Evolutionary Analysis of the Zinc Finger and Homeoboxes Family of Proteins Identifies Multiple Conserved Domains and a Common Early Chordate Ancestor.

The Zinc Fingers and Homeoboxes (Zhx) proteins, Zhx1, Zhx2, and Zhx3, comprise a small family of proteins containing two amino-terminal C2-H2 zinc fingers and four or five carboxy-terminal homeodomains. These multiple homeodomains make Zhx proteins unusual because the majority of homeodomain-containing proteins contain a single homeodomain. Studies in cultured cells and mice suggest that Zhx proteins can function as positive or negative transcriptional regulators. Zhx2 regulates numerous hepatic genes, and all three Zhx proteins have been implicated in different cancers. Because Zhx proteins contain multiple predicted homeodomains, are associated with interesting physiological traits, and seem to be only present in the vertebrate lineage, we investigated the evolutionary history of this small family by comparing Zhx homologs from a wide range of chordates. This analysis indicates that the zinc finger motifs and homeodomains are highly similar among all Zhx proteins and also identifies additional Zhx-specific conserved regions, including a 13 amino acid amino-terminal motif that is nearly identical among all gnathostome Zhx proteins. We found single Zhx proteins in the sea lamprey (Petromyzon marinus) and in the nonvertebrate chordates sea squirt (Ciona intestinalis) and lancelet (Branchiostoma floridae); these Zhx proteins are most similar to gnathostome Zhx3. Based on our analyses, we propose that a duplication of the primordial Zhx gene gave rise to Zhx3 and the precursor to Zhx1 and Zhx2. A subsequent tandem duplication of this precursor generated Zhx1 and Zhx2 found in gnathostomes.

A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development.

KEY MESSAGE: Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

Genome-wide identification and analysis of the WUSCHEL-related homeobox (WOX) gene family in allotetraploid Brassica napus reveals changes in WOX genes during polyploidization.

BACKGROUND: WUSCHEL-related homeobox (WOX) genes encoding plant-specific homeobox (HB) transcription factors play important roles in the growth and development of plants. To date, WOX genes has been identified and analyzed in many polyploids (such as cotton and tobacco), but the evolutionary analysis of them during polyploidization is rare. With the completion of genome sequencing, allotetraploid Brassica napus and its diploid progenitors (B. rapa and B. oleracea) are a good system for studying this question. RESULTS: In this study, 52, 25 and 29 WOX genes were identified in allotetraploid B. napus (2n = 4x = 38, AnCn), the An genome donor B. rapa (2n = 2x = 20, Ar) and the Cn genome donor B. oleracea (2n = 2x = 18, Co), respectively. All identified WOX genes in B. napus and its diploid progenitors were divided into three clades, and these genes were selected to perform gene structure and chromosome location analysis. The results showed that at least 70 and 67% of WOX genes maintained the same gene structure and relative position on chromosomes, respectively, indicating that WOX genes in B. napus were highly conserved at the DNA level during polyploidization. In addition, the analysis of duplicated genes and transposable elements (TEs) near WOX genes showed that whole-genome triplication (WGT) events, segmental duplication and abundant TEs played important roles in the expansion of the WOX gene family in B. napus. Moreover, the analysis of the expression profiles of WOX gene pairs with evolutionary relationships suggested that the WOX gene family may have changed at the transcriptional regulation level during polyploidization. CONCLUSIONS: The results of this study increased our understanding of the WOX genes in B. napus and its diploid progenitors, providing a rich resource for further study of WOX genes in these species. In addition, the changes in WOX genes during the process of polyploidization were discussed from the aspects of gene number, gene structure, gene relative location and gene expression, which provides a reference for future polyploidization analysis.

Diversification of four human HOX gene clusters by step-wise evolution rather than ancient whole-genome duplications.

