Massively parallel reporter assays (MPRAs) represent a set of high-throughput technologies that measure the functional effects of thousands of sequences/variants on gene regulatory activity. There are several different variations of MPRA technology and they are used for numerous applications, including regulatory element discovery, variant effect measurement, saturation mutagenesis, synthetic regulatory element generation or characterization of evolutionary gene regulatory differences. Despite their many designs and uses, there is no comprehensive database that incorporates the results of these experiments. To address this, we developed MPRAbase, a manually curated database that currently harbors 130 experiments, encompassing 17,718,677 elements tested across 35 cell types and 4 organisms. The MPRAbase web interface serves as a centralized user-friendly repository to examine online the activity of regulatory elements across cell types and organisms, and to download MPRA data for independent analysis.

Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is sexually dimorphic, with increased incidence in females. A genome-wide association study identified a female-specific AIS susceptibility locus near the PAX1 gene. Here, we use mouse enhancer assays, three mouse enhancer knockouts, and subsequent phenotypic analyses to characterize this region. Using mouse enhancer assays, we characterize a sequence, PEC7, which overlaps the AIS-associated variant, and find it to be active in the tail tip and intervertebral disc. Removal of PEC7 or Xe1, a known sclerotome enhancer nearby, or deletion of both sequences lead to a kinky tail phenotype only in the Xe1 and combined (Xe1+PEC7) knockouts, with only the latter showing a female sex dimorphic phenotype. Extensive phenotypic characterization of these mouse lines implicates several differentially expressed genes and estrogen signaling in the sex dimorphic bias. In summary, our work functionally characterizes an AIS-associated locus and dissects the mechanism for its sexual dimorphism.

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.

The gene regulatory code and grammar remain largely unknown, precluding our ability to link phenotype to genotype in regulatory sequences. Here, using a massively parallel reporter assay (MPRA) of 209,440 sequences, we examine all possible pair and triplet combinations, permutations and orientations of eighteen liver-associated transcription factor binding sites (TFBS). We find that TFBS orientation and order have a major effect on gene regulatory activity. Corroborating these results with genomic analyses, we find clear human promoter TFBS orientation biases and similar TFBS orientation and order transcriptional effects in an MPRA that tested 164,307 liver candidate regulatory elements. Additionally, by adding TFBS orientation to a model that predicts expression from sequence we improve performance by 7.7%. Collectively, our results show that TFBS orientation and order have a significant effect on gene regulatory activity and need to be considered when analyzing the functional effect of variants on the activity of these sequences.

Alternative DNA conformations, termed non-B DNA structures, can affect transcription, but the underlying mechanisms and their functional impact have not been systematically characterized. Here, we used compu-tational genomic analyses coupled with massively parallel reporter assays (MPRAs) to show that certain non -B DNA structures have a substantial effect on gene expression. Genomic analyses found that non-B DNA structures at promoters harbor an excess of germline variants. Analysis of multiple MPRAs, including a pro-moter library specifically designed to perturb non-B DNA structures, functionally validated that Z-DNA can significantly affect promoter activity. We also observed that biophysical properties of non-B DNA motifs, such as the length of Z-DNA motifs and the orientation of G-quadruplex structures relative to transcriptional direction, have a significant effect on promoter activity. Combined, their higher mutation rate and functional effect on transcription implicate a subset of non-B DNA motifs as major drivers of human gene-expression -associated phenotypes.

ABSTRACT The majority of autism spectrum disorder (ASD) risk genes are associated with ASD due to haploinsufficiency, where only one gene copy is functional. Here, using SCN2A haploinsufficiency, a major risk factor for ASD, we show that increasing the expression of the existing functional SCN2A allele with CRISPR activation (CRISPRa) can provide a viable therapeutic approach. We first demonstrate therapeutic potential by showing that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency. Next, using an rAAV-CRISPRa based treatment, we restore electrophysiological deficits in both Scn2a heterozygous mice and human stem-cell-derived neurons. Our results provide a novel therapeutic approach for numerous ASD-associated genes and demonstrate that rescue of Scn2a haploinsufficiency, even at adolescent stages, can ameliorate neurodevelopmental phenotypes.

Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development. How these deletions lead to this phenotype is unknown. Using whole-genome sequencing, we fine-mapped the acheiropodia-associated region to 12 kb and show that it does not function as an enhancer. CTCF and RAD21 ChIP-seq together with 4C-seq and DNA FISH identify three CTCF sites within the acheiropodia-deleted region that mediate the interaction between the ZRS and the SHH promoter. This interaction is substituted with other CTCF sites centromeric to the ZRS in the disease state. Mouse knockouts of the orthologous 12 kb sequence have no apparent abnormalities, showcasing the challenges in modelling CTCF alterations in animal models due to inherent motif differences between species. Our results show that alterations in CTCF motifs can lead to a Mendelian condition due to altered enhancer–promoter interactions.

