A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species.

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

Ancestry of the major long-range regulatory site of the α-globin genes in the Portuguese population with the common 3.7 kb α-thalassemia deletion.

BACKGROUND: The α-Major Regulatory Element (α-MRE), also known as HS-40, is located upstream of the α-globin gene cluster and has a crucial role in the long-range regulation of the α-globin gene expression. This enhancer is polymorphic and several haplotypes were identified in different populations, with haplotype D almost exclusively found in African populations. The purpose of this research was to identify the HS-40 haplotype associated with the 3.7 kb α-thalassemia deletion (-α3.7del) in the Portuguese population, and determine its ancestry and influence on patients' hematological phenotype. METHODS AND RESULTS: We selected 111 Portuguese individuals previously analyzed by Gap-PCR to detect the presence of the -α3.7del: 50 without the -α3.7del, 34 heterozygous and 27 homozygous for the -α3.7del. The HS-40 region was amplified by PCR followed by Sanger sequencing. Four HS-40 haplotypes were found (A to D). The distribution of HS-40 haplotypes and genotypes are significantly different between individuals with and without the -α3.7del, being haplotype D and genotype AD the most prevalent in patients with this deletion in homozygosity. Furthermore, multiple correspondence analysis revealed that individuals without the -α3.7del are grouped with other European populations, while samples with the -α3.7del are separated from these and found more closely related to the African population. CONCLUSION: This study revealed for the first time an association of the HS-40 haplotype D with the -α3.7del in the Portuguese population, and its likely African ancestry. These results may have clinical importance as in vitro analysis of haplotype D showed a decrease in its enhancer activity on α-globin gene.

Using a comprehensive atlas and predictive models to reveal the complexity and evolution of brain-active regulatory elements.

Most genetic variants associated with psychiatric disorders are located in noncoding regions of the genome. To investigate their functional implications, we integrate epigenetic data from the PsychENCODE Consortium and other published sources to construct a comprehensive atlas of candidate brain cis-regulatory elements. Using deep learning, we model these elements' sequence syntax and predict how binding sites for lineage-specific transcription factors contribute to cell type-specific gene regulation in various types of glia and neurons. The elements' evolutionary history suggests that new regulatory information in the brain emerges primarily via smaller sequence mutations within conserved mammalian elements rather than entirely new human- or primate-specific sequences. However, primate-specific candidate elements, particularly those active during fetal brain development and in excitatory neurons and astrocytes, are implicated in the heritability of brain-related human traits. Additionally, we introduce PsychSCREEN, a web-based platform offering interactive visualization of PsychENCODE-generated genetic and epigenetic data from diverse brain cell types in individuals with psychiatric disorders and healthy controls.

Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Genome-wide profiling of angiogenic cis-regulatory elements unravels cis-regulatory SNPs for vascular abnormality.

Angiogenesis is extensively involved in embryonic development and requires complex regulation networks, whose defects can cause a variety of vascular abnormalities. Cis-regulatory elements control gene expression at all developmental stages, but they have not been studied or profiled in angiogenesis yet. In this study, we exploited public DNase-seq and RNA-seq datasets from a VEGFA-stimulated in vitro angiogenic model, and carried out an integrated analysis of the transcriptome and chromatin accessibility across the entire process. Totally, we generated a bank of 47,125 angiogenic cis-regulatory elements with promoter (marker by H3K4me3) and/or enhancer (marker by H3K27ac) activities. Motif enrichment analysis revealed that these angiogenic cis-regulatory elements interacted preferentially with ETS family TFs. With this tool, we performed an association study using our WES data of TAPVC and identified rs199530718 as a cis-regulatory SNP associated with disease risk. Altogether, this study generated a genome-wide bank of angiogenic cis-regulatory elements and illustrated its utility in identifying novel cis-regulatory SNPs for TAPVC, expanding new horizons of angiogenesis as well as vascular abnormality genetics.

CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Regulatory landscape enrichment analysis (RLEA): a computational toolkit for non-coding variant enrichment and cell type prioritization.

