In all organisms, cell differentiation and survival depend on differential transcription, which initiates
at so called core promoters (CPs) <U+F02D> short sequences around the transcription start sites (TSSs) of
genes. CPs are often described as gateways to transcription due to their central role in enabling
precise and highly regulated transcription of genes. They are sufficient to direct the assembly of the
pre-initiation complex (PIC), to drive low levels of transcription. In addition, CPs are also able to
receive regulatory information from distal regulators, which can strongly boost transcription.
Despite the importance of CPs, only a few well-defined CPs have been studied in detail, even though
it is clear that various types of CPs exist that differ in sequence and function. For example, scientists
including our group found that different types of fruit fly (Drosophila melanogaster or D.
melanogaster) CPs display distinct specificities towards regulatory input at the level of enhancers
and cofactor proteins.
For our project, we propose to systematically identify protein factors that are bound to different
types of CPs and are required for these CPs functions. We use D. melanogaster cells as our main
model system and propose to extend the work to human cells. We plan to identify candidate
proteins that bind to the DNA of different CP types by affinity purification and mass spectrometry.
We expect to observe both shared and differential binding of canonical PIC components at the
different core promoter types, as well as novel protein factors potentially important for core-
promoter function, especially of non-canonical, non-TATA type core promoters. We will functionally
test the requirement of differentially bound canonical PIC components and the role of novel protein
factors enriched at specific CP types by measuring transcription upon targeted protein degradation.
This project will provide the first systematic identification of proteins bound to distinct types of CPs
in both fly and human and uncover these proteins role in transcriptional regulation. This will provide
important insights into which factors are required for PIC assembly and transcription from the
different promoter types. We will for example test the presumed universality of the canonical PIC
components, the importance of novel factors, and the different factors requirement for the
different steps of transcription. Together, they will provide the basis for understanding the
molecular mechanisms that underlie the observed regulatory specificities, as well as architectural
and functional diversity of CPs, a decade-old question at the very center of transcriptional
regulation. Therefore, the expected results are not only important for our basic understanding of
transcription, but also timely and important for applied medical research today, when the
development of new therapeutic approaches based on targeted alteration of gene expression relies
on well-understood molecular mechanisms that govern transcription of specific genes.