The homeobox

Hox genes are defined by a DNA sequence known as the homeobox, which is a sequence of 180 nucleotides that code for aprotein domain known as the homeodomain.

The first genes found to encode homeodomain proteins were Drosophila developmental control genes, in particular Hom-C genes, from which the name homeobox was derived. However, many homeobox genes are not homeotic genes; the homeobox is a sequence motif, while "homeotic" is a functional description for genes that cause homeotic transformations.^[1]

Sequence conservation

The homeodomain protein motif is highly conserved across vast evolutionary distances. The functional equivalence of Hox proteins can be demonstrated by the fact that a fly can function perfectly well with a chicken Hox protein in place of its own.^[2] This means that, despite having a last common ancestor that lived over 670 million years ago^[3], a given Hox protein in chickens and the homologous gene in flies are so similar that they can actually take each other's places.

Although the protein sequence is highly conserved, the DNA sequence from which it is made is slightly less so, a result of codon degeneracy (i.e., more than one codon codes for the same amino acid). The reason for this high level of conservation is related to the function of these proteins. Hox genes set up the basic regional layout of an organism, so that eyes form on the head and not on the abdomen, and limbs form at the sides and not on the head. Even a single mutation in the DNA of these genes can have drastic effects on the organism (see Homeotic mutations, below), and so these genes have changed relatively little over time.

The homeodomain

The protein products of Hox genes belong to a class of proteins known as transcription factors, all of which are capable of binding to DNA, thereby regulating the transcription of genes. The homeobox sequence codes for a 61 amino acid helix-turn-helix protein known as the homeodomain. The homeodomain acts as an "on/off" switch for gene transcription by binding to specific sequenceenhancers of a gene, which either activates or represses the gene. The same Hox protein can act as a repressor at one gene and an activator at another. For example, in flies (Drosophila melanogaster) the protein product of the Hox gene Antennapediaactivates genes that specify the structures of the 2nd thoracic segment, which contains a leg and a wing, and represses genes involved in eye and antenna formation^[4]. Thus, legs and wings, but not eyes and antennae, will form wherever the Antennapedia protein is located.

Genes Regulated by Hox Genes

Hox genes act at many levels within developmental gene hierarchies: at the "executive" level they regulate genes that in turn regulate large networks of other genes (like the gene pathway that forms an appendage). They also directly regulate what are called realisator genes or effector genes that act at the bottom of such hierarchies to ultimately form the tissues, structures, and organs of each segment. Segmentation involves such processes as morphogenesis (differentiation of precursor cells into their terminal specialized cells), the tight association of groups of cells with similar fates, the sculpting of structures and segment boundaries via programmed cell death, and the movement of cells from where they are first born to where they will ultimately function, so it is not surprising that the target genes of Hox genes promote cell division, cell adhesion, apoptosis, and cell migration^[5].

Enhancer sequences that bind the homeodomain

The DNA sequence that is bound by the homeodomain protein contains the nucleotide sequence TAAT, with the 5' terminal T being the most important for binding^[13]. This sequence is conserved in nearly all sites recognized by homeodomains, and probably distinguishes such locations as DNA binding sites. The base pairs following this initial sequence are used to distinguish between homeodomain proteins, all of which have similar recognition sites. For instance, the nucleotide following the TAAT sequence is recognized by the amino acid at position 9 of the homeodomain protein. In the maternal protein Bicoid, this position is occupied by lysine, which recognizes and binds to the nucleotide guanine. In Antennapedia, this position is occupied byglutamine, which recognizes and binds to adenine. If the lysine in Bicoid is replaced by glutamine, the resulting protein will recognize Antennapedia-binding enhancer sites^[14]

Regulation of Hox genes

Just as Hox genes regulate realisator genes, they are in turn regulated themselves by gap genes and pair-rule genes, which are in their turn regulated by maternally-supplied mRNA. This results in a transcription factor cascade: maternal turns on gap or pair-rule genes; gap and pair-rule genes turn on Hox genes; then, finally, Hox genes turn on realisator genes that cause the segments in the developing embryo to differentiate.

Regulation is achieved via protein concentration gradients, called morphogenic fields. For example, high concentrations of one maternal protein and low concentrations of others will turn on a specific set of gap or pair-rule genes. In flies, stripe 2 in the embryo is activated by the maternal proteins Bicoid and Hunchback, but repressed by the gap proteins Giant and Kruppel. Thus, stripe 2 will only form wherever there is Bicoid and Hunchback, but not where there is Giant and Kruppel^[15].

