Project description:Recent genome-wide association studies have established that most complex disease-associated loci are found in noncoding regions where defining their function is nontrivial. In this study, we leverage a modular massively parallel reporter assay (MPRA) to uncover sequence features linked to context-specific regulatory activity. We screened enhancer activity across a panel of 198-bp fragments spanning over 10k type 2 diabetes- and metabolic trait-associated variants in the 832/13 rat insulinoma cell line, a relevant model of pancreatic beta cells. We explored these fragments’ context sensitivity by comparing their activities when placed up- or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin gene (INS). We identified clear effects of MPRA construct design on measured fragment enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. A separate set of fragments exhibited promoter bias (n = 698/11,656), mostly towards the cell-specific INS promoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments with INS promoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory variants. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted the INS promoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.

Project description:Genome-wide association studies (GWAS) have successfully identified 145 genomic regions that contribute to schizophrenia risk, but linkage disequilibrium (LD) makes it challenging to discern causal variants. Computational finemapping prioritized thousands of credible variants, ~98% of which lie within poorly characterized non-coding regions. To functionally validate their regulatory effects, we performed a massively parallel reporter assay (MPRA) on 5,173 finemapped schizophrenia GWAS variants in primary human neural progenitors (HNPs). We identified 440 variants with allelic regulatory effects (MPRA-positive variants), with 72% of GWAS loci containing at least one MPRA-positive variant. Transcription factor binding had modest predictive power for predicting the allelic activity of MPRA-positive variants, while GWAS association, finemap posterior probability, enhancer overlap, and evolutionary conservation failed to predict MPRA-positive variants. Furthermore, 64% of MPRA-positive variants did not exhibit eQTL signature, suggesting that MPRA could identify yet unexplored variants with regulatory potentials. MPRA-positive variants differed from eQTLs, as they were more frequently located in distal neuronal enhancers. Therefore, we leveraged neuronal 3D chromatin architecture to identify 273 genes that physically interact with MPRA-positive variants. These genes annotated by chromatin interactome displayed higher mutational constraints and regulatory complexity than genes annotated by eQTLs, recapitulating a recent finding that eQTL- and GWAS-detected variants map to genes with different properties. Finally, we propose a model in which allelic activity of multiple variants within a GWAS locus can be aggregated to predict gene expression by taking chromatin contact frequency and accessibility into account. In conclusion, we demonstrate that MPRA can effectively identify functional regulatory variants and delineate previously unknown regulatory principles of schizophrenia.

Project description:Low copy number plasmids must encode maintenance mechanisms, such as partitioning systems, to ensure that the plasmid is sustained through host generations. Plasmid partition systems segregate sister plasmid copies and are subdivided into different types based on the NTPase they encode. The characterisation and distribution of partition system types is well understood in Enterobacteriaceae plasmids. However, how these systems maintain plasmids and are distributed across wider bacterial diversity is poorly understood. We searched a large and diverse plasmid database to identify the distribution of partition system types and found that plasmids encoding multiple partition systems are more widespread than previously realised, composing ~19% of all plasmids predicted to encode partition systems. Many of these plasmids were predicted to encode multiple partition systems of the same type, which had never been studied previously. We used the Streptomyces coelicolor A3(2) plasmid SCP1, which encodes two type Ia partition systems, as a model to investigate this. Sequence analysis of the SCP1 partition systems revealed that both ParB proteins contain less conserved CTP-binding pockets, suggesting one or both proteins may not behave like canonical ParB proteins. However, using a combination of chromatin immunoprecipitation with deep sequencing (ChIP-seq), biochemistry and mutagenesis we show that both ParB proteins behave like bone fide ParB proteins: they bound to distinct parS sites on SCP1, both proteins bound and hydrolysed CTP and both proteins required CTP and their cognate parS site to accumulate, or spread, on DNA. Intriguingly, however, only ParB1 was critical for SCP1 stability. Together, our findings further our understanding of Streptomyces plasmid maintenance by providing the first functional characterisation of two type Ia partition systems coexisting on a single plasmid and offer new insights into the diversity and distribution of plasmid partition systems.

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Massively parallel reporter assay reveals promoter-, position-, and strand-specific effects in transcription start sites

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