Functional role of HMG-box factors in transcriptional regulation during early

3.2 Functional role of HMG-box factors in transcriptional regulation during early embryonic development in regard to the early onset of the ic-CRE expression

One mechanism involved in transcriptional control underlying patterning during early embryonic development is regulation of chromatin structure in order to facilitate assembly of multiple protein-DNA complexes and protein-DNA-bound protein-protein interactions. Members of the High Mobility Group (HMG)-domain superfamily are involved in modulating chromatin structure by displaying a functional architectural role; although they may be either incapable of direct transactivation (in the case of several SOX subfamily members; Kamachi et al., 1995; Yuan et al., 1995) or they may contain a transactivation domain functional only in a specific context of protein-protein interactions (in the case of Lef-1; Giese et al., 1993), the property of the HMG proteins to induce strong DNA bending upon binding (Giese et al., 1997; Dragan et al., 2004) suggests that they can facilitate assembly and stabilization of transcription factor-DNA complexes.

The HMG domain constitutes a discrete DNA-binding structure of ~80 aa. The superfamily can be divided in two subgroups; members of the TCF/SOX/MATA group contain a single sequence-specific HMG domain which binds in the minor groove of DNA recognizing variants of the motif sequence WWCAAAG (Laudet et al., 1993). Members of the HMG/UBF subgroup have multiple HMG domains which bind DNA in a less sequence-specific manner (Grosschedl et al., 1994;

Soullier et al., 1999). Both sequence-specific (SS) and non-sequence-specific (NSS) HMG-box proteins can modify chromatin structure by bending and unwinding DNA (Giese et al., 1997; Dragan et al. 2004), meaning that their functional role is basically architectural, i.e., to facilitate simultaneous binding of other sequence-specific transcription factors. An example stands for sequence-specific binding of the Sox2 HMG-box protein which participates in complexes with POU domain factors to facilitate transcription during early embryonic development (Dailey and Basilico, 2001). A consequence of their architectural role is that DNA-recognition and binding by the HMG-box factors leads to transcriptional activation only in a specific transcription factor-binding site context (Giesse and Grosshedi, 1993).

SOX proteins (Sry-related High-mobility-group Box) are related to the mammalian testis determining factor SRY, sharing at least 60% identities in their HMG-box DNA-binding domains (Laudet et al., 1993). Sox family members have been implicated in cell fate specification and differentiation, in processes such as male differentiation, neurogenesis and skeletogenesis (reviewed in Lefebvre et al., 2007). In Drosophila eight Sox genes have been identified and their expression patterns determined (Cremazy et al., 2001).

In §2.5.2, results supporting the role of HMG-related activity in temporal control of the early onset of the ic-CRE expression are presented. After 5’ dissection of the enhancer sequence [-4085_-3799 bp] it came up that fragment [-4014_-3985 bp] is essential for ensuring early onset of reporter expression in the intercalary anlage at stage 8 (Fig 2_35_B). This 30 bp element consists of two highly conserved (12 sp.) blocks GGATCAAAaGG and GTTGACAAAt (Capitals represent conserved residues). Both conform to the general HMG-box binding consensus [WCAAAS] (NCBI Conserved Domain Database; ‘cd01388 Sox-TCF_HMG-box’; Love et al., 1995; Werner et al., 1995) with one mismatch being in both cases a non-conserved nucleotide. Additionally, 3’

juxtaposed to the second block one more putative HMG-box recognition site TACAAAC lies in reverse complement orientation (-3977_-3983) matching the WCAAAS consensus. This sequence is filtered through 11 species phylogenetic conservation, with the D. yakuba species sequence being divergent (i.e. not in capitals in Fig. 2_37).

In particular the first conservation block GGATCAAAaGG scores the binding matrix of dTCF/Pangolin [WTCAAAS](MatInspector; Lee and Frasch 2000). It also strongly resembles the consensus binding sequence of dTCF determined by PCR-based site selection [GATCAAAGG] (van de Wetering et al., 1997), which matches the canonical binding site consensus of the mammalian Lef1/TCF [WWTCAAAGG] (van de Wetering et al., 1993). The predicted site displays only one mismatch to dTCF binding consensus which is the additional non-conserved A. Indeed, the wild type sequence is recognized in vitro by dTCF with very low affinity, but a mutated version of the probe removing the extra A leads to specific shift complex formation with in vitro expressed dTCF (Fig.

