Periodic Reporting for period 1 - Eprom (Epromoters in health and disease)
Periodo di rendicontazione: 2022-08-01 al 2024-12-31
Regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site proximal (promoters) and -distal (enhancers) regulatory elements. It is well established that enhancers play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. The classical definition of enhancers implies the property to activate gene expression at a distance, while promoters induce local gene expression. However, this basic dichotomy has been challenged by broad similarities between them, which leads to the question what distinguishes enhancer and promoter function?
Using a technique called CapSTARR-seq, which captures regions of interest (promoters) which are then tested for enhancer function in a high-throughput manner, the host team demonstrated that a subset of promoters, termed Epromoters, not only work as promoters, but also as bona fide enhancers, regulating expression of a distal gene as well. These Epromoters seem to regulate gene expression in several organisms from drosophila to humans. It was also found that Epromoters might play an essential role in the coordination of rapid gene induction during the inflammatory response. Therefore, we hypothesize that Epromoters work as a hub for recruiting the essential transcription factors (TFs) required for gene activation in different stress conditions and thus ensure a rapid coordinated expression response of several distal genes simultaneously (Figure 1). In addition to Epromoters, we are interested in human variation, or single nucleotide polymorphisms (SNPs). In recent years, common SNPs have been associated with susceptibility to several diseases and traits by genome-wide association studies (GWAS). Interestingly, most SNPs associated to pathology lie in the non-coding or regulatory genome, i.e. in enhancer or promoter regions. The discovery of Epromoters opens a new paradigm in the study of regulatory variants, as a SNP in a promoter could potentially influence the expression of several genes or change the relative ratio of promoter versus enhancer activity, thus potentially elucidating new disease mechanisms. This led to our project, identifying new Epromoters in the immune response, finding common (disease-associated) variants that are located in Epromoter, and characterizing the impact of these variants on enhancer versus promoter function.
Using a technique called CapSTARR-seq, which captures regions of interest (promoters) which are then tested for enhancer function in a high-throughput manner, the host team demonstrated that a subset of promoters, termed Epromoters, not only work as promoters, but also as bona fide enhancers, regulating expression of a distal gene as well. These Epromoters seem to regulate gene expression in several organisms from drosophila to humans. It was also found that Epromoters might play an essential role in the coordination of rapid gene induction during the inflammatory response. Therefore, we hypothesize that Epromoters work as a hub for recruiting the essential transcription factors (TFs) required for gene activation in different stress conditions and thus ensure a rapid coordinated expression response of several distal genes simultaneously (Figure 1). In addition to Epromoters, we are interested in human variation, or single nucleotide polymorphisms (SNPs). In recent years, common SNPs have been associated with susceptibility to several diseases and traits by genome-wide association studies (GWAS). Interestingly, most SNPs associated to pathology lie in the non-coding or regulatory genome, i.e. in enhancer or promoter regions. The discovery of Epromoters opens a new paradigm in the study of regulatory variants, as a SNP in a promoter could potentially influence the expression of several genes or change the relative ratio of promoter versus enhancer activity, thus potentially elucidating new disease mechanisms. This led to our project, identifying new Epromoters in the immune response, finding common (disease-associated) variants that are located in Epromoter, and characterizing the impact of these variants on enhancer versus promoter function.
A reporter assay (CapSTARR-seq) was performed in the GM12878 cell line, in order to generate a new dataset - in addition to the ones publicly available - on which to predict Epromoters. We selected 28 reporter assay datasets to find candidate promoters which show enhancer function, i.e. a comprehensive resource of candidate Epromoters, in 11 cell lines. We further characterized the candidate Eprom for epigenomic markers, showing that they are distinct from a set of control promoters. Then, we overlapped the candidate Epromoter regions with over 180,000 SNPs that are associated with diverse traits and diseases, yielding 4,330 GWAS-SNPs in 2,301 Epromoters, showing that Epromoters are in fact enriched for variants that are associated with more GWAS traits that even fall into different categories, indicating the potential pleiotropic effect of Epromoters. Here, pleiotropy is defined as a single element affecting more than one trait independently. Using eQTL data, we show that the candidate variant Epromoters are in fact associated with the altered expression of multiple target genes. Additionally, using massive parallel reporter assay data, we find that the GWAS-SNPs with validated allelic impact are present more often in Epromoters (with higher pleiotropy) than in control promoters, further validating the potential pleiotropic function of Epromoters. We experimentally validated one of these SNPs associated with COVID19 risq. These SNP lied within the OAS3 Epromoter and we showed that genetic variation impact on both the promoter and enhancer activity of this Epromoter.
These results beg the question, how do variants influence the enhancer versus promoter function of Epromoters? A number of candidate variant Epromoters were selected for genome editing using CRISPR/Cas9, and later prime editing to introduce the minor and major alleles to determine the effect of the variants on enhancer versus promoter function and thus the transcriptional changes induced in the target genes. Unfortunately, no clones were obtained that incorporated the selected variants into the genome, despite thorough efforts with multiple techniques and cell lines. Therefore, we pursued a different, more high-throughput approach; MPRA. Using the same synthesized sequences of the two alleles of variants in candidate Epromoters, we cloned the sequences in two orientations in a vector to test separately enhancer or promoter activity. Encountering a number of obstacles to data interpretation along the way, we are currently analyzing the data.
These results beg the question, how do variants influence the enhancer versus promoter function of Epromoters? A number of candidate variant Epromoters were selected for genome editing using CRISPR/Cas9, and later prime editing to introduce the minor and major alleles to determine the effect of the variants on enhancer versus promoter function and thus the transcriptional changes induced in the target genes. Unfortunately, no clones were obtained that incorporated the selected variants into the genome, despite thorough efforts with multiple techniques and cell lines. Therefore, we pursued a different, more high-throughput approach; MPRA. Using the same synthesized sequences of the two alleles of variants in candidate Epromoters, we cloned the sequences in two orientations in a vector to test separately enhancer or promoter activity. Encountering a number of obstacles to data interpretation along the way, we are currently analyzing the data.
Results of the project included three open-access published articles as listed below, as well as a number of publicly accessible datasets (CapSTARR-seq, candidate Epromoters). Indeed, the produced variant promoter and enhancer MPRA datasets are currently being analyzed, and additional resources will be needed to validate the outcomes of these analyses, as well as to publish these results.
“Comprehensive mapping of genetic variation at Epromoters reveals pleiotropic association with multiple disease traits” Nucleic Acids Research, Wan & van Ouwerkerk et al., 2024, PMID: 39727170
“Severe COVID-19-associated variants linked to chemokine receptor gene control in monocytes and macrophages” Genome Biology, Stikker, Stik & van Ouwerkerk et al., 2022, PMID: 35421995
“How the dark genome enlightens the molecular mechanisms of diseases”, Open Access Government, van Ouwerkerk & Spicuglia, 2024, https://doi.org/10.56367/OAG-043-11421(si apre in una nuova finestra)
“Comprehensive mapping of genetic variation at Epromoters reveals pleiotropic association with multiple disease traits” Nucleic Acids Research, Wan & van Ouwerkerk et al., 2024, PMID: 39727170
“Severe COVID-19-associated variants linked to chemokine receptor gene control in monocytes and macrophages” Genome Biology, Stikker, Stik & van Ouwerkerk et al., 2022, PMID: 35421995
“How the dark genome enlightens the molecular mechanisms of diseases”, Open Access Government, van Ouwerkerk & Spicuglia, 2024, https://doi.org/10.56367/OAG-043-11421(si apre in una nuova finestra)