transcriptional activator of sulphur metabolism, is reported. metR mutants are tight auxotrophs requiring methionine or homocysteine for growth. Mutations in the metR gene are epistatic to mutations in the negative-acting sulphur regulatory scon genes. The metR coding sequence is interrupted by a single intron of 492 bp which is unusually long for fungi. Aspergillus nidulans METR is a member of bZIP family of DNA-binding proteins. The bZIP domains of METR and the Neurospora crassa CYS3 transcriptional activator of sulphur genes are highly similar. Although Neurospora cys-3 gene does not substitute for the metR function, a chimeric metR gene with a cys-3 bZIP domain is able to transform the DeltametR mutant to methionine prototrophy. This indicates that METR recognizes the same regulatory sequence as CYS3. The metR gene is not essential, as deletion mutants are viable and have similar phenotype as point mutants. In contrast to the Neurospora cys-3, transcription of the metR gene was found to be regulated neither by METR protein nor by sulphur source. Transcription of metR gene is derepressed in the sconB2 mutant. Transcription of genes encoding sulphate permease, homocysteine synthase, cysteine synthase, ATP-sulphurylase, and sulphur controller--sconB is strongly regulated by the metR gene product and depends on the character of the metR mutation and sulphur supplementation.

Homez, a homeobox leucine zipper gene specific to the vertebrate lineage.

This work describes a vertebrate homeobox gene, designated Homez (homeodomain leucine zipper-encoding gene), that encodes a protein with an unusual structural organization. There are several regions within Homez, including three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously expressed in human and murine tissues, although the expression pattern is more restricted during mouse development. Genomic analysis revealed that human and mouse genes are located at 14q11.2 and 14C, respectively, and are composed of two exons. The zebrafish and pufferfish homologs share high similarity to mammalian sequences, particularly within the homeodomain sequences. Based on homology of homeodomains and on the similarity in overall protein structure, we delineate Homez and members of ZHX family of zinc finger homeodomain factors as a subset within the superfamily of homeobox-containing proteins. The type and composition of homeodomains in the Homez subfamily are vertebrate-specific. Phylogenetic analysis indicates that Homez lineage was separated from related genes >400 million years ago before separation of ray- and lobe-finned fishes. We apply a duplication-degeneration-complementation model to explain how this family of genes has evolved.