HOX genes encode transcriptional factors that play a pivotal role in specifying regional identity in nearly every bilateral animal. The birth of HOX gene cluster and its subsequent evolution, either in regulation or function, underlie the evolution of many bilaterian features and hence to the evolutionary radiation of this group. Despite of this importance, evolution of HOX cluster in vertebrates remains largely obscure because the phylogenetic history of these genes is poorly resolved. This has led to the controversy about whether four HOX clusters in human originated through two rounds (2R) of whole-genome duplications or instead evolved by small-scale events early in vertebrate evolution. Recently, the large-scale phylogenetic analysis of triplicate and quadruplicate paralogous regions residing on human HOX-bearing chromosomes provided an unprecedented insight into events that shaped vertebrate genome early in their history. Based on these data and comparative genomic analysis of fruit fly, red floor beetle, and human, this study infers the genic content of minimal HOX locus in the Urbilaterian and reconstructs its duplication history. It appears that four HOX clusters of humans are not remnants of polyploidy events in vertebrate ancestry. Rather, current evidence suggests that one-to-four transition in HOX cluster number occurred by three-step sequential process involving regional duplication events. Therefore, it is concluded that the evolutionary origin of vertebrate novelties, including the complexity of their body, is the consequence of small-scale genetic changes at widely different times over their history.

Embryo development. A cysteine-clamp gene drives embryo polarity in the midge Chironomus.

In the fruit fly Drosophila, head formation is driven by a single gene, bicoid, which generates head-to-tail polarity of the main embryonic axis. Bicoid deficiency results in embryos with tail-to-tail polarity and no head. However, most insects lack bicoid, and the molecular mechanism for establishing head-to-tail polarity is poorly understood. We have identified a gene that establishes head-to-tail polarity of the mosquito-like midge, Chironomus riparius. This gene, named panish, encodes a cysteine-clamp DNA binding domain and operates through a different mechanism than bicoid. This finding, combined with the observation that the phylogenetic distributions of panish and bicoid are limited to specific families of flies, reveals frequent evolutionary changes of body axis determinants and a remarkable opportunity to study gene regulatory network evolution.

Comprehensive characterization and RNA-Seq profiling of the HD-Zip transcription factor family in soybean (Glycine max) during dehydration and salt stress.

BACKGROUND: The homeodomain leucine zipper (HD-Zip) transcription factor family is one of the largest plant specific superfamilies, and includes genes with roles in modulation of plant growth and response to environmental stresses. Many HD-Zip genes are characterized in Arabidopsis (Arabidopsis thaliana), and members of the family are being investigated for abiotic stress responses in rice (Oryza sativa), maize (Zea mays), poplar (Populus trichocarpa) and cucumber (Cucmis sativus). Findings in these species suggest HD-Zip genes as high priority candidates for crop improvement. RESULTS: In this study we have identified members of the HD-Zip gene family in soybean cv. 'Williams 82', and characterized their expression under dehydration and salt stress. Homology searches with BLASTP and Hidden Markov Model guided sequence alignments identified 101 HD-Zip genes in the soybean genome. Phylogeny reconstruction coupled with domain and gene structure analyses using soybean, Arabidopsis, rice, grape (Vitis vinifera), and Medicago truncatula homologues enabled placement of these sequences into four previously described subfamilies. Of the 101 HD-Zip genes identified in soybean, 88 exist as whole-genome duplication-derived gene pairs, indicating high retention of these genes following polyploidy in Glycine ~13 Mya. The HD-Zip genes exhibit ubiquitous expression patterns across 24 conditions that include 17 tissues of soybean. An RNA-Seq experiment performed to study differential gene expression at 0, 1, 6 and 12 hr soybean roots under dehydration and salt stress identified 20 differentially expressed (DE) genes. Several of these DE genes are orthologs of genes previously reported to play a role under abiotic stress, implying conservation of HD-Zip gene functions across species. Screening of HD-Zip promoters identified transcription factor binding sites that are overrepresented in the DE genes under both dehydration and salt stress, providing further support for the role of HD-Zip genes in abiotic stress responses. CONCLUSIONS: We provide a thorough description of soybean HD-Zip genes, and identify potential candidates with probable roles in dehydration and salt stress. Expression profiles generated for all soybean genes, under dehydration and salt stress, at four time points, will serve as an important resource for the soybean research community, and will aid in understanding plant responses to abiotic stress.

Composition and interrelationships of a large Neotropical freshwater fish group, the subfamily Cheirodontinae (Characiformes: Characidae): a case study based on mitochondrial and nuclear DNA sequences.

Characidae is the most species-rich family of freshwater fishes in the order Characiformes, with more than 1000 valid species that correspond to approximately 55% of the order. Few hypotheses about the composition and internal relationships within this family are available and most fail to reach an agreement. Among Characidae, Cheirodontinae is an emblematic group that includes 18 genera (1 fossil) and approximately 60 described species distributed throughout the Neotropical region. The taxonomic and systematic history of Cheirodontinae is complex, and only two hypotheses about the internal relationships in this subfamily have been reported to date. In the present study, we test the composition and relationships of fishes assigned to Cheirodontinae based on a broad taxonomic sample that also includes some characid incertae sedis taxa that were previously considered to be part of Cheirodontinae. We present phylogenetic analyses of a large molecular dataset of mitochondrial and nuclear DNA sequences. Our results reject the monophyly of Cheirodontinae as previously conceived, as well as the tribes Cheirodontini and Compsurini, and the genera Cheirodon, Compsura, Leptagoniates, Macropsobrycon, Odontostilbe, and Serrapinnus. On the basis of these results we propose: (1) the exclusion of Amazonspinther and Spintherobolus from the subfamily Cheirodontinae since they are the sister-group of all remaining Characidae; (2) the removal of Macropsobrycon xinguensis of the genus Macropsobrycon; (3) the removal of Leptagoniates pi of the genus Leptagoniates; (4) the inclusion of Leptagoniates pi in the subfamily Cheirodontinae; (5) the removal of Cheirodon stenodon of the genus Cheirodon and its inclusion in the subfamily Cheirodontinae under a new genus name; (6) the need to revise the polyphyletic genera Compsura, Odontostilbe, and Serrapinnus; and (7) the division of Cheirodontinae in three newly defined monophyletic tribes: Cheirodontini, Compsurini, and Pseudocheirodontini. Our results suggest that our knowledge about the largest Neotropical fish family, Characidae, still is incipient.

Lycodon and Dinodon: one genus or two? Evidence from molecular phylogenetics and morphological comparisons.

Based on a molecular phylogeny and a comparison of maxillary dentition and morphology, the relationship between the genera Lycodon and Dinodon was investigated. Bayesian Inference and Maximum Likelihood analysis of two mitochondrial genes (cyt b and ND4) and two nuclear genes (c-mos and Rag1) suggested that the two genera shared a most recent common ancestor. However, Dinodon was paraphyletic and Lycodon was polyphyletic, each with respect to the other. The results from counts of maxillary teeth indicated that the diagnostic characters used by previous authors to separate Dinodon and Lycodon were not reliable. Taking the molecular and morphological evidence together, we synonymized Dinodon with Lycodon. In addition, the validity of the species L. futsingensis was confirmed to be distinctly different from the other species of Dinodon and Lycodon.

Molecular analysis and its expression of a pou homeobox protein gene during development and in response to salinity stress from brine shrimp, Artemia sinica.

Brine shrimps of the genus Artemia are aquatic species of economic importance because of their important significance to aquaculture and are used as a model species in physiology and developmental biology. Research on Artemia POU homeobox gene function will enhance our understanding of the physiological and developmental processes of POU homeobox gene in animals. Herein, a full-length cDNA encoding an Artemia POU homeobox protein gene 1 (APH-1) from Artemia sinica (designated as As-APH-1) was cloned and characterized by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. The As-APH-1 gene encoded a protein of 388 amino acid polypeptide with a calculated molecular mass of 42.85kDa and an isoelectric point of 6.90 and the protein belongs to the POU III family. Multiple sequence alignments revealed that A. sinica As-APH-1 protein sequence shared a conserved POU homeobox domain with other species. The early and persistent expression of As-APH-1 in the naupliar stages by semi-quantitative RT-PCR and whole-mount embryonic immunohistochemistry suggest that As-APH-1 functions very early in the salt gland and may be required continuously in this organ. Later in development, expression of As-APH-1 begins to dramatically decrease and disappear in salt gland of the sub-adult Artemia. In addition, we also discovered that As-APH-1 increased obviously as the salinity increased, indicating that As-APH-1 might be used as a good indicator of salinity stress. In summary, we are the first to identify the As-APH-1 gene and to determine its gene expression patterns in early embryogenesis stages and in different salinity stress in brine shrimp, A. sinica. The result of expression of As-APH-1 affected by salinity changes will provide us further understanding of the underlying mechanisms of osmoregulation in Artemia early embryonic development.

