Structural studies of function and regulation of microtubules and transcriptional gene expression machinery

项目摘要

Project Abstract Our lab is dedicated to the mechanistic understanding of macromolecular function through the visualization of structure, dynamics, and regulatory interactions. Towards that goal, we use cryo-EM, together with biochemical and biophysical assays. Our areas of study are centered on the characterization of the regulatory molecular mechanisms governing the function of microtubules and of human transcription/epigenetic complexes. Microtubules (MTs) are essential polymers in eukaryotic cells s built of -tubulin dimers. Dynamic instability, the switching between growing and shrinking phases due to the coupling of the assembly process to the exchange and hydrolysis of GTP in -tubulin, is an essential property for MT function. Many MT cellular partners modulate MT dynamics or utilize it to carry out specific functions. In the past. In the last 5 years, we confirmed and complemented our past studies, which used non-hydrolyzable GTP analogs to characterize conformational changes in MTs that accompany GTP hydrolysis, now using of GTP-hydrolysis tubulin mutants. We also defined the mode of binding and action of cellular factors that regulate MT assembly, dynamics and organization in the cell and described the effect of tubulin acetylation on MT structure and function. We will continue this work with the central theme of adding complexity to our studies in order to bring us closer to the regulated function of microtubule cellular systems as we also add techniques complementary to our major tool, cryo-EM. Transcriptional regulation of gene expression is critical for growth and survival, and of obvious significance to human health. Its initiation involves RNA polymerase II (Pol II) together with TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Regulation is achieved by sequence-specific activators or repressors, co-factors, and chromatin remodeling/modifying complexes. During the last funding period we defined the structures of human TFIID[17] and TFIIH[18], as well as other large transcriptional coactivators like the human SAGA[19], and the yeast NuA4 (in preparation) and RSC complexes[20]. We will now characterize the human TIP60 complex, the binding of transcriptional co-activators to chromatin substrates, and pursue the dynamic visualization of TFIID engagement with promoter DNA to gain further knowledge of how these complexes work in the nucleus to regulate gene expression. Polycomb repressive complex 2 (PRC2) is an epigenetic gene silencer that methylates lysine 27 of histone H3 and is essential for cellular differentiation and development. After obtaining the structure of human PRC2 with its cofactors JARID2 and AEBP2 and its interaction with a di-nucleosome, we have now defined how PRC2 recognizes mono-ubiquitylated nucleosome, the substrate created by the other major Polycomb complex, PRC1. We will now further characterize the regulatory landscape of PRC2 looking into other histone modifications, different forms of PRC2 (i.e., other cofactors), the role of PRC2 auto-methylation, and binding of PRC2 to RNA.