2_36). It is interesting that the one extra A in the sequence that distorts matching to dTCF consensus (WTCAAAS) and abolishes in vitro recognition and binding, is not phylogenetically filtered and disturbs the conservation block. Nevertheless, 11 Drosophila species carry an A at position 7 and it’s only D. grimshawi that carries a C instead. In vivo, it is likely that the site is recognized by another member of the HMG family as it still strongly conforms to the WWCAAW HMG-box binding consensus (Lee and Frasch, 2000).

Although in vitro expressed dTCF does not efficiently recognize in EMSAs any of the wild-type HMG-box putative binding sequences (found within the DNA stretch that confers the early onset of ic-CRE expression), it cannot currently be excluded that in vivo the ubiquitously expressed dTCF contributes to regulation of the ic-CRE expression outcome through direct binding. It was not however possible to detect a delay in the onset of reporter expression in the intercalary segment, controlled by the ‘γ1_mF3_hh R4 550 bp’ enhancer fragment (Fig. 2_35_I) in a loss-of-function background; dTCF activity was down-regulated by RNAi (UAS-pangolin hairpin (VDRC) driven from the maternal driver {pnos-GAL4/GCN4_bcd 3’ UTR}, §5.3). Regarding the resolved function

of dTCF/Pangolin as a wingless signaling effector (van de Wetering et al., 1997; Lee and Frasch, 2000), even if dTCF activity is involved in the early establishment of intercalary-specific expression of hh in vivo, then it is certainly independent of upstream wingless activity, since onset of wingless expression in the intercalary segment succeeds hh.

Furthermore, both highly conserved block sequences of the 30 bp fragment conferring early onset of the ic-CRE expression, plus the 3’ juxtaposed site (found at reverse-complement orientation), they all strongly resemble consensus binding sequence of the Sox-subfamily of HMG-box proteins [WWCAAW](reviewed in Lefebvre et al., 2007 and references therein; Churchill et al., 1995).

Specificity of DNA sequence, which consists of a cluster of three putative HMG-box recognition sites, in combination with previously reported critical functions of HMG-box factors in transcriptional regulation during early embryonic development (for example, Dailey and Basilico, 2001), supports that Drosophila HMG-box activity, perhaps other than dTCF, may indeed be involved in the establishment of hh expression in the procephalic intercalary segment anlage, ensuring the early onset of expression at stage 8.

Interestingly, one of the previously characterized Drosophila Sox homologues (Cremazy et al., 2001), Dichaete/Fish/Sox70D displays an early embryonic expression pattern that would recruit it as a very good candidate for being directly involved in the early activation steps of the ic-CRE expression. At stages 5/6 it is expressed in a procephalic ventral-lateral region, corresponding to the presumptive intercalary anlage, overlapping the early anterior procephalic expression domain of hh (Nambu and Nambu, 1996; Russell et al., 1996). During stages 7-10 strong expression is detected in the procephalic neuroectoderm including the intercalary segment. Defects appearing in the tritocerebrum anlage (Sorriano and Russell, 2000) of homozygous mutants indicate that the early embryonic function of Dichaete is involved in the establishment of cell-fates within the intercalary segment.

Moreover, Dichaete null mutants exhibit severe segmentation defects, including organization of head structures (Nambu and Nambu, 1996), and Sox-HMG-box function of Dichaete has been implicated in segment-specific transcriptional regulation of pair-rule as well as independently segment polarity gene expression. For example, loss of wg expression in the maxillary and labial segments was reported. Although it was suggested that it is rather unlikely that Dichaete is required for the initial activation of segment polarity gene expression, at least not in the gnathal and trunk segments, still a potential involvement in temporal control of the ic-CRE early onset of expression remains to be clarified. Nevertheless, it was proposed that Dichaete may directly modulate regulatory function of transcriptional complexes required for segment polarity gene expression. In addition, Sox-domain proteins can physically associate with transcription factors from divergent

families (reviewed in Wilson and Koopman, 2002) and by providing their strong DNA-bending properties they facilitate the assembly and stabilization of transcription factors-DNA binding complexes, thus providing a crucial architectural role in the establishment and coordination of transcriptional regulatory interactions.

Finding of a cluster of three HMG-box sites in the DNA stretch conferring early onset of the ic-CRE expression, all conforming to the SOX DNA-binding sequence consensus, as well as early procephalic ectodermal expression pattern of Dichaete Sox-encoding gene overlapping the anterior procephalic broad expression domain of hh and reported function of Dichaete in the development of the intercalary segment and transcriptional regulation of segment polarity genes wg and en, would all in a concert constitute reasonable directions to examine if Sox function of the Dichaete candidate is involved in ensuring early onset of the ic-CRE expression. This would subsequently lead to examine whether the Sox function of Dichaete is also involved in early procephalic transcriptional regulation of hh expression.