La Reintegración Económica en la Alta Consejería para la Reintegración, ACR

There are many lines of evidence that demonstrate that HOX proteins function as regulators of gene expression. First, HOX proteins have a DNA binding HD

(MCGINNISet al. 1984). Second, many experiments have demonstrated that HD containing proteins can control transcriptional activation or repression of target gene activity (TOURet al. 2005; CAPOVILLAet al. 1994; JAYNES and O’FARRELL 1988). Last, gain of function and loss of function alleles of Hox genes have demonstrated that these genes have defined functions. For example, the Hox gene Antp is required for establishing T2 leg identity and Scr is required for T1 leg identity. Microarray analyses of downstream target genes of Hox expression have shown that each Hox gene, with perhaps the exception of Abd-B, regulates a unique set of target genes (HUEBERet al. 2007). Despite unique in vivo expression profiles, Drosophila HOX proteins share a high degree of sequence similarity in the HD and bind the same

DNA sequences in vitro; therefore, it is unclear how HOX proteins achieve specificity in vivo.

To understand HOX specificity, studies first aimed to identify the regions of HOX proteins required for functional specificity. Examination of chimeric ANTP and SCR proteins narrowed down the region required for specificity to residues 1, 4, 6 and 7 of the HD (FURUKUBO-TOKUNAGAet al. 1993; GIBSONet al. 1990). This particular region of the HD interacts with the minor groove of DNA, and was proposed to alter

specificity by selective protein-protein interactions with other regulatory proteins and/or by affecting DNA binding. One mechanism that may affect specificity by this latter mode is post-translational modification. The HOX protein SCR was reported to be phosphorylated at residues 6 and 7 of the HD, which are threonine and serine residues, by serine-threonine protein phosphatase 2A (dPP2A,B’; BERRY and GEHRING 2000). In ectopic expression experiments, expression of SCR molecules mimicking constitutively dephosphorylation at residues 6 and 7 were active and could induce transformations typical of overexpression of wild-type SCR, such as the formation of ectopic T1 beards and embryonic salivary glands. The version of SCR mimicking constitutively phosphorylation was found to be inactive due to impaired DNA binding (BERRY and GEHRING 2000). However, in another study, a null mutation in PP2A,B’ did not result in the expected decrease of SCR activity (MOAZZENet al. 2009). Despite the loss of PP2A,B’ activity, there was no significant change in the

development of three SCR dependent structures: the sex comb bristles on T1 leg, the proboscis and the larval salivary glands. This latter observation questions the role of phosphorylation as a mechanism for determining SCR specificity.

Another potential solution is the regulation of HOX specificity through interaction with HOX cofactors. The best-studied HOX cofactor is Extradenticle (EXD). exd is a zygotic, X-linked embryonic lethal allele that causes homeotic transformation of segments (WIESCHAUSet al. 1984; WIESCHAUS and NOELL 1986). In exd loss of function mutants the expression pattern of Hox genes is not altered, though the segments themselves have different segmental identities (PEIFER and WEISCHAUS 1990). The

ectopic expression of UBX in exd null embryos also demonstrates the role of EXD in conferring segmental identity. Where ectopic UBX expression normally results in the transformation of head and thoracic segments towards A1 identity, ectopic expression of UBX in exd null mutants results in the transformation of these

segments towards A3, rather than A1, identity (MANN and HOGNESS 1990; PEIFER and WEISCHAUS 1990).

The interaction between EXD and HOX proteins is direct. Using yeast two hybrid assays it was determined that this interaction is dependent upon the EXD HD and the region of UBX encoding the HD plus a 15aa sequence upstream, which includes the highly conserved YPWM motif (JOHNSONet al. 1995). A direct interaction

between EXD and another HOX protein, ABD-A, has also been observed in vivo using bimolecular florescence complementation (BiFC; HUDRYet al. 2011). The interaction of EXD with HOX proteins confers specificity by altering the sites to which HOX proteins bind. HOX monomers bind indiscriminately to 8-10bp sequences in vitro; however, HOX-EXD heterodimers bind sequences that are specific to a particular HOX protein (SLATTERYet al. 2011; MULLERet al. 1988, CHOet al. 1988; HOEY and LEVINE 1988. One example of EXD dependent specificity is the role of SCR and EXD in salivary gland development (RYOO and MANN 1999). Together, SCR and EXD

regulate the expression of a number of salivary gland genes including forkhead (fkh). It has been shown with in vivo and vitro experiments that SCR and EXD bind

cooperatively to the fkh[250] enhancer; whereas, other HOX-EXD heterodimers do not bind this enhancer in vivo and bind with a low affinity in vitro (RYOO and MANN 1999). The principle EXD interaction domain in HOX proteins, the YPWM motif, is highly conserved and found in all HOX proteins. The YPWM motif of SCR makes a protein-protein interaction with the hydrophobic pocket of the EXD HD (JOSHIet al. 2007). Deleting the YPWM motif of the mammalian homologue, HOXA5, results in an inability to induce ectopic Forkhead (FKH) expression (ZHAOet al. 1996);

however, this deletion of 16 amino acids includes a His residue important for minor groove interactions between SCR and EXD with the fkh enhancer element (JOSHIet

al. 2007). This latter observation suggests the presence of regions in addition to the YPWM motif that are required for interaction with EXD.

There are, however, many examples of EXD independent HOX function. Though EXD is required with SCR for salivary gland development, EXD is not required with SCR for the formation of sex comb bristles and pseudotrachea (PERCIVAL-SMITH and HAYDEN 1998; JULIAet al. 2006). This is similar to the observation that UBX does not require EXD for haltere development (GALANT and CARROLL 2002). An explanation for the observation that EXD is not required for all HOX dependent functions is that the YPWM of SCR interacts with a protein other than EXD. The YPWM motif of ANTP binds BIP2, a TATA binding protein associated factor linked with the basal

transcriptional machinery (PRINCEet al. 2008). The co-ectopic expression of BIP2 and ANTP promotes the formation of ectopic wing tissue in Drosophila (GANGLOFFet

al. 2001; PRINCEet al. 2008). Since BIP2 is expressed widely throughout all of the imaginal discs of third instar larvae, there is a strong possibility that BIP2 may interact with the YPWM motifs of other HOX proteins as well (GANGLOFFet al. 2001). Despite the isolation of HOX genes more than 30 years ago, there are still major gaps in our knowledge of how these genes function as transcription factors in the cell, because few target genes have been identified and little is understood about general functional domain structure.