Origins of Cdx1 regulatory elements suggest roles in vertebrate evolution.

Cdx1, an upstream regulator of Hox genes, is best characterized for its homeotic effects upon the developing axial skeleton, particularly in the neck. It responds to retinoic acid (RA) in both mouse embryos and embryonal carcinoma (EC) cells. By use of beta-galactosidase chemiluminescence, we show that a mouse Cdx1/lacZ reporter expressed in P19 EC cells responds to RA by the combined activities of an intron retinoic acid response element (RARE) and an upstream RARE. In contrast, a chicken Cdx1/lacZ reporter responds only by activity of the intron RARE. Database analyses upon Cdx1 from twenty three vertebrate species reveal that the intron RARE is structurally conserved in amniotes (eutherian mammals, marsupials, birds and Anole lizard), but not in Xenopus or fish. The upstream RARE is structurally conserved only in eutherian mammals. We conclude that the intron RARE originated at around the amphibian/amniote division, and the upstream RARE appeared around the marsupial/eutherian mammal division. In view of the site of action of Cdx1, we propose that acquisition of the intron RARE may have facilitated the substantial changes that occurred in the neck and anterior thorax at the advent of the amniotes. We present evidence that Cdx1 is also a developmental regulator of the female urogenital system, and we suggest that acquisition of the upstream RARE may have contributed to morphological divergence of marsupial and eutherian mammals.

Involvement of Prop1 homeobox gene in the early development of fish pituitary gland.

When mutated in mammals, paired-like homeobox Prop1 gene produces highly variable pituitary phenotypes with impaired regulation of Pit1 and eventually defective synthesis of Pit1-regulated pituitary hormones. Here we have identified fish prop1 orthologs, confirmed their pituitary-specific expression, and blocked the splicing of zebrafish prop1 transcripts using morpholino oligonucleotides. Very early steps of the gland formation seemed unaffected based on morphology and expression of early placodal marker pitx. Prop1 knock-down reduced the expression of pit1, prl (prolactin) and gh (growth hormone), as expected if the function of Prop1 is conserved throughout vertebrates. Less expectedly, lim3 was down regulated. This gene is expressed from early stages of vertebrate pituitary development but is not known to be Prop1-dependent. In situ hybridizations on prop1 morphants using probes for the pan pituitary gene pitx3 and for the hormone gene markers prl, gh and tshß, revealed abnormal shape, growth and cellular organization of the developed adenohypophysis. Strikingly, the effects of prop1 knock-down on adenohypophysis morphology and gene expression were gradually reversed during late development, despite persistent splice-blocking of transcripts. Therefore, prop1 function appears to be conserved between mammals and fish, at least for the mediation of hormonal cell type differentiation via pit1, but the existence of other fish-specific pathways downstream of prop1 are suggested by our observations.

The zebrafish genes encoding the Polycomb repressive complex (PRC) 1.

Polycomb repression controls regulation of hundreds of genes involved in development, signalling or cancer and is mediated by essentially two classes of chromatin-associated protein complexes, the Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 at Lysine 27 and this H3K27me3 epigenetic mark serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates. In particular, mammalian genomes encode five Pc family members (CBX2, 4, 6, 7 and 8), six Psc family members (BMI1, PCGF1, 2, 3, 5, and 6), three Ph family members (PHC1, 2 and 3) and two Sce family members (RING1 and RNF2) generating an enormous scope for potential combinatorial diversity. In order to identify the corresponding PRC1 genes in zebrafish, homology searches were undertaken and allowed the identification of a total of 19 genes. Using phylogenetic, gene organization and gene location analyses, these genes were classified. The zebrafish genes encoding the PRC1 protein complex include 8 Pc orthologs (cbx2, cbx4, cbx6a, cbx6b, cbx7a, cbx7b, cbx8a and cbx8b), 6 Psc orthologs (bmi1a, bmi1b, pcgf1, pcgf5a, pcgf5b and pcgf6), 4 Ph orthologs (phc1, phc2a, phc2b and phc3) and a single Sce ortholog (rnf2). Our results indicate that the potentially high number of distinct PRC1 protein complexes generated by the components combinatorial appeared early in the vertebrate evolution. In addition to conserved gene organization and syntenies, transcript analyses revealed that transcriptional regulation leading to various isoforms syntheses is also conserved at genes encoding the PRC1 components, highlighting a possible important biological role of these isoforms.

