Muñoz Centeno, María de la CruzChávez de Diego, SebastianFontalva Ostio, Sara2024-10-082024-10-082024-07-08Fontalva Ostio, S. (2024). Human prefoldin regulates gene Transcription through FACT-mediated chromatin Dynamics. (Tesis Doctoral Inédita). Universidad de Sevilla, Sevilla.https://hdl.handle.net/11441/163313The primary function of prefoldin is well-established in the cytoplasm, where it serves as a cochaperone, favouring the co-translational folding of actin and tubulin monomers by transferring them to the ATP-dependent class II chaperonin CCT. The canonical form of prefoldin comprises six subunits known as PFDN1, 2, 3, 4, 5, and 6. This complex is ubiquitous across archaea and all eukaryotic organisms, and exhibits a highly conserved structure, consisting of two α and four β subunits. While prefoldin's role in the cytoplasm is widely recognized, it is also found in the nucleus, where it has been associated with transcription regulation and pre-mRNA splicing. However, the precise molecular mechanisms connecting human prefoldin to the control of gene expression remain elusive; prompting us to make it the aim of this doctoral thesis. To unravel these mechanisms, we followed different proteomic and genetic approaches. We employed mass spectrometry and genome wide sequencing techniques to identify prefoldin interactors in chromatin, and to delineate the changes that occur in chromatin under prefoldin perturbation conditions. We also analysed the composition of elongating RNA polymerase-associated factors to visualize how prefoldin contributes to the gene transcription. Our findings reveal that the whole prefoldin complex is indeed present in human chromatin, localizes to active genes, exhibiting a distribution pattern similar to RNA polymerase II, and locally influences the expression of these genes. We found that this chromatinic prefoldin interacts with the FACT complex, enhancing the presence of this H2A/H2B histone chaperone at transcriptionally active genes. Accordingly, we found that prefoldin was required for the optimal co-transcriptional dynamic of histones, and more specifically, allowing the removal of non-canonical H2A-H2B dimers. In agreement with FACT´s role in preserving chromatin integrity, we found that lack of prefoldin provoked abnormal accessibility to intragenic regions of transcribed genes and activation of repressed repetitive elements. Furthermore, we also found that prefoldin is required for the recruitment of the protein kinase CDK9 to active genes, the phosphorylation of elongating RNA polymerase II in the Ser2 residues of its CTD domain, and the subsequent association of pre-mRNA splicing and cleavage factors. These findings highlight the role of prefoldin in ensuring accurate gene expression, regulating chromatin dynamics and maintaining chromatin integrity. This is consistent with previous conclusion on the nuclear functions of canonical prefoldin in budding yeast, and underscores its significance for gene expression throughout evolution.application/pdf133 p.engAttribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccess