TheAntennapedia homeodomain protein fromDrosophila melanogaster bound to a fragment ofDNA.[1] The recognition helix and unstructured N-terminus are bound in the major and minor grooves respectively.
Ahomeobox is aDNA sequence, around 180base pairs long, that regulates large-scale anatomical features in the early stages of embryonic development. Mutations in a homeobox may change large-scale anatomical features of the full-grown organism.
Homeoboxes are found withingenes that are involved in the regulation of patterns of anatomical development (morphogenesis) inanimals,fungi,plants, and numerous single celleukaryotes.[2] Homeobox genes encodehomeodomainprotein products that aretranscription factors sharing a characteristicprotein fold structure that bindsDNA to regulate expression of target genes.[3][4][2] Homeodomain proteins regulate gene expression and cell differentiation during early embryonic development, thus mutations in homeobox genes can cause developmental disorders.[5]
Homeosis is a term coined byWilliam Bateson to describe the outright replacement of a discrete body part with another body part, e.g.antennapedia—replacement of the antenna on the head of a fruit fly with legs.[6] The "homeo-" prefix in the words "homeobox" and "homeodomain" stems from thismutational phenotype, which is observed when some of these genes are mutated inanimals. The homeobox domain was first identified in a number ofDrosophilahomeotic and segmentation proteins, but is now known to be well-conserved in many other animals, includingvertebrates.[3][7][8]
Drosophila with theantennapedia mutant phenotype exhibit homeotic transformation of the antennae into leg-like structures on the head.
The existence of homeobox genes was first discovered inDrosophila by isolating the gene responsible for a homeotic transformation where legs grow from the head instead of the expected antennae. Walter Gehring identified a gene calledantennapedia that caused this homeotic phenotype.[9] Analysis ofantennapedia revealed that this gene contained a 180 base pair sequence that encoded a DNA binding domain, which William McGinnis termed the "homeobox".[10] The existence of additionalDrosophila genes containing theantennapedia homeobox sequence was independently reported by Ernst Hafen,Michael Levine,William McGinnis, andWalter Jakob Gehring of theUniversity of Basel inSwitzerland andMatthew P. Scott and Amy Weiner ofIndiana University inBloomington in 1984.[11][12] Isolation of homologous genes byEdward de Robertis and William McGinnis revealed that numerous genes from a variety of species contained the homeobox.[13][14] Subsequentphylogenetic studies detailing the evolutionary relationship between homeobox-containing genes showed that these genes are present in allbilaterian animals.
The vnd/NK-2 homeodomain-DNA complex. Helix 3 of the homeodomain binds in the major groove of the DNA and the N-terminal arm binds in the minor groove, in analogy with other homeodomain-DNA complexes.
Helix 2 and helix 3 form a so-calledhelix-turn-helix (HTH) structure, where the two alpha helices are connected by a short loop region. TheN-terminal two helices of the homeodomain areantiparallel and the longerC-terminal helix is roughly perpendicular to the axes of the first two. It is this third helix that interacts directly withDNA via a number of hydrogen bonds and hydrophobic interactions, as well as indirect interactions via water molecules, which occur between specificside chains and the exposedbases within themajor groove of the DNA.[7]
Homeodomain proteins are found ineukaryotes.[2] Through the HTH motif, they share limited sequence similarity and structural similarity to prokaryotic transcription factors,[16] such aslambda phage proteins that alter the expression of genes inprokaryotes. The HTH motif shows some sequence similarity but a similar structure in a wide range of DNA-binding proteins (e.g.,cro andrepressor proteins, homeodomain proteins, etc.). One of the principal differences between HTH motifs in these different proteins arises from the stereochemical requirement forglycine in the turn which is needed to avoidsteric interference of the beta-carbon with the main chain: for cro and repressor proteins the glycine appears to be mandatory, whereas for many of the homeotic and other DNA-binding proteins the requirement is relaxed.
