Identification of the components of the E-hox DNA-binding complexes in HDFs

The first approach was to use antisera against class A bHLH proteins to determine whether they super-shift or disrupt the complex. Preliminary indications were that a commercial anti serum against E2A (Santa Cruz, sc-416) did not quantitatively affect the complex, in line with unpublished findings of Fiji Hara (Figure 3.7a).

Since there are few dependable antisera available to other members of the E2A family (antisera against E2-2 and HEB proteins were not available from commercial services), polyclonal rabbit antisera to the C-terminal peptides of human E2-2 and HEB were generated and evaluated for their ability to recognise the cognate proteins synthesised in vitro or in labelled cell lysates (data not shown). For example, it was important to show that these sera specifically affect the E2-2 and HEB homodimers formed in the model in vitro bandshift assay (Figures 3.8 and 3.9). Full length cDNAs for E2-2 and HEB, obtained from Tom Kadesch and Robert Kingston respectively, were transferred into appropriate vectors for coupled in vitro transcription and

Hours after serum addition

0 4 8 12 16 20 22 24 28 30 32 36

c - Specific com plex

<3= Free probe

Figure 3.6 Cell cycle-dependent expression of an E-box complex in HDFs.

Quiescent TIG-3 cells were stimulated by the addition of serum and whole-cell extracts were prepared at the indicated times after serum addition. The whole cell extracts were used in EMSA assays using a 26bp oligonucleotide E-box probe from the MCK promoter. The specific high molecular weight protein-DNA complex is arrowed. Flow cytometric analysis of parallel cell cultures indicated that the cells began to enter S phase after «24 h.

Chapter 3 - The role o f Id and bHLH proteins in cell cycle control w Specific complex Free probe

II

Figure 3.7 Identification of the components of the E-box DNA-binding

complex in human primary diploid fibroblasts (TIG-3 cells).

a, Electrophoretic mobility shift assays were performed using extracts prepared from TIG-3 cells (at 16 h after serum addition). In lane 1, the specific high molecular weight complex (arrowed) was competed by 20 ng of hacterially expressed Id2. In lanes 3 and 4, either 0.2 pi or 2 p 1 of E2A anti serum was added to the reaction mixture, b, A similar assay was performed using, in lane I, 2 pi of HEB anti serum and, in lane2, 2 pi of E2-2 anti serum added to the reaction mixture. The specific high molecular weight complex is arrowed.

translation. The corresponding human HEB (Hu et a l, 1992) and B2-2 (Bain et a l, 1994) proteins were synthesised in reticulocyte lysates and their sizes and yields were verified by ^^S-methionine labelling and SDS-PAGE (data not shown). Equivalent amounts were then used for EMSA experiments, either individually or in combination with MyoD, with two different oligonucleotide probes containing respectively the pE2 site and the pE5 site from the human IgH gene enhancer region (Figures 3.8 and 3.9). Preliminary experiments have indicated a modest binding preference for HEB on the pE2 site and for E2-2 on the pE5 site (Henthom et a l, 1990; Ruezinsky et a l, 1991; Hu et al., 1992; Bain et a l, 1993). Full-length HEB protein formed a complex on the pE2 ^^P-labelled DNA probe consistent with the formation of an HEB homodimer (Figure 3.9, lane 2). As previously described, a bandshift was detected using MyoD alone (Figure 3.9, lane 3). A 1:1 mixture of HEB and MyoD proteins produced an additional DNA-binding complex, presumably a heterodimer, that migrated at an intermediate position between the HEB-HEB and MyoD-MyoD homodimer complexes (Figure 3.9, lane 4). As a control for the specificity of these interactions, an excess of unlabelled oligonucleotide completely abolished the formation of complexes on the labelled probe whereas an oligonucleotide containing a mutated pE2 site did not (Figure 3.9, lanes 5 and 6). As previously described, addition of MyoD anti serum decreased the mobility of the MyoD-MyoD complexes (Figure 3.9, lanes 7 and 8) as well as affecting the mobility of the presumed HEB-MyoD heterodimer whereas the HEB-HEB homodimer complex was unaffected by the MyoD antiserum (Figure 3.9, lanes 9 and 10). Addition of the polyclonal anti serum against human HEB was able to super-shift the HEB-HEB homodimer complex (Figure 3.9, lanes 11 and 12) as well as affecting the mobility of the presumed HEB-MyoD heterodimer. As anticipated, the MyoD-MyoD complexes were unaffected even by a higher concentration (2 pi) of HEB antibody (Figure 3.9, lanes 13 and 14).

Similar results were obtained for E2-2 and MyoD binding to the pE5 E-box oligonucleotide (Figure 3.8, lanes 2-10). A polyclonal antibody against human E2-2 that

Chapter 3 - The role o f Id and bHLH proteins in cell cycle control

MyoD heterodimer, leaving the MyoD-MyoD complexes unaffected (Figure 3.8, lanes 13 and 14).

Having validated HEB and B2-2 antibodies in model in vitro EMSA using HEB and E2-2 bHLH proteins and pE2 and pE5 E-box oligonucleotides, we then asked whether these antibodies would super-shift the specific E-box in HDFs. However, the specific E-box DNA binding complex observed in human primary fibroblasts did not seem to be affected by either antisera against HEB or antisera against E2-2 (Figure 3.7b). Either the in vivo complexes contain additional proteins that mask the epitopes recognised by the antibodies, or not enough antibody has been used. An alternative but rather surprising explanation would be that none of the known class A proteins are involved in the fibroblast-specific complex.

3.8.Cell-cycle dependent expression of an E-box DNA-binding complex in Wt and