Periodic Reporting for period 2 - CHROMATRANS (Structural biology of chromatin transcription)
Periodo di rendicontazione: 2022-01-01 al 2023-06-30
The research project CHROMATRANS funded by the ERC addresses the fundamental biomedical question of how mammalian genes are regulated in their natural context. The issue being addressed is how genes are switched on in cells within their natural chromatin environment in the nucleus. Genes are copied by an enzyme called RNA polymerase, which uses DNA as a template to produce RNA. The RNA then can be used as an instruction plan for the synthesis of proteins that bring about cellular function. Cells regulate which genes are switched on by regulating access of polymerase to genes. Genes are made from DNA but embedded in a large complex called chromatin that contains proteins that wrap up DNA and condense it. Other proteins can open such dense chromatin structures and allow access of polymerase to their target genes. This is a major mechanism of gene regulation that we wish to understand. During this project we will provide major new insights into the molecular mechanism of how genes are activated by changing chromatin structure and how polymerase can copied genes in the context of chromatin. This is achieved by combining several techniques of molecular and structural biology. Since the dysregulation of genes occurs during many diseases, including cancer, it is of great biomedical importance and interest to the society to understand the mechanisms of gene regulation uncovered during this work. We hope that later this understanding can help in the development of therapeutic intervention.
During the first project period, we have prepared biochemically complexes of the Pol II pre-initiation on a ‘+1’ nucleosome at the beginning of a gene. We have also prepared biochemically complexes of mammalian Pol II elongation complexes with factors that are required for chromatin transcription, and study them structurally. We have now arrived at several Pol II structures in complex with nucleosomes and chromatin transcription factors that have elucidated many aspects of the mechanism of nucleosome passage and chromatin transcription. The work culminated with a molecular movie that showed how Pol II transcribes a nucleosome, the fundamental unit of chromatin. In the remaining time, we were able to extend our studies to the characterization of an intermediate of nucleosome transcription, which contains a hexasome and shows the polymerase in a backtracked state. These results have refined the obtained movie and led to a deeper understanding of chromatin transcription.
Transcription of protein-coding genes governs organism development, cell differentiation, and cell identity. We had previously provided the mechanisms of transcription initiation and elongation by RNA polymerase II, but these studies were carried out on naked DNA, not on the natural template, which is chromatin. With the ERC Advanced Grant project CHROMATRANS we were able to elucidate transcription initiation and elongation in a chromatin context on a structural level. In particular, we obtained three-dimensional structures of the RNA polymerase II pre-initiation complex on a nucleosome and several structures of RNA polymerase II elongation complexes transcribing a nucleosome. Since several functional states were trapped and several different factors were present in these structures, we could combine them into a movie of the polymerase passing through a nucleosome that now includes also the backtracking, arrest and reactivation of polymerase during this process. These studies provide a basis for chromatin transcription and its regulation by cellular cues. Several junior researchers could base their career on these studies and have taken principal investigator positions within Europe, the USA and Asia.