A hypothetical relationship between the nuclear reprogramming factors for induced pluripotent stem (iPS) cells generation--bioinformatic and algorithmic approach.

A hypothetical evolutionary relationship was generated between the nuclear reprogramming factors for induced pluripotent stem (iPS) cells generation. Utilizing bioinformatics techniques, sequence analyses and phylogenetic tree algorithms, a comparative study has been performed to understand the evolutionary relationship of human nuclear reprogramming factors of induced pluripotent stem cells (iPSCs) generation. Among the total six nuclear reprogramming factors, the four reprogramming factors (SOX2, C-MYC, KLF4, and LIN28) have significant evolutionary origin. Our study shows SOX2 and C-MYC have evolutionary relationship and common point of origin. Likewise, KLF4 and LIN28 are having evolutionary relationship and have common point of origin. Based on these evidences, we propose that our study may be a great help to the future researchers to understand the mechanism(s) as well as pathway of nuclear reprogramming process.

Early evolution of the LIM homeobox gene family.

BACKGROUND: LIM homeobox (Lhx) transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known. RESULTS: We identified Lhx genes in two cnidarians and a placozoan and report the expression of Lhx genes during embryonic development in Nematostella and the demosponge Amphimedon. Members of the six major LIM homeobox subfamilies are represented in the genomes of the starlet sea anemone, Nematostella vectensis, and the placozoan Trichoplax adhaerens. The hydrozoan cnidarian, Hydra magnipapillata, has retained four of the six Lhx subfamilies, but apparently lost two others. Only three subfamilies are represented in the haplosclerid demosponge Amphimedon queenslandica. A tandem cluster of three Lhx genes of different subfamilies and a gene containing two LIM domains in the genome of T. adhaerens (an animal without any neurons) indicates that Lhx subfamilies were generated by tandem duplication. This tandem cluster in Trichoplax is likely a remnant of the original chromosomal context in which Lhx subfamilies first appeared. Three of the six Trichoplax Lhx genes are expressed in animals in laboratory culture, as are all Lhx genes in Hydra. Expression patterns of Nematostella Lhx genes correlate with neural territories in larval and juvenile polyp stages. In the aneural demosponge, A. queenslandica, the three Lhx genes are expressed widely during development, including in cells that are associated with the larval photosensory ring. CONCLUSIONS: The Lhx family expanded and diversified early in animal evolution, with all six subfamilies already diverged prior to the cnidarian-placozoan-bilaterian last common ancestor. In Nematostella, Lhx gene expression is correlated with neural territories in larval and juvenile polyp stages. This pattern is consistent with a possible role in patterning the Nematostella nervous system. We propose a scenario in which Lhx genes play a homologous role in neural patterning across eumetazoans.

Classification and nomenclature of all human homeobox genes.

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 repetitive DUX1 to DUX5 homeobox 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.

The zinc finger transcription factor ZFHX1A is linked to cell proliferation by Rb-E2F1.

ZFHX1A is expressed in proliferating cells in the developing embryo, and in the present study we provide evidence that its expression is confined to proliferating cells through dependence on the Rb (retinoblastoma protein) family/E2F cell cycle pathway. Mutation of the Rb or E2F1 genes lead to induction of ZFHX1A mRNA, implying that the Rb-E2F1 repressor complex is important for repression of ZFHX1A. This repression is associated with recruitment of an E2F-Rb-histone deacetylase repressor complex to the promoter. A dominant-negative form of E2F1 inhibited ZFHX1A expression in p16INK4a- cells where Rb is constitutively hyperphosphorylated and inactive, suggesting that E2F can contribute to ZFHX1A transactivation in the absence of functional Rb. ZFHX1A is an E-box-binding transcription factor whose binding sites overlap with those bound by Snail1 and 2, and ZFHX1B/SIP1 (leading to at least partially overlapping function; for example, each of the proteins can repress E-cadherin expression). We found that expression of Snail1 and ZFHX1B/SIP1 is also regulated by E2Fs, but in contrast with ZFHX1A this regulation is Rb-family-independent. Snail2 expression was unaffected by either E2F or the Rb family. We propose that the differential effects of the Rb family/E2F pathway on expression of these E-box-binding proteins are important in maintaining their distinct patterns (and thus distinct functions) during embryogenesis.

