MODULATORS OF MAD FAMILY FUNCTION, MLX AND MSIN3

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Myc and mad are transcription factors of the basic region helix-loop- helix leucine zipper (BHLHZip) family that play a role in controlling proliferation, differentiation and cell death. Myc and Mad both require dimerization with Max, another member of the bHLHZip family for function. Myc:Max complexes are transcriptional activators and their activity is associated with proliferation. Mad:Max complexes are transcriptional repressors and their activity is associated with cell cycle exit and differentiation. Therefore Mad is an antagonist of Myc. The goal of this study is to gain a better understanding two modulators of Mad function, M1x and mSin3. M1x is a new member of the BHLHZip family which we have identified as binding partner for Mad1. It is most similar to Max and may have similar functions. mSin3A is a transcriptional corepressor required by Mad to function as a transcriptional repressor. By studying these two proteins insight into the mechanism of Mad biological activity and its modulation of the Myc oncogene will be gained. Mix activity will be studied on several levels. M1x protein will be examined in a variety of cells by western blotting and immunoprecipitation. The half-life, subcellular localization and association with Mad family proteins will be determined. It will be determined if M1X is a transcriptional activator or repressor and how it effects the transcriptional activities of Myc and Mad. The DNA binding specificity of M1x will be examined by the electrophoretic mobility shift assay and specific contacts by chemical modification. Because M1x can function to inhibit cell growth, the effect of M1x overexpression on cell cycle behavior will be examined by expressing M1x using retroviral vectors. mSin3A is a component of a multiprotein complex. To gain a better understanding of mSin3A driven transcription repression the mSin3 complex will be purified by standard and affinity chromatographic methods. The mSin3A associated proteins will be microsequenced and using this peptide sequence the cDNAs encoding them will be identified, cloned and sequenced. The mechanism of transcription repression will be studied in vitro using a reconstituted in vitro transcription system and the purified mSin3A complex. To understand the molecular connectivity of the complex intermolecular associations between the components of the complex will be investigated using directed two-hybrid assays.