Nicolas Mermod

BACKGROUND: PCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. In this study, we evaluated the performance of known methods, and compared them with newly developed data processing strategies in terms of resolution, precision and robustness. RESULTS: Our results indicate that simple methods that do not rely on the estimation of the efficiency of the PCR amplification may provide reproducible and sensitive data, but that they do not quantify DNA with precision. Other evaluated methods based on sigmoidal or exponential curve fitting were generally of both poor resolution and precision. A statistical analysis of the parameters that influence efficiency indicated that it depends mostly on the selected amplicon and to a lesser extent on the particular biological sample analyzed. Thus, we devised various strategies based on individual or averaged efficiency values, which were used to assess the regulated expression of several genes in response to a growth factor. CONCLUSION: Overall, qPCR data analysis methods differ significantly in their performance, and this analysis identifies methods that provide DNA quantification estimates of high precision, robustness and reliability. These methods allow reliable estimations of relative expression ratio of two-fold or higher, and our analysis provides an estimation of the number of biological samples that have to be analyzed to achieve a given precision.

Enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to the symmetrical DNA sequence, CAGCTG. Here, we report the molecular cloning and characterization of human AP-4 cDNAs. The deduced amino acid sequence reveals that AP-4 is a helix-loop-helix (HLH) protein. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary and sufficient to confer site-specific DNA binding. However, unlike other HLH proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. The analysis of various deletion and point mutants for their ability to dimerize in the presence or absence of DNA reveals several unusual features. Although the HLH basic region is sufficient for DNA recognition and binding, dimer formation between different truncated versions of AP-4 in solution requires an intact LR1 or LR2 domain. AP-4 is unable to form heterodimers with other helix-loop-helix family members such as the immunoglobulin enhancer binding factor, E12. In contrast, an AP-4 derivative, delta C222, which lacks LR1 and LR2 but retains an intact HLH, can form heterodimers with E12. Moreover, AP-4 molecules containing LR2 or LR1 are unable to form mixed dimers with carboxy-terminally truncated AP-4 molecules such as delta C222, but retain the ability to form complexes with longer versions of AP-4 that contain LR1 and/or LR2. Our findings strongly suggest that AP-4 contains multiple protein-protein interfaces that function to promote homodimer formation and restrict heterocomplexes. These findings provide a mechanism by which different members of the helix-loop-helix family of transcription factors can form functional dimers in a specific fashion with their appropriate partners to control transcriptional networks during cellular differentiation.