Ancient origin of the new developmental superfamily DANGER.

Developmental proteins play a pivotal role in the origin of animal complexity and diversity. We report here the identification of a highly divergent developmental protein superfamily (DANGER), which originated before the emergence of animals (approximately 850 million years ago) and experienced major expansion-contraction events during metazoan evolution. Sequence analysis demonstrates that DANGER proteins diverged via multiple mechanisms, including amino acid substitution, intron gain and/or loss, and recombination. Divergence for DANGER proteins is substantially greater than for the prototypic member of the superfamily (Mab-21 family) and other developmental protein families (e.g., WNT proteins). DANGER proteins are widely expressed and display species-dependent tissue expression patterns, with many members having roles in development. DANGER1A, which regulates the inositol trisphosphate receptor, promotes the differentiation and outgrowth of neuronal processes. Regulation of development may be a universal function of DANGER family members. This family provides a model system to investigate how rapid protein divergence contributes to morphological complexity.

ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation.

Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosis, and DNA repair as important cofactors of p53. ING1 and ING3 are stable components of the mSin3A HDAC and Tip60/NuA4 HAT complexes, respectively. We now report the purification of the three remaining human ING proteins. While ING2 is in an HDAC complex similar to ING1, ING4 associates with the HBO1 HAT required for normal progression through S phase and the majority of histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs. These ING5 HAT complexes interact with the MCM helicase and are essential for DNA replication to occur during S phase. Our data also indicate that ING subunits are crucial for acetylation of chromatin substrates. Since INGs, HBO1, and MOZ/MORF contribute to oncogenic transformation, the multisubunit assemblies characterized here underscore the critical role of epigenetic regulation in cancer development.

LASS6, an additional member of the longevity assurance gene family.

Longevity assurance genes (LAGs) represent a subgroup of the homeobox gene family. Five mammalian homologs have been reported, and the corresponding proteins have previously been investigated with respect to their key role in ceramide synthesis. However, members of the LAG family have been shown to be involved in cell growth regulation and cancer differentiation. In an effort to characterize additional members of the LAG family, we have screened the latest releases of genomic databases and report on the bioinformatic characterization of yet another member, LAG1 longevity assurance homolog 6 (LASS6). Like other LAG family members, the LASS6 protein contained a homeodomain and LAG1 domain. In phylogenetic analyses, it displayed highest homology to LASS5. The corresponding gene was localized to human chromosome 2q24.3, spanning a rather large genomic region of 318 kb. Orthologous sequences in mouse and zebrafish suggested a conservation of LASS6 in vertebrates as the protein and corresponding genomic sequences were highly conserved. LASS6 expression was analyzed in silico, and the gene was shown to be broadly expressed in a wide range of tissues. Furthermore, available microarray data suggested a role in cancer differentiation and early embryonic development.

Zic4, a zinc-finger transcription factor, is expressed in the developing mouse nervous system.

Zic genes comprise a family of transcription factors, characterized by the presence of a zinc-finger domain containing two cysteines and two histidines (C2-H2). Whereas the embryonic expression patterns of Zic1, 2, 3, and 5 have been described in detail, Zic4 has not yet received close attention. We studied the expression of Zic4 by in situ hybridization during mouse embryogenesis. Zic4 mRNA was first detected at low intensity at embryonic day (E) 9 and, by E10.5, expression was up-regulated in the dorsal midline of the forebrain with a strong, expanded expression domain at the boundary between the diencephalon and telencephalon, the septum, and the lamina terminalis. The choroid plexus of the third ventricle expresses Zic4, as does the dorsal part of the spinal neural tube, excluding the roof plate. The dorsal sclerotome and the dorsomedial lip of the dermomyotome also express Zic4 whereas dorsal root ganglia are negative. At E12.5, Zic4 continues to be expressed in the midline of the forebrain and in the dorsal spinal neural tube. Postnatally, Zic4 is expressed in the granule cells of the postnatal day 2 cerebellum, and in the periventricular thalamus and anterior end of the superior colliculus. We conclude that Zic4 has an expression pattern distinct from, but partly overlapping with, other members of the Zic gene family.