Cross-talk between the Paired Domain and the Homeodomain of Pax3

Abstract

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.Pax3 is a member of a family of 9 transcription factors (1), defined by a DNA binding module, the Paired domain (PD), 1 that was first identified in the Drosophila protein Paired (Prd) (2). Pax proteins play critical roles during normal embryonic development, and inactivating mutations cause major defects in development of skeleton, muscles, nervous system, eyes, kidneys, and the immune system (3-12). Pax3 is expressed in developing somites and in neural tube and neural crest cell derivatives and plays a role in the proliferation, migration, and differentiation of cells involved in neurogenesis and myogenesis (13, 14). The Pax3 mouse mutant (splotch, Sp) displays spina bifida and exencephaly and lacks limb muscles; likewise, mutations in human PAX3 cause Waardenburg syndrome (WS), a condition characterized by pigmentary disturbances, craniofacial abnormalities and sensorineuronal deafness (14 -21). PAX3 also plays a role in cell transformation as shown by the translocation t(2;13) (q35;q14) involving PAX3 (22) and forkhead-related transcription factor (23) associated with the solid tumor alveolar rhabdomyosarcoma. The translocation leads to the expression of a fusion protein containing the Ntermi...

Nephroblastoma overexpressed (NOV/CCN3) gene: a paired-domain-specific PAX3-FKHR transcription target that promotes survival and motility in alveolar rhabdomyosarcoma cells

“…Our previous and current studies have revealed a major distinction between the transcriptional targets of PAX3 and PAX3-FKHR. Although the PD and HD DBDs of PAX3, either as individual entities or in the context of the full-length protein, can bind their cognate DNA sites independently of one another in vitro (Underhill and Gros, 1997; Apuzzo et al , 2004), it is generally accepted that PAX3 transcriptional activation requires the simultaneous engagement of both DBDs in vivo . Those studies have identified a canonical PAX3 responsive element as a composite consensus sequence of ATTAN 1 – 13 CT(G/T)(A/C)(C/T), where ATTA is a HD-binding site and GT(G/T)(A/C)(C/T) is a PD-binding site (Underhill and Gros, 1997; Phelan and Loeken, 1998).…”

“…Indeed, selective transactivation by PAX3-FKHR but not PAX3 has been detected for a handful of genes (Epstein et al , 1998; Begum et al , 2005; Zhang and Wang, 2007). PAX3 can only activate transcription when both DBDs act synergistically and cooperatively (Chalepakis et al , 1994; Chalepakis and Gruss, 1995; Underhill and Gros, 1997; Apuzzo et al , 2004). Although this specificity toward PAX3-dependent targets is conserved in PAX3-FKHR, the fusion protein can use either DBD independently to activate genes that are not PAX3 targets (Epstein et al , 1998; Zhang and Wang, 2007).…”

The Diabetes-Linked Transcription Factor PAX4: From Gene to Functional Consequences

“…The N-terminal subdomain, termed PAI, is a highly conserved region that confers DNA sequence recognition. The C-terminal subdomain, the RED subdomain, is more divergent and, despite presenting lower affinity for DNA in some PAX proteins, can modify the binding specificity of the PD [45,46,47,48,49]. The flexible linker between these two HTH motifs also establishes contact with DNA [47].…”

“…The flexible linker between these two HTH motifs also establishes contact with DNA [47]. The second DBD, the HD, can also bind independently and with high affinity to specific DNA sequences; nevertheless, a cooperative action of the PD and the HD on DNA recognition has been documented [50], and functional interdependence between these two domains regulates PAX proteins binding to their target gene regulatory elements [48,51,52,53]. This double DBD with possibility of independent vs. cooperative action confers extraordinary flexibility of sequence recognition to each PAX protein hindering the correct prediction of recognition sites by sequence analysis of the putative target genes.…”

Pax7 pioneer factor action requires both paired and homeo DNA binding domains

“…However, close interaction of the two DBDs appears important for optimal DNA binding ( 18 , 19 ). Indeed, DNA binding by either the PD or HD causes a structural change in the other DBD ( 20 ). In this context, recognition of a unique composite DNA sequence containing binding motifs for both PD and HD enriched at pioneered targets suggest that Pax7 pioneer action may involve a unique conformation or binding requirement.…”