Homeodomains can bind both specifically and nonspecifically toB-DNA with the C-terminal recognition helix aligning in the DNA's major groove and the unstructured peptide "tail" at the N-terminus aligning in the minor groove. The recognition helix and the inter-helix loops are rich inarginine andlysine residues, which formhydrogen bonds to the DNA backbone.Conservedhydrophobic residues in the center of the recognition helix aid in stabilizing the helix packing. Homeodomain proteins show a preference for the DNA sequence 5'-TAAT-3'; sequence-independent binding occurs with significantly lower affinity. The specificity of a single homeodomain protein is usually not enough to recognize specific target gene promoters, making cofactor binding an important mechanism for controlling binding sequence specificity and target gene expression. To achieve higher target specificity, homeodomain proteins form complexes with other transcription factors to recognize thepromoter region of a specific target gene.
Homeodomain proteins function astranscription factors due to the DNA binding properties of the conserved HTH motif. Homeodomain proteins are considered to be master control genes, meaning that a single protein can regulate expression of many target genes. Homeodomain proteins direct the formation of the body axes and body structures duringearly embryonic development.[17] Many homeodomain proteins inducecellular differentiation by initiating the cascades of coregulated genes required to produce individualtissues andorgans. Other proteins in the family, such asNANOG are involved in maintainingpluripotency and preventing cell differentiation.
Hox genes and their associatedmicroRNAs are highly conserved developmental master regulators with tight tissue-specific, spatiotemporal control. These genes are known to be dysregulated in several cancers and are often controlled by DNA methylation.[18][19] The regulation of Hox genes is highly complex and involves reciprocal interactions, mostly inhibitory.Drosophila is known to use thepolycomb andtrithorax complexes to maintain the expression of Hox genes after the down-regulation of the pair-rule and gap genes that occurs during larval development.Polycomb-group proteins can silence the Hox genes by modulation ofchromatin structure.[20]
Mutations to homeobox genes can produce easily visiblephenotypic changes in body segment identity, such as the Antennapedia and Bithorax mutant phenotypes inDrosophila. Duplication of homeobox genes can produce new body segments, and such duplications are likely to have been important in theevolution of segmented animals.
Phylogenetic analysis of homeobox gene sequences and homeodomain protein structures suggests that the last common ancestor of plants, fungi, and animals had at least two homeobox genes.[21] Molecular evidence shows that some limited number of Hox genes have existed in theCnidaria since before the earliest trueBilatera, making these genes pre-Paleozoic.[22] It is accepted that the three major animal ANTP-class clusters, Hox, ParaHox, and NK (MetaHox), are the result of segmental duplications. A first duplication created MetaHox and ProtoHox, the latter of which later duplicated into Hox and ParaHox. The clusters themselves were created by tandem duplications of a single ANTP-class homeobox gene.[23] Gene duplication followed byneofunctionalization is responsible for the many homeobox genes found in eukaryotes.[24][25] Comparison of homeobox genes and gene clusters has been used to understand the evolution of genome structure and body morphology throughout metazoans.[26]
Hox genes are the most commonly known subset of homeobox genes. They are essentialmetazoan genes that determine the identity of embryonic regions along the anterior-posterior axis.[27] The first vertebrate Hox gene was isolated inXenopus byEdward De Robertis and colleagues in 1984.[28] The main interest in this set of genes stems from their unique behavior and arrangement in the genome. Hox genes are typically found in an organized cluster. The linear order of Hox genes within a cluster is directly correlated to the order in which they are expressed in both time and space during development. This phenomenon is called colinearity.
Mutations in thesehomeotic genes cause displacement of body segments during embryonic development. This is calledectopia. For example, when one gene is lost the segment develops into a more anterior one, while a mutation that leads to a gain of function causes a segment to develop into a more posterior one. Famous examples areAntennapedia andbithorax inDrosophila, which can cause the development of legs instead of antennae and the development of a duplicated thorax, respectively.[29]
In vertebrates, the fourparalog clusters are partially redundant in function, but have also acquired several derived functions. For example, HoxA and HoxD specify segment identity along thelimb axis.[30][31] Specific members of the Hox family have been implicated in vascular remodeling,angiogenesis, and disease by orchestrating changes in matrix degradation, integrins, and components of the ECM.[32] HoxA5 is implicated in atherosclerosis.[33][34] HoxD3 and HoxB3 are proinvasive, angiogenic genes that upregulate b3 and a5 integrins and Efna1 in ECs, respectively.[35][36][37][38] HoxA3 inducesendothelial cell (EC) migration by upregulating MMP14 and uPAR. Conversely, HoxD10 and HoxA5 have the opposite effect of suppressing EC migration and angiogenesis, and stabilizing adherens junctions by upregulating TIMP1/downregulating uPAR and MMP14, and by upregulating Tsp2/downregulating VEGFR2, Efna1, Hif1alpha and COX-2, respectively.[39][40] HoxA5 also upregulates the tumor suppressor p53 and Akt1 by downregulation of PTEN.[41] Suppression of HoxA5 has been shown to attenuatehemangioma growth.[42] HoxA5 has far-reaching effects on gene expression, causing ~300 genes to become upregulated upon its induction in breast cancer cell lines.[42] HoxA5 protein transduction domain overexpression prevents inflammation shown by inhibition of TNFalpha-inducible monocyte binding to HUVECs.[43][44]
LIM genes (named after the initial letters of the names of three proteins where the characteristic domain was first identified) encode two 60 amino acid cysteine and histidine-rich LIM domains and a homeodomain. The LIM domains function in protein-protein interactions and can bind zinc molecules. LIM domain proteins are found in both the cytosol and the nucleus. They function in cytoskeletal remodeling, at focal adhesion sites, as scaffolds for protein complexes, and as transcription factors.[45]
Most Pax genes contain a homeobox and a paired domain that also binds DNA to increase binding specificity, though some Pax genes have lost all or part of the homeobox sequence.[46] Pax genes function in embryosegmentation,nervous system development, generation of thefrontal eye fields,skeletal development, and formation of face structures.Pax 6 is a master regulator of eye development, such that the gene is necessary for development of the optic vesicle and subsequent eye structures.[47]
Proteins containing a POU region consist of a homeodomain and a separate,structurally homologous POU domain that contains twohelix-turn-helix motifs and also binds DNA. The two domains are linked by a flexible loop that is long enough to stretch around the DNA helix, allowing the two domains to bind on opposite sides of the target DNA, collectively covering an eight-base segment withconsensus sequence 5'-ATGCAAAT-3'. The individual domains of POU proteins bind DNA only weakly, but have strong sequence-specific affinity when linked. The POU domain itself has significant structural similarity with repressors expressed inbacteriophages, particularlylambda phage.
As in animals, the plant homeobox genes code for the typical 60 amino acid long DNA-binding homeodomain or in case of the TALE (three amino acid loop extension) homeobox genes for an atypical homeodomain consisting of 63 amino acids. According to their conserved intron–exon structure and to unique codomain architectures they have been grouped into 14 distinct classes: HD-ZIP I to IV, BEL, KNOX, PLINC, WOX, PHD, DDT, NDX, LD, SAWADEE and PINTOX.[24] Conservation of codomains suggests a common eukaryotic ancestry for TALE[48] and non-TALE homeodomain proteins.[49]
ParaHox genes are analogously found in four areas. They includeCDX1,CDX2,CDX4;GSX1,GSX2; andPDX1. Other genes considered Hox-like includeEVX1,EVX2;GBX1,GBX2;MEOX1,MEOX2; andMNX1. The NK-like (NKL) genes, some of which are considered "MetaHox", are grouped with Hox-like genes into a large ANTP-like group.[50][51]
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