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The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.

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.

Summary Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. In Arabidopsis, two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in a dgat1‐1/pdat1‐2 double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified. We transformed a dgat1‐1/dgat1‐1//PDAT1/pdat1‐2 parent with transgenic constructs containing the Arabidopsis DGAT1 promoter fused to the AtDGAT1 open reading frame either with or without the first intron. Triple homozygous plants were obtained, however, in the absence of the DGAT1 first intron anthers fail to fill with pollen, seed yield is c. 10% of wild‐type, seed oil content remains reduced (similar to dgat1‐1/dgat1‐1), and non‐Mendelian segregation of the PDAT1/pdat1‐2 locus occurs. Whereas plants expressing the AtDGAT1pro:AtDGAT1 transgene containing the first intron mostly recover phenotypes to wild‐type. This study establishes that a combination of the promoter and first intron of AtDGAT1 provides the proper context for temporal and tissue‐specific expression of AtDGAT1 in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes in Arabidopsis. [ABSTRACT FROM AUTHOR]

Lipoprotein lipase (LPL) is a multifunctional protein that catalyzes the hydrolysis of plasma triglycerides, releasing free fatty acids, which play critical roles in the metabolism and transport of lipids. The transcription of LPL in response to cell types and regulatory factors is a complex process that starts with its promoter. In previous studies, several proximal regulatory elements within the human LPL promoter were individually characterized. This study was designed to characterize the effect of 12 proximal regulatory elements as a combined unit on the transcriptional activity of the LPL promoter. The hypothesis was that these proximal regulatory elements collectively result in the optimal transcriptional activity of the human LPL promoter. Full and partial LPL promoter sequences, which contained and excluded the 12 regulatory elements, respectively, were cloned and inserted into a promoterless luciferase reporter vector. The functional activities of these constructs were tested in vitro using a dual-luciferase reporter assay. Our results showed that HEK-293 cells transfected with the full LPL promoter exhibited significantly greater luciferase activity than cells transfected with partial LPL promoters. Our results indicate that the proximal regulatory elements within the LPL promoter, including four TATA boxes, two Oct-1 sites, one CT element, two C/EBPα sites, one SP1 site, and two cis-acting regions (LP-α and LP-β), are essential for its transcriptional activity.

Summary: Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. In Arabidopsis, two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in a dgat1‐1/pdat1‐2 double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified.We transformed a dgat1‐1/dgat1‐1//PDAT1/pdat1‐2 parent with transgenic constructs containing the Arabidopsis DGAT1 promoter fused to the AtDGAT1 open reading frame either with or without the first intron.Triple homozygous plants were obtained, however, in the absence of the DGAT1 first intron anthers fail to fill with pollen, seed yield is c. 10% of wild‐type, seed oil content remains reduced (similar to dgat1‐1/dgat1‐1), and non‐Mendelian segregation of the PDAT1/pdat1‐2 locus occurs. Whereas plants expressing the AtDGAT1pro:AtDGAT1 transgene containing the first intron mostly recover phenotypes to wild‐type.This study establishes that a combination of the promoter and first intron of AtDGAT1 provides the proper context for temporal and tissue‐specific expression of AtDGAT1 in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes in Arabidopsis. [ABSTRACT FROM AUTHOR]

Abstract Background Winter wheat undergoes vernalization, a process activated by prolonged exposure to low temperatures. During this phase, flowering signals are generated and transported to the apical meristems, stimulating the transition to the inflorescence meristem while inhibiting tiller bud elongation. Although some vernalization genes have been identified, the key cis-regulatory elements and precise mechanisms governing this process in wheat remain largely unknown. Results In this study, we construct extensive epigenomic and transcriptomic profiling across multiple tissues—leaf, axillary bud, and shoot apex—during the vernalization of winter wheat. Epigenetic modifications play a crucial role in eliciting tissue-specific responses and sub-genome-divergent expressions during vernalization. Notably, we observe that H3K27me3 primarily regulates vernalization-induced genes and has limited influence on vernalization-repressed genes. The integration of these datasets enables the identification of 10,600 putative vernalization-related regulatory elements including distal accessible chromatin regions (ACRs) situated 30Kb upstream of VRN3, contributing to the construction of a comprehensive regulatory network. Furthermore, we discover that TaSPL7/15, integral components of the aging-related flowering pathway, interact with the VRN1 promoter and VRN3 distal regulatory elements. These interactions finely regulate their expressions, consequently impacting the vernalization process and flowering. Conclusions Our study offers critical insights into wheat vernalization’s epigenomic dynamics and identifies the putative regulatory elements crucial for developing wheat germplasm with varied vernalization characteristics. It also establishes a vernalization-related transcriptional network, and uncovers that TaSPL7/15 from the aging pathway participates in vernalization by directly binding to the VRN1 promoter and VRN3 distal regulatory elements.

Abstract Malaria transmission and endemicity in Africa remains hugely disproportionate compared to