Nuclear compartments are thought to play a role in three-dimensional genome organization and gene expression. In mammalian brain, the architecture and dynamics of nuclear compartment-associated genome organization is not known. In this study, we developed Genome Organization using CUT and RUN Technology (GO-CaRT) to map genomic interactions with two nuclear compartments-the nuclear lamina and nuclear speckles-from different regions of the developing mouse, macaque and human brain. Lamina-associated domain (LAD) architecture in cells in vivo is distinct from that of cultured cells, including major differences in LADs previously considered to be cell type invariant. In the mouse and human forebrain, dorsal and ventral neural precursor cells have differences in LAD architecture that correspond to their regional identity. LADs in the human and mouse cortex contain transcriptionally highly active sub-domains characterized by broad depletion of histone-3-lysine-9 dimethylation. Evolutionarily conserved LADs in human, macaque and mouse brain are enriched for transcriptionally active neural genes associated with synapse function. By integrating GO-CaRT maps with genome-wide association study data, we found speckle-associated domains to be enriched for schizophrenia risk loci, indicating a physical relationship between these disease-associated genetic variants and a specific nuclear structure. Our work provides a framework for understanding the relationship between distinct nuclear compartments and genome function in brain development and disease.

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Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide. While this is primarily attributed to trauma and aging, genetic variation is associated with disc degeneration in humans. However, the precise mechanisms driving the initiation and progression of disease remain elusive due to a paucity of genetic animal models. Here, we discuss a novel genetic mouse model of endplate-oriented disc degeneration. We show that the adhesion G-protein coupled receptor G6 (ADGRG6) mediates several anabolic and catabolic factors, fibrotic collagen genes, pro-inflammatory pathways, and mechanical properties of the IVD, prior to the onset of overt histopathology of these tissues. Furthermore, we found increased IL-6/STAT3 activation in the IVD and demonstrate that treatment with a chemical inhibitor of STAT3 activation ameliorates disc degeneration in these mutant mice. These findings establish ADGRG6 as a critical regulator of homeostasis of adult disc homeostasis and implicate ADGRG6 and STAT3 as promising therapeutic targets for degenerative joint diseases. Author summary Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide. While this is primarily attributed to trauma and aging, genetic variation is associated with disc degeneration in humans. However, the precise mechanisms driving the initiation and progression of disease remain elusive due to a paucity of genetic animal models. Here, we discuss a novel genetic mouse model of endplate-oriented disc degeneration. We show that the adhesion G-protein coupled receptor G6 (ADGRG6) mediates fibrotic collagen expression, causing increased mechanical stiffness of the IVD prior to the onset of histopathology in adult mice. Furthermore, we found increased IL-6/STAT3 activation in the IVD and demonstrate that treatment with a chemical inhibitor of STAT3 activation ameliorates disc degeneration in these mutant mice. Our results demonstrate that ADGRG6 regulation of STAT3 signaling is important for IVD homeostasis, indicating potential therapeutic targets for degenerative joint disorders.

Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide and has been attributed primarily to trauma and the accumulation of pathology during aging. While genetic defects have also been associated with disc degeneration, the precise mechanisms driving the initiation and progression of disease have remained elusive due to a paucity of genetic animal models. Here, we discuss a novel conditional mouse genetic model of endplate-oriented disc herniations in adult mice. Using conditional mouse genetics, we show increased mechanical stiffness and reveal dysregulation of typical gene expression profiles of the IVD in adhesion G-protein coupled receptor G6 (Adgrg6) mutant mice prior to the onset of endplate-oriented disc herniations in adult mice. We observed increased STAT3 activation prior to IVD defects and go on to demonstrate that treatment of Adgrg6 conditional mutant mice with a small molecule inhibitor of STAT3 activation ameliorates endplate-oriented herniations. These findings establish ADGRG6 and STAT3 as novel regulators of IVD endplate and growth plate integrity in the mouse, and implicate ADGRG6/STAT3 signaling as promising therapeutic targets for endplate-oriented disc degeneration.