BACKGROUND: As genomic studies continue to implicate non-coding sequences in disease, testing the roles of these variants requires insights into the cell type(s) in which they are likely to be mediating their effects. Prior methods for associating non-coding variants with cell types have involved approaches using linkage disequilibrium or ontological associations, incurring significant processing requirements. GaiaAssociation is a freely available, open-source software that enables thousands of genomic loci implicated in a phenotype to be tested for enrichment at regulatory loci of multiple cell types in minutes, permitting insights into the cell type(s) mediating the studied phenotype. RESULTS: In this work, we present Regulatory Landscape Enrichment Analysis (RLEA) by GaiaAssociation and demonstrate its capability to test the enrichment of 12,133 variants across the cis-regulatory regions of 44 cell types. This analysis was completed in 134.0 ± 2.3 s, highlighting the efficient processing provided by GaiaAssociation. The intuitive interface requires only four inputs, offers a collection of customizable functions, and visualizes variant enrichment in cell-type regulatory regions through a heatmap matrix. GaiaAssociation is available on PyPi for download as a command line tool or Python package and the source code can also be installed from GitHub at https://github.com/GreallyLab/gaiaAssociation . CONCLUSIONS: GaiaAssociation is a novel package that provides an intuitive and efficient resource to understand the enrichment of non-coding variants across the cis-regulatory regions of different cells, empowering studies seeking to identify disease-mediating cell types.

Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Massively parallel characterization of regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Loci cg06256735 and cg15815843 in the MFAP5 gene regulatory regions are hypomethylated in varicose veins apparently due to active demethylation.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

Evolutionary Rate Shifts in Coding and Regulatory Regions Underpin Repeated Adaptation to Sulfidic Streams in Poeciliid Fishes.

Adaptation to extreme environments often involves the evolution of dramatic physiological changes. To better understand how organisms evolve these complex phenotypic changes, the repeatability and predictability of evolution, and possible constraints on adapting to an extreme environment, it is important to understand how adaptive variation has evolved. Poeciliid fishes represent a particularly fruitful study system for investigations of adaptation to extreme environments due to their repeated colonization of toxic hydrogen sulfide-rich springs across multiple species within the clade. Previous investigations have highlighted changes in the physiology and gene expression in specific species that are thought to facilitate adaptation to hydrogen sulfide-rich springs. However, the presence of adaptive nucleotide variation in coding and regulatory regions and the degree to which convergent evolution has shaped the genomic regions underpinning sulfide tolerance across taxa are unknown. By sampling across seven independent lineages in which nonsulfidic lineages have colonized and adapted to sulfide springs, we reveal signatures of shared evolutionary rate shifts across the genome. We found evidence of genes, promoters, and putative enhancer regions associated with both increased and decreased convergent evolutionary rate shifts in hydrogen sulfide-adapted lineages. Our analysis highlights convergent evolutionary rate shifts in sulfidic lineages associated with the modulation of endogenous hydrogen sulfide production and hydrogen sulfide detoxification. We also found that regions with shifted evolutionary rates in sulfide spring fishes more often exhibited convergent shifts in either the coding region or the regulatory sequence of a given gene, rather than both.

The Long Non-Coding RNA MALAT1 Modulates NR4A1 Expression through a Downstream Regulatory Element in Specific Cancer Cell Types.

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci.

BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.

Functional differentiation and genetic diversity of rice cation exchanger (CAX) genes and their potential use in rice improvement.

Cation exchanger (CAX) genes play an important role in plant growth/development and response to biotic and abiotic stresses. Here, we tried to obtain important information on the functionalities and phenotypic effects of CAX gene family by systematic analyses of their expression patterns, genetic diversity (gene CDS haplotypes, structural variations, gene presence/absence variations) in 3010 rice genomes and nine parents of 496 Huanghuazhan introgression lines, the frequency shifts of the predominant gcHaps at these loci to artificial selection during modern breeding, and their association with tolerances to several abiotic stresses. Significant amounts of variation also exist in the cis-regulatory elements (CREs) of the OsCAX gene promoters in 50 high-quality rice genomes. The functional differentiation of OsCAX gene family were reflected primarily by their tissue and development specific expression patterns and in varied responses to different treatments, by unique sets of CREs in their promoters and their associations with specific agronomic traits/abiotic stress tolerances. Our results indicated that OsCAX1a and OsCAX2 as general signal transporters were in many processes of rice growth/development and responses to diverse environments, but they might be of less value in rice improvement. OsCAX1b, OsCAX1c, OsCAX3 and OsCAX4 was expected to be of potential value in rice improvement because of their associations with specific traits, responsiveness to specific abiotic stresses or phytohormones, and relatively high gcHap and CRE diversity. Our strategy was demonstrated to be highly efficient to obtain important genetic information on genes/alleles of specific gene family and can be used to systematically characterize the other rice gene families.

Divergence in regulatory mechanisms of GR-RBP genes in different plants under abiotic stress.