MicroRNA strands located in hox clusters have been shown to inhibit more anterior hox genes ("posterior prevalence phenomenon"), possibly to better fine tune its expression pattern. ^[16]

Non-coding RNA (ncRNA) has been shown to be abundant in Hox clusters. In humans, 231 ncRNA may be present. One of these, HOTAIR, silences in transcription (it is transcribed from the HOXC cluster and inhibits late HOXD genes) by binding toPolycomb-group proteins (PRC2). ^[17]

The chromatin structure is essential for transcription but it also requires the cluster to loop out of the chromosomal territory.^[18]

Quantitative PCR has shown several trends regarding colinearity: the system is in equlibrium and the total number of transcripts depends on the number of genes present according to a linear relationship ^[19].

Homeotic mutations

Incorrect expression of Hox genes can lead to major changes in the morphology of the individual. Homeotic mutations were first identified in 1894, when William Bateson noticed that floral stamens occasionally appeared in the wrong place; he found for example flowers in which the stamens would grow in the place where petals normally grow.

In the late 1940s, Edward Lewis began studying homeotic mutation on Drosophila melanogaster which caused bizarre rearrangements of body parts. Mutations in the genes that code for limb development can cause deformity or lead to death. For an example, mutations in the Antennapedia gene cause legs to develop on the head of a fly instead of the antenna.^[20]

Another famous example in the Drosophila melanogaster is the mutation of the Ultrabithorax Hox gene, which specifies the 3rd thoracic segment. Normally, this segment displays a pair of legs and a pair of halteres (a reduced pair of wings used for balancing). In the mutant lacking functional Ultrabithorax protein, the 3rd thoracic segment now expresses the same structures found on the segment to its immediate anterior, the 2nd thoracic segment, which contains a pair of legs and a pair of (fully developed) wings. These mutants sometimes occur in wild populations of flies, and it was these mutants that led to the discovery of Hox genes.

Colinearity of Hox genes

The various Hox genes are situated very close to one another on the chromosome in groups or clusters.

Classification of Hox genes

Hox genes in different phyla have been given different names, which has led to confusion about nomenclature. The complement of Hox genes of the Ecdysozoa (arthropods,nematodes, etc) is made up of two clusters, the Antennapedia complex and the Bithorax complex, which together are referred to as the HOM-C (for Homeotic Complex). Hox genes in deuterostomes (echinoderms, chordates) are correctly referred to as Hox genes, and are arranged in four clusters: Hoxa, Hoxb, Hoxc, and Hoxd. Although it is technically incorrect to refer to homeotic genes in non-deuterostome phyla as "Hox genes", the practice of using "Hox" in place of "Hom-C" is now acceptable even in the scientific literature.

Phylogenetic distribution of Hox genes

In Ecdysozoa, there are approximately ten Hox genes. Vertebrates have four duplicates (paralogues) of these ten genes, known as Hoxa, Hoxb, Hoxc, and Hoxd. These four paralogous clusters are a consequence of the ancestral vertebrate genome being twice duplicated in its entirety^[21]. The first occurred before the Cnidaria-Bilateria split, the second during the evolution of the fishes.[1] The arrows represent Hox genes arrayed along a chromosome. The bottom line represents the ten Hox genes seen in most invertebrates, and is the ancestral complement of the vertebrates. The top four lines represent the four duplicated clusters of these ten genes seen in vertebrates. In order from left to right (anterior to posterior), they are: labial, proboscipedia, zerknullt, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, Abdominal-A and Abdominal B. Arrows with the same color came from the same ancestral gene.

Although these vertebrate genes are duplicates of the same genes seen in the Ecdysozoans, the four copies are not actually identical. Each copy has accumulated its own unique mutations over time, producing proteins with distinct functions. Some have actually been deleted entirely or duplicated again in certain vertebrate groups.

For example, Hoxa and Hoxd are involved in the segment identity along the limb axis. Hox expression in the limb has two phases, an early wave of expression for the arm and a late wave for the digits, which involves Hoxd 8 – 13 and has a separate regulatory region 5’ of Hoxd 13 which is not found in teleost fish ^[22].

Human genes

Humans contain Hox genes in four clusters: