Structure and mechanism of the oocyte-specific transcription pre-initiation machinery

Proteins in all eukaryotes are transcribed from encoding genes by RNA polymerase II (Pol II). Pol II transcription initiation requires a complex interplay of transcription factors, transcriptional co-activators and enhancers to achieve ordered assembly of protein complexes on the DNA template, forming the preinitiation complex (PIC). The traditional text-book view suggests that transcription is initiated by a defined set of general transcription factors (GTFs), step-wise assembled on a core promoter recognized by the TATA-box binding protein (TBP). This 'monolithic' view has been challenged by the discovery of specialized paralogues of PIC components, including three distinct TBP homologs, revealing a level of variability in the mechanism of core promoter recognition that is poorly understood.

To date, transcription initiation mechanisms have mainly been deciphered in dividing cells. Our multidisciplinary proposal aims, for the first time, to provide a step-change in our understanding of transcription initiation mechanisms present in a non-dividing cell type, the growing oocyte. Here, TBP has been replaced by a paralogue, TBPL2, and sets of GTF components are absent, providing a unique model system. How transcription is initiated in the growing oocyte in absence of these factors, and how a functional oocyte-specific PIC is assembled in this essential cell-type, remains elusive. We aim to fill this vital knowledge gap, with implications for development, disease states, and infertility - a growing concern particularly in developed societies.

Our joint proposal stems from successful collaborations on the structure and mechanism of eukaryotic transcription complexes between the Berger and Schaffitzel groups in the Schools of Biochemistry and Chemistry at Bristol University, and our international network of collaborators including the Tora and Vincent groups (IGBMC France), the Grohmann group (Regensburg, Germany) and the Taatjes group (Boulder USA).

Our work combines the disciplines of structural biology, biochemistry and cell biology, with each informing the other. The recent discovery of a unique transcription pre-initiation complex in the growing oocyte sets the stage for the present project. Together, we aim to elucidate the structure and mechanisms of the oocyte-specific transcription machinery at the molecular level.

This proposal synergistically integrates molecular and structural biology (X-ray, Cryo-EM), biochemistry, single-molecule analysis, proteomics data and cell-based in vivo assays to elucidate the structure and mechanisms of the oocyte-specific transcription machinery. It stems from time tested successful collaborations on the structure and mechanism of basal transcription complexes between the Berger and Schaffitzel groups in the Schools of Biochemistry and Chemistry at University of Bristol (UoB), and our international network of collaborators including the Tora and Vincent groups (IGBMC France), the Grohmann group (Regensburg, Germany) and the Taatjes group (Boulder USA).

We address fundamental questions in transcription regulation of the growing oocyte, a non-dividing cell blocked at the end of prophase I. In the mouse, TBPL2 completely replaces TBP during oocyte growth. We will analyse the interactions of TBPL2 with associated factors and DNA by structural and biochemical approaches and validate our findings in cell-based assays in vivo. We will reconstitute a nucleosomal complex comprising the minimal oocyte PIC, for high-resolution structural analysis by electron cryo-microscopy (Cryo-EM) to provide chromatin context. We further aim to obtain a structural snapshot of oocyte-specific preinitiation of an RNA polymerase II (Pol II)-bound minimal oocyte PIC on DNA by Cryo-EM. Finally, by combining quantitative proteomics with in vivo and in vitro screening, we aim to discover novel proteins engaging the oocyte PIC.

To date, transcription initiation mechanisms have mainly been deciphered in dividing cells. Our proposal aims, for the first time, to provide a step-change in our understanding of transcription initiation mechanisms present in a non-dividing cell type, the growing oocyte, filling a vital knowledge gap with implications for development, disease states, and infertility - a growing concern particularly in developed societies.