How Hox proteins attain specificity in vivo

By | May 2, 2021
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In one of the previous posts on iflybio  we talked about Hox genes ,about their properties ,importance in anterior posterior patterning axis in Drosophila and we also seen how these Hox genes come into picture during early embryogenesis.Today we will briefly overview into one of fundamental question in the field of biology “How do Hox Proteins or transcription factors in general attain specificity invivo.

Hox genes codes for transcription factors which controls a development fate by regulating a cascade of downstream target genes.Hox proteins bind to DNA as monomer through a very conserved 60 amino acid long motif called as Homeodomain. The Homeodomain consists of an N-terminal arm that lies in the DNA minor groove, and three helices, the second and third of which form a helix-turn-helix motif. The third helix inserts into the major groove and makes base-specific contacts, thereby getting the name “recognition helix” . Several experiments on Hox proteins specificity yielded two disconcerting results were obtained. First being that these Hox proteins bind to specific 4-6 pairs sequence (numerous such sequences will exists on DNA),making it more common for ensuring enough biological specificity.Secondly all hox proteins have similar and sometimes identical binding properties invitro.
These in vitro results cannot reflect the state of affairs in vivo, where each homeotic gene product confers a unique segmental identity.

Hox specificity and activity can be largely attributed to other proteins which act as co factors.Hox proteins mostly doesnot act alone in regulation of target genes.Co factors along with Hox proteins forms a complex at promoter region of the gene and bring in the regulation.First evidence for the presence of co factors came from genetic experiments of Peifer and Eric Wieschaus(1990) which led to identification of extradenticle (exd) gene. Mutations of this gene caused homeotic transformation without effecting the exression patterns of Hox genes suggesting role of exd in refining Hox selection for target gene enhancer. Exd is also a Homeodomain containing protein,belongs to PBC class of protein (vastly Diverged from Hox like Homeodomain).Homeodomain of PBC belongs to a much broader class called TALE group (Tri aminoacids loop extensions) which differs by having three amino acids more in Homeodomain between helices 1 and 2.

Genetic experiments from the lab of Eric wieschaus’s lab showed that in the absence of sufficient extradenticle product, altered segmental morphology results from alteration of the functional consequences of specific homeodomain proteins, possibly through alterations in their target gene specificity.They also proposed that Exd could alter the DNA-binding specificity of the Hox proteins by forming heterodimers with them.The identification of PBC proteins in a variety of other animal species suggesting that a similar mechanism may underlie Hox function throughout the animal kingdom.Later from the work done in the lab of Richard Mann and C.Murre in 1994 provided the biochemical evidence for heterodimeriation of Hox proteins with PBC class of proteins on spcific DNA sequences which harbors Hox and PBC binding sites side by side.In this way the consensus for Hox-PBC binding becomes much longer then 4-6 bases recognised by Hox alone which will be not common in genome ,offering explanation for invivo specificity.Later another Hox cofactor Homothorax(Hth) was also identified (again from Richard mann’s lab ) in drosophila which is required for exd translocation into the nucleus.In the absence of Homothorax exd is blocked in cytoplasam.Hth is also Homeodomain containing protein belonging to Tale class interacts with exd through HM domain.Mammalian Hth homologs Meis1 and Prep1 have also been implicated as PBC partners. Hth heterodimerizes with Exd in solution, and trimeric complexes of HD proteins from the Hox.

Structural biologists also looked at structure of Hox protein and exd dimer on short DNA sequence to see how does this interaction occur.

Four research articles on Hox exd interaction on DNA sequence have been published :

1) Hox B1 -PBC Piper et al 1999
Piper, D.E., Batchelor, A.H., Chang, C.-P., Cleary, M.L. & Wolberger, C. Cell 96, 587–597 (1999).

2) ubx -exd Passner et al 1999
Passner, J.M., Ryoo, H.D., Shen, L., Mann, R.S. & Aggarwal, A.K. Nature 397, 714–719 (1999).

3)HOXB9-PBC: LaRonde-LeBlanc NA and Wolberger C.

Structure of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide and DNA recognition anterior to posterior.
Genes Dev. 2003 Aug 15;17(16):2060-72.

4) SCR-exd Joshi et al

Joshi R, Passner JM, Rohs R, Jain R, Sosinsky A, Crickmore MA,Jacob V, Aggarwal AK, Honig B, Mann RS.
Functional specificity of a Hox protein mediated by the recognition of minor groove structure.
Cell. 2007 Nov 2;131(3):530-43

One common thing from all is that sequences outisde the homeodomain in hox protein is important for interaction with exd.In addition to having similar homeodomains, most HOX proteins have a short conserved stretch of amino acids to the N-terminal side of their homeodom,called as YPWM motif or hexapeptide .There is little if any direct contact between the two HDs themselves; rather, contact is made through the hexapeptide motif of the Hox protein, which fits into a hydrophobic cavity on the surface of the PBC homeodomain.The hydrophobic pockets in HD’s of PBC/exd class is due to three excess amino acids in the HD ,This what separates normal HD and TALE HD.The tryptophan’W’ in the YPWM makes a major contribution in interaction by making hydrophobic contacts.The importance of the tryptophan residue further proved by its absolute conservation in the more than 70 YPWM motifs.A conserved Tryptophan has also been shown to make contacts with exd in AbdB class of protein which lack a proper hexapeptide.

Hox proteins in presence of various cofactors by binding to their respective sites on DNA imparts all important specificity invivo for proper patterning during development.

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