Massively parallel jumping assay decodes Alu retrotransposition activity.

The human genome contains millions of retrotransposons, several of which could become active due to somatic mutations having phenotypic consequences, including disease. However, it is not thoroughly understood how nucleotide changes in retrotransposons affect their jumping activity. Here, we developed a novel massively parallel jumping assay (MPJA) that can test the jumping potential of thousands of transposons en masse. We generated nucleotide variant library of selected four Alu retrotransposons containing 165,087 different haplotypes and tested them for their jumping ability using MPJA. We found 66,821 unique jumping haplotypes, allowing us to pinpoint domains and variants vital for transposition. Mapping these variants to the Alu-RNA secondary structure revealed stem-loop features that contribute to jumping potential. Combined, our work provides a novel high-throughput assay that assesses the ability of retrotransposons to jump and identifies nucleotide changes that have the potential to reactivate them in the human genome.

Massively parallel characterization of transcriptional regulatory elements in three diverse human cell types.

The human genome contains millions of candidate cis-regulatory elements (CREs) with cell-type-specific activities that shape both health and myriad disease states. However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these CREs. Here, we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of over 680,000 sequences, representing a nearly comprehensive set of all annotated CREs among three cell types (HepG2, K562, and WTC11), finding 41.7% to be functional. By testing sequences in both orientations, we find promoters to have significant strand orientation effects. We also observe that their 200 nucleotide cores function as non-cell-type-specific 'on switches' providing similar expression levels to their associated gene. In contrast, enhancers have weaker orientation effects, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict CRE function with high accuracy and delineate regulatory motifs. Testing an additional lentiMPRA library encompassing 60,000 CREs in all three cell types, we further identified factors that determine cell-type specificity. Collectively, our work provides an exhaustive catalog of functional CREs in three widely used cell lines, and showcases how large-scale functional measurements can be used to dissect regulatory grammar.

Characterization of De Novo Promoter Variants in Autism Spectrum Disorder with Massively Parallel Reporter Assays.

Autism spectrum disorder (ASD) is a common, complex, and highly heritable condition with contributions from both common and rare genetic variations. While disruptive, rare variants in protein-coding regions clearly contribute to symptoms, the role of rare non-coding remains unclear. Variants in these regions, including promoters, can alter downstream RNA and protein quantity; however, the functional impacts of specific variants observed in ASD cohorts remain largely uncharacterized. Here, we analyzed 3600 de novo mutations in promoter regions previously identified by whole-genome sequencing of autistic probands and neurotypical siblings to test the hypothesis that mutations in cases have a greater functional impact than those in controls. We leveraged massively parallel reporter assays (MPRAs) to detect transcriptional consequences of these variants in neural progenitor cells and identified 165 functionally high confidence de novo variants (HcDNVs). While these HcDNVs are enriched for markers of active transcription, disruption to transcription factor binding sites, and open chromatin, we did not identify differences in functional impact based on ASD diagnostic status.

Co-option of the lineage-specific LAVA retrotransposon in the gibbon genome.

Co-option of transposable elements (TEs) to become part of existing or new enhancers is an important mechanism for evolution of gene regulation. However, contributions of lineage-specific TE insertions to recent regulatory adaptations remain poorly understood. Gibbons present a suitable model to study these contributions as they have evolved a lineage-specific TE called LAVA (LINE-AluSz-VNTR-AluLIKE), which is still active in the gibbon genome. The LAVA retrotransposon is thought to have played a role in the emergence of the highly rearranged structure of the gibbon genome by disrupting transcription of cell cycle genes. In this study, we investigated whether LAVA may have also contributed to the evolution of gene regulation by adopting enhancer function. We characterized fixed and polymorphic LAVA insertions across multiple gibbons and found 96 LAVA elements overlapping enhancer chromatin states. Moreover, LAVA was enriched in multiple transcription factor binding motifs, was bound by an important transcription factor (PU.1), and was associated with higher levels of gene expression in cis We found gibbon-specific signatures of purifying/positive selection at 27 LAVA insertions. Two of these insertions were fixed in the gibbon lineage and overlapped with enhancer chromatin states, representing putative co-opted LAVA enhancers. These putative enhancers were located within genes encoding SETD2 and RAD9A, two proteins that facilitate accurate repair of DNA double-strand breaks and prevent chromosomal rearrangement mutations. Co-option of LAVA in these genes may have influenced regulation of processes that preserve genome integrity. Our findings highlight the importance of considering lineage-specific TEs in studying evolution of gene regulatory elements.