Unlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) are considered non-conserved. Although lncRNAs have been shown to function in diverse pathophysiological processes in mice, it remains largely unknown whether human lncRNAs have such in vivo functions. Here, we describe an integrated pipeline to define the in vivo function of non-conserved human lncRNAs. We first identify lncRNAs with high function potential using multiple indicators derived from human genetic data related to cardiometabolic traits, then define lncRNA’s function and specific target genes by integrating its correlated biological pathways in humans and co-regulated genes in a humanized mouse model. Finally, we demonstrate that the in vivo function of human-specific lncRNAs can be successfully examined in the humanized mouse model, and experimentally validate the predicted function of an obesity-associated lncRNA, LINC01018, in regulating the expression of genes in fatty acid oxidation in humanized livers through its interaction with RNA-binding protein HuR.

In embryonic stem cells (ESCs), a core transcription factor (TF) network establishes the gene expression program necessary for pluripotency. To address how interactions between four key TFs contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of binding sites for SOX2, POU5F1 (OCT4), KLF4, and ESRRB. Comparisons between synthetic cis-regulatory elements and genomic sequences with comparable binding site configurations revealed some aspects of a regulatory grammar. The expression of synthetic elements is influenced by both the number and arrangement of binding sites. This grammar plays only a small role for genomic sequences, as the relative activities of genomic sequences are best explained by the predicted occupancy of binding sites, regardless of binding site identity and positioning. Our results suggest that the effects of transcription factor binding sites (TFBS) are influenced by the order and orientation of sites, but that in the genome the overall occupancy of TFs is the primary determinant of activity.

Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with different human blinding diseases that vary in their severity and age of onset. How different variants in a single TF cause a range of pathological phenotypes is not understood. We deployed massively parallel reporter assays (MPRAs) to measure changes to CRX cis-regulatory function in live mouse retinas carrying knock-ins of two phenotypically distinct human disease-causing Crx variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). We found that the effects of CRX variants on global cis-regulatory activity patterns correspond with the severity of their phenotypes. The variants affect similar sets of enhancers but to different degrees. A subset of silencers were converted to enhancers in retinas lacking a functional CRX effector domain, but were unaffected by p.R90W. Episomal MPRA activities of CRX-bound sequences showed some correspondence with chromatin environments at their original genomic loci, including an enrichment of silencers and depletion of strong enhancers among distal elements whose accessibility increases later in retinal development. Many distal silencers were de-repressed by p.E168d2, but not by p.R90W, suggesting that loss of developmentally timed silencing caused by p.E168d2 may contribute to phenotypic differences between the two variants. Our findings indicate that phenotypically distinct disease variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to mis-regulation of similar sets of enhancers, while having a qualitatively different impact on silencers.