In order to detect the disease-associated genes and their gene interaction function and association with melanoma mechanisms, we identified a total of 1,310 differentially expressed genes (DEGs) from the Gene Expression Omnibus database GSE3189 with FDR <0.01 and |logFC| >2 using the R package. After constructing the gene interaction network by STRING with the selected DEGs, we applied a statistical approach to identify the topological hub genes with PageRank score. Forty-four genes were identified in this network and AKT1 was selected as the most important hub gene. The AKT1 gene encodes a serine‑threonine protein kinase (AKT). High expression of AKT is involved in the resistance of cell apoptosis as well as adaptive resistance to treatment in melanoma. Our results indicated that AKT1 with a higher expression in melanoma showed enriched binding sites in the negative regulation of response to external stimulus, which enables cells to adapt to changes in external stimulation for survival. Another finding was that AKT regulated the lipid metabolic process and may be involved in melanoma progression and promotion of tumor growth through gene enrichment function analysis. Two highlighted pathways were detected in our study: i) the estrogen signaling pathway modulates the immune tolerance and resistance to cell apoptosis, which contributes to the growth of melanoma and ii) the RAP1 signaling pathway which regulates focal adhesion (FA) negative feedback to cell migration and invasion in melanoma. Our studies highlighted the top differentially expressed gene AKT1 and its correlation with the estrogen signaling and RAP1 signaling pathways to alter the proliferation and apoptosis of melanoma cells. Analysis of the enrichment functions of genes associated with melanoma will help us find the exact mechanism of melanoma and advance the full potential of newly targeted cancer therapy.

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor, but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing.

Psoriasis is an inflammatory skin disease. Deceleration in keratinocyte apoptosis is the most significant pathological change observed in psoriasis. To detect a meaningful correlation between the genes and gene functions associated with the mechanism underlying psoriasis, 927 differentially expressed genes (DEGs) were identified using the Gene Expression Omnibus database, GSE13355 [false discovery rate (FDR) <0.01; |log fold change >1] with the package in R langue. The selected DEGs were further constructed using the search tool for the retrieval of interacting genes, in order to analyze the interaction network between the DEGs. Subsequent to PageRank analysis, 14 topological hub genes were identified, and the functions and pathways in the hub genes network were analyzed. The top‑ranked hub gene, estrogen receptor‑1 (ESR1) is downregulated in psoriasis, exhibited binding sites enriched with genes possessing anti‑apoptotic functions. The ESR1 gene encodes estrogen receptor α (ERα); a reduced level of ERα expression provides a crucial foundation in response to the anti‑apoptotic activity of psoriatic keratinocytes by activating the expression of anti‑apoptotic genes. Furthermore, it was detected that the pathway that is associated most significantly with psoriasis is the pathways in cancer. Pathways in cancer may protect psoriatic cells from apoptosis by inhibition of ESR1 expression. The present study provides support towards the investigation of ESR1 gene function and elucidates that the interaction with anti‑apoptotic genes is involved in the underlying biological mechanisms of resistance to apoptosis in psoriasis. However, further investigation is required to confirm the present results.

In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.

Tissue-specific gene expression is thought to be one of the major forces shaping mammalian gene order. A recent study that used whole-genome chromosome conformation assays has shown that the mammalian genome is divided into specific topological domains that are shared between different tissues and organisms. Here, we wanted to assess whether gene expression and regulation are involved in shaping these domains and can be used to classify them. We analyzed gene expression and regulation levels in these domains by using RNA-seq and enhancer-associated ChIP-seq datasets for 17 different mouse tissues. We found 162 domains that are active (high gene expression and regulation) in all 17 tissues. These domains are significantly shorter, contain less repeats, and have more housekeeping genes. In contrast, we found 29 domains that are inactive (low gene expression and regulation) in all analyzed tissues and are significantly longer, have more repeats, and gene deserts. Tissue-specific active domains showed some correlation with tissue-type and gene ontology. Domain temporal gene regulation and expression differences also displayed some gene ontology terms fitting their temporal function. Combined, our results provide a catalog of shared and tissue-specific topological domains and suggest that gene expression and regulation could have a role in shaping them.

Enhancers are essential gene regulatory elements whose alteration can lead to morphological differences between species, developmental abnormalities, and human disease. Current strategies to identify enhancers focus primarily on noncoding sequences and tend to exclude protein coding sequences. Here, we analyzed 25 available ChIP-seq data sets that identify enhancers in an unbiased manner (H3K4me1, H3K27ac, and EP300) for peaks that overlap exons. We find that, on average, 7% of all ChIP-seq peaks overlap coding exons (after excluding for peaks that overlap with first exons). By using mouse and zebrafish enhancer assays, we demonstrate that several of these exonic enhancer (eExons) candidates can function as enhancers of their neighboring genes and that the exonic sequence is necessary for enhancer activity. Using ChIP, 3C, and DNA FISH, we further show that one of these exonic limb enhancers,Dync1i1exon 15, has active enhancer marks and physically interacts withDlx5/6promoter regions 900 kb away. In addition, its removal by chromosomal abnormalities in humans could cause split hand and foot malformation 1 (SHFM1), a disorder associated withDLX5/6. These results demonstrate that DNA sequences can have a dual function, operating as coding exons in one tissue and enhancers of nearby gene(s) in another tissue, suggesting that phenotypes resulting from coding mutations could be caused not only by protein alteration but also by disrupting the regulation of another gene.