The IVa subfamily of glycine-rich proteins (GRPs) comprises a group of glycine-rich RNA binding proteins referred to as GR-RBPa here. Previous studies have demonstrated functions of GR-RBPa proteins in regulating stress response in plants. However, the mechanisms responsible for the differential regulatory functions of GR-RBPa proteins in different plant species have not been fully elucidated. In this study, we identified and comprehensively studied a total of 34 GR-RBPa proteins from five plant species. Our analysis revealed that GR-RBPa proteins were further classified into two branches, with proteins in branch I being relatively more conserved than those in branch II. When subjected to identical stresses, these genes exhibited intensive and differential expression regulation in different plant species, corresponding to the enrichment of cis-acting regulatory elements involving in environmental and internal signaling in these genes. Unexpectedly, all GR-RBPa genes in branch I underwent intensive alternative splicing (AS) regulation, while almost all genes in branch II were only constitutively spliced, despite having more introns. This study highlights the complex and divergent regulations of a group of conserved RNA binding proteins in different plants when exposed to identical stress conditions. These species-specific regulations may have implications for stress responses and adaptations in different plant species.

Mutant FOXO1 controls an oncogenic network via enhancer accessibility.

Transcriptional dysregulation is a hallmark of diffuse large B cell lymphoma (DLBCL), as transcriptional regulators are frequently mutated. However, our mechanistic understanding of how normal transcriptional programs are co-opted in DLBCL has been hindered by a lack of methodologies that provide the temporal resolution required to separate direct and indirect effects on transcriptional control. We applied a chemical-genetic approach to engineer the inducible degradation of the transcription factor FOXO1, which is recurrently mutated (mFOXO1) in DLBCL. The combination of rapid degradation of mFOXO1, nascent transcript detection, and assessment of chromatin accessibility allowed us to identify the direct targets of mFOXO1. mFOXO1 was required to maintain accessibility at specific enhancers associated with multiple oncogenes, and mFOXO1 degradation impaired RNA polymerase pause-release at some targets. Wild-type FOXO1 appeared to weakly regulate many of the same targets as mFOXO1 and was able to complement the degradation of mFOXO1 in the context of AKT inhibition.

Deep learning the cis-regulatory code for gene expression in selected model plants.

Elucidating the relationship between non-coding regulatory element sequences and gene expression is crucial for understanding gene regulation and genetic variation. We explored this link with the training of interpretable deep learning models predicting gene expression profiles from gene flanking regions of the plant species Arabidopsis thaliana, Solanum lycopersicum, Sorghum bicolor, and Zea mays. With over 80% accuracy, our models enabled predictive feature selection, highlighting e.g. the significant role of UTR regions in determining gene expression levels. The models demonstrated remarkable cross-species performance, effectively identifying both conserved and species-specific regulatory sequence features and their predictive power for gene expression. We illustrated the application of our approach by revealing causal links between genetic variation and gene expression changes across fourteen tomato genomes. Lastly, our models efficiently predicted genotype-specific expression of key functional gene groups, exemplified by underscoring known phenotypic and metabolic differences between Solanum lycopersicum and its wild, drought-resistant relative, Solanum pennellii.

Probabilistic association of differentially expressed genes with cis-regulatory elements.

Differential gene expression in response to perturbations is mediated at least in part by changes in binding of transcription factors (TFs) and other proteins at specific genomic regions. Association of these cis-regulatory elements (CREs) with their target genes is a challenging task that is essential to address many biological and mechanistic questions. Many current approaches rely on chromatin conformation capture techniques or single-cell correlational methods to establish CRE-to-gene associations. These methods can be effective but have limitations, including resolution, gaps in detectable association distances, and cost. As an alternative, we have developed DegCre, a nonparametric method that evaluates correlations between measurements of perturbation-induced differential gene expression and differential regulatory signal at CREs to score possible CRE-to-gene associations. It has several unique features, including the ability to use any type of CRE activity measurement, yield probabilistic scores for CRE-to-gene pairs, and assess CRE-to-gene pairings across a wide range of sequence distances. We apply DegCre to six data sets, each using different perturbations and containing a variety of regulatory signal measurements, including chromatin openness, histone modifications, and TF occupancy. To test their efficacy, we compare DegCre associations to Hi-C loop calls and CRISPR-validated CRE-to-gene associations, establishing good performance by DegCre that is comparable or superior to competing methods. DegCre is a novel approach to the association of CREs to genes from a perturbation-differential perspective, with strengths that are complementary to existing approaches and allow for new insights into gene regulation.

Emx2 underlies the development and evolution of marsupial gliding membranes.

Phenotypic variation among species is a product of evolutionary changes to developmental programs1,2. However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis-regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis-regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty.