Diverse molecular mechanisms contribute to differential expression of human duplicated genes

ABSTRACTEmerging evidence links genes within human-specific segmental duplications (HSDs) to traits and diseases unique to our species. Strikingly, despite being nearly identical by sequence (>98.5%), paralogous HSD genes are differentially expressed across human cell and tissue types, though the underlying mechanisms have not been examined. Comparing cross-tissue mRNA levels between humans and chimpanzees, we determined that broadly, HSD paralogs (75 genes from 30 families) display expression patterns consistent with pseudo- or neofunctionalization. In general, the ancestral paralog exhibited greatest expression conservation with the chimpanzee ortholog, though exceptions suggest duplicate paralogs that may retain or supplant ancestral functions. To understand mechanisms underlying this observed regulatory divergence, we reanalyzed data from human lymphoblastoid cell lines (LCLs) (n=445), showing that ~75% of derived HSD paralogs exhibit significant differential expression and a greater than two-fold difference from their ancestral counterpart. To identify active cis-regulatory elements (CREs) in HSDs, we reanalyzed ENCODE data to recover hundreds of candidate CREs in these regions. Further, we generated ChIP-seq data for active chromatin features in an LCL using longer Illumina reads to better distinguish peaks in paralogous regions. Some of these duplicated CREs are sufficient to drive differential reporter activity, suggesting they may contribute to divergent cis-regulation of paralogs. This work provides evidence that cis-regulatory divergence contributes to novel expression patterns of recent gene duplicates in humans.

Population-scale study of eRNA transcription reveals bipartite functional enhancer architecture

Enhancer RNAs (eRNA) are unstable non-coding RNAs, transcribed bidirectionally from active regulatory sequences, whose expression levels correlate with enhancer activity. We use capped-nascent-RNA sequencing to efficiently capture bidirectional transcription initiation across several human lymphoblastoid cell lines (Yoruba population) and detect ~75,000 eRNA transcription sites with high sensitivity and specificity. The use of nascent-RNA sequencing sidesteps the confounding effect of eRNA instability. We identify quantitative trait loci (QTLs) associated with the level and directionality of eRNA expression. High-resolution analyses of these two types of QTLs reveal distinct positions of enrichment at the central transcription factor (TF) binding regions and at the flanking eRNA initiation regions, both of which are associated with mRNA expression QTLs. These two regions—the central TF-binding footprint and the eRNA initiation cores—define a bipartite architecture of enhancers, inform enhancer function, and can be used as an indicator of the significance of non-coding regulatory variants.

Population-scale study of eRNA transcription reveals bipartite functional enhancer architecture

Enhancer RNAs (eRNA) are non-coding RNAs transcribed bidirectionally from active regulatory sequences. Their expression levels correlate with the activating potentials of the enhancers, but due to their instability, eRNAs have proven difficult to quantify in large scale. To overcome this, we use capped-nascent-RNA sequencing to efficiently capture the bidirectional initiation of eRNAs. We apply this in large scale to the human lymphoblastoid cell lines from the Yoruban population, and detected nearly 75,000 eRNA transcription sites with high sensitivity and specificity. We identify genetic variants significantly associated with overall eRNA initiation levels, as well as the transcription directionality between the two divergent eRNA pairs, namely the transcription initiation and directional initiation quantitative trait loci (tiQTLs and diQTLs) respectively. High-resolution analyses of these two types of eRNA QTLs reveal distinct positions of enrichment not only at the central transcription factor (TF) binding regions but also at the flanking eRNA initiation regions, both of which are equivalently associated with mRNA expression QTLs. These two regions - the central TF binding footprint and the eRNA initiation cores - define the bipartite architecture and the function of enhancers, and may provide further insights into interpreting the significance of non-coding regulatory variants.