Multimodal single cell analysis infers widespread enhancer co-activity in a lymphoblastoid cell line.

Non-coding regulatory elements such as enhancers are key in controlling the cell-type specificity and spatio-temporal expression of genes. To drive stable and precise gene transcription robust to genetic variation and environmental stress, genes are often targeted by multiple enhancers with redundant action. However, it is unknown whether enhancers targeting the same gene display simultaneous activity or whether some enhancer combinations are more often co-active than others. Here, we take advantage of recent developments in single cell technology that permit assessing chromatin status (scATAC-seq) and gene expression (scRNA-seq) in the same single cells to correlate gene expression to the activity of multiple enhancers. Measuring activity patterns across 24,844 human lymphoblastoid single cells, we find that the majority of enhancers associated with the same gene display significant correlation in their chromatin profiles. For 6944 expressed genes associated with enhancers, we predict 89,885 significant enhancer-enhancer associations between nearby enhancers. We find that associated enhancers share similar transcription factor binding profiles and that gene essentiality is linked with higher enhancer co-activity. We provide a set of predicted enhancer-enhancer associations based on correlation derived from a single cell line, which can be further investigated for functional relevance.

Shared regulation and functional relevance of local gene co-expression revealed by single cell analysis.

Most human genes are co-expressed with a nearby gene. Previous studies have revealed this local gene co-expression to be widespread across chromosomes and across dozens of tissues. Yet, so far these studies used bulk RNA-seq, averaging gene expression measurements across millions of cells, thus being unclear if this co-expression stems from transcription events in single cells. Here, we leverage single cell datasets in >85 individuals to identify gene co-expression across cells, unbiased by cell-type heterogeneity and benefiting from the co-occurrence of transcription events in single cells. We discover >3800 co-expressed gene pairs in two human cell types, induced pluripotent stem cells (iPSCs) and lymphoblastoid cell lines (LCLs) and (i) compare single cell to bulk RNA-seq in identifying local gene co-expression, (ii) show that many co-expressed genes - but not the majority - are composed of functionally related genes and (iii) using proteomics data, provide evidence that their co-expression is maintained up to the protein level. Finally, using single cell RNA-sequencing (scRNA-seq) and single cell ATAC-sequencing (scATAC-seq) data for the same single cells, we identify gene-enhancer associations and reveal that >95% of co-expressed gene pairs share regulatory elements. These results elucidate the potential reasons for co-expression in single cell gene regulatory networks and warrant a deeper study of shared regulatory elements, in view of explaining disease comorbidity due to affecting several genes. Our in-depth view of local gene co-expression and regulatory element co-activity advances our understanding of the shared regulatory architecture between genes.