Ascl1 binds in vitro to the E-box in enhancer MSB4

First, I asked whether native Ascl1 was able to bind to the E-box consensus in the enhancer MSB4. I chose enhancer MSB4 for the first EMSA experiments because of its simple architecture, with only one E-box and one Sox motif present in the sequence. Furthermore, MSB4 showed an interesting feature in the regulation by Ascl1 and Sox factors, more precisely the requirement of the Sox motif for the activation of the element by exogenous Ascl1. A 63 bp 32P-labelled DNA probe corresponding to the region of MSB4 enhancer containing both the E-box and the Sox motif, named MSB4

wt probe, was incubated with a whole cell lysate from 293T cells overexpressing Ascl1. The expression vector contained a fusion protein Ascl1 tethered to E47, which is known to bind strongly to an E-box consensus sequence (Castro et al., 2006). It is known that proneural proteins bind the DNA as heterodimers formed with the ubiquitously expressed E proteins, such as E12 and E47 in mammals (Massari and Murre, 2000, Castro et al., 2006). Therefore, the use of the fusion protein Ascl1-E47 has the advantage of strong and specific binding to the E-box in the sequence of the probe in the in vitro binding assay. The binding mixture was resolved on a native polyacrylamide gel. A major complex between the DNA probe and the Ascl1-293T cell lysate was detected (Figure 5.2 A, lane 3 vs 2 with Mock-293T cell lysate). To prove the identity of Ascl1 as binding protein in the complex with the DNA, a supershift assay was performed. In this control experiment, the use of an antibody against the protein of interest should recognise and bind specifically the protein. Two are the possible outcomes of the control experiment if the antibody detects and binds the protein of interest in the complex: either it further retards the migration of the complex causing a “supershift” or it competes with the DNA probe for binding to the protein and therefore weakens the intensity of the band due to reduced amount of the specific protein-DNA complex in the binding mixture. In this experiment, the addition of a mouse Ascl1 antibody led to the supershift of the complex, while the control mouse IgG antibody did not affect its mobility (Figure 5.2 A, lanes 7 and 8). Therefore, this indicates that it is specifically Ascl1-E47 to bind to the probe and to form a complex with the DNA. To prove that Ascl1-E47 binds specifically to the sequence of enhancer MSB 4, a specific competitor analysis was also carried out. The addition of a 200-fold excess of unlabelled specific probe (thus referred to as cold probe), MSB 4 cold, with the same sequence than the 32P labelled one, successfully competed for Ascl1 binding, strongly

reducing the detection of the band, as showed in lane 4 (Figure 5.2 A, compare lane 4 and 3). This proves that Ascl1 tethered to E47 binds specifically to the MSB4 enhancer sequence. Next, I examined the requirement of an intact E-box and Sox motifs within the MSB4 enhancer in the formation of the Ascl1-DNA complex. Mutation of the E-box prevented Ascl1-E47 from binding when MSB4 M1 (E-box mutant probe) was added to the binding reaction mixture instead of MSB4 wt; while mutation of the Sox motif did not affect formation of the complex and detection of the band when MSB4 M2 (Sox mutant probe) was used instead of the wt probe (Figure 5.2 A, lanes 5 and 6). This proved that Ascl1 tethered to E47 needs an intact E-box for binding, and it can bind independently from the Sox motif.

Consistently, a similar experiment where the Ascl1-E47 fusion protein was expressed using the “cell-free” rabbit reticulocyte lysate TNT system for in vitro translation led to the same results, as showed in Figure 5.2B. In this experiment also an unrelated probe corresponding to the Nestin enhancer sequence, Nes probe, without known Ascl1 binding motif in the sequence, was used alternatively to the MSB4 probe to further prove sequence-specificity of the binding (Tanaka et al., 2004). Indeed, no band was observed in the absence of an Ascl1 binding motif when Nes probe was added to the binding reaction instead of MSB4 wt (Figure 5.2 B, lane 7). In conclusion, these results showed that Ascl1 binds to the MSB4 enhancer specifically through an intact E-box, and it is able to bind independently from the Sox motif.

Figure 5.2. Ascl1 binds in vitro to the MSB 4 enhancer sequence.

(A) EMSA experiments were used to analyse the interaction between native Ascl1 and a 63 bp 32_P-

labelled probe corresponding to MSB 4 wt sequence containing the E box and the Sox motif. Whole cell lysate from 293T cells transfected with a Ascl1-tethered to E47 construct was used as source of the protein. Fusion protein Ascl1-E47 was capable to form a specific complex with the wt probe as confirmed by supershift analysis with mouse anti-Ascl1 Antibody (α-Ascl1) (lanes 3, 7 and 8). 200x-fold excess of cold specific competitor probe, MSB 4 wt cold, could compete for binding with Ascl1, as proved by strong fainting of the band (lane 4). Use of a MSB 4 probe mutated in the E box, MSB 4 M1, could prevent the binding, showing requirement of an intact E box (lane 5) while the use of a MSB 4 probe mutated in the Sox motif, MSB 4 M2, did not affect the binding, proving that Ascl1 can bind independently from the Sox motif on the MSB 4 enhancer sequence (lane 6). Lane 1 shows the migration pattern of the free unbound wt probe, MSB 4 WT. Lane 2 is the control mock lysate from untransfected 293T cells. (B) The same EMSA experiment as in figure A was carried out using rabbit reticulocyte lysate TNT system as source of the fusion protein Ascl1-E47. Results were consistent in both cases. In lane 7 an unrelated probe was used, Nes probe, to confirm sequence-specificity of the binding, as no band was detected when using this probe, according to predictions.

5.3 Discussion

The results of the EMSA experiments proved that the TF Ascl1 binds directly to the MSB4 enhancer sequence. The use of mutant probes carrying aberrant E-box and Sox motif proved that binding of Ascl1 requires specifically an intact E-box and is independent from the Sox site. The results for Ascl1 are consistent with its transcriptional activation of the same enhancer, as shown in luciferase assay. Furthermore, in luciferase assay loss of activation of the mutant enhancer with disrupted E-box by Ascl1 correlates with loss of binding of the same TF to the mutated probe observed in EMSA.

In the future, it will be important to optimise the experimental condition of EMSA to determine if the Sox factors analysed in this study, in particular Sox2 and the SoxC factors Sox4 and Sox11, bind directly to the Sox motif of the same neuronal enhancer MSB4. It will be important to determine if they bind independently or simultaneously and cooperatively with Ascl1. Results from luciferase assays so far suggest that Ascl1 and Sox2 might not bind to neuronal enhancer MSB4 simultaneously since they counteract each other in the transcriptional regulation of this element. Conversely, results from luciferase assays can be compatible with cooperative binding of SoxC factors with Ascl1 to this enhancer to explain their synergistic activation of this element. In conclusion, the functionality of the Sox binding to this enhancer and the requirement of the Sox motif for the transcriptional activation of this element by exogenous Ascl1 needs to be further understood.