Identifying proteins required at distinct promoter types

In all organisms, cell differentiation and survival depend on differential transcription, which initiates at so called core promoters (CPs) 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.

In all organisms, cell differentiation and survival depend on differential gene expression that is tightly regulated by an intricate interplay of specialized DNA elements and regulatory proteins. One class of DNA elements - so called promoters - are located at the start of genes and initiate gene transcription, i.e. the copying of the gene's DNA into RNA. Promoters function via regulatory proteins that bind to promoters and recruit polymerase II that performs the DNA-to-RNA copying. While it has been recognized since decades that promoters are diverse in form and function, it has remained unknown if the different promoters function via similar or distinct proteins and mechanisms. In this project, we have characterized proteins that bind to different promoter types in the model system Drosophila melanogaster. We found that indeed, different promoter types bind - and functionally depend upon - different sets of proteins. For example, when we rapidly depleted the protein factor TBP, only TATA-box-type promoters were affected. Overall, the distinct dependencies on different proteins could also explain a long-standing conundrum of transcription initiation patters that - across all animals - can be focused or dispersed. We also extended the work to mammalian cells by developing a functional screen that allows the determination of factors that can activate transcription from different promoters. Both parts were recently published in peer-reviewed journals (Serebreni et al., EMBO Journal 2023 and Nemcko et al., Nature Methods 2024). This project provided the basis for a comprehensive annotation of Drosophila promoters and promoter-bound proteins and as an extension to mammalian cells lead to the development of a high-throughput method that is used by us and others. Overall, our work lead to novel insights into the sequence and function of promoters and our understanding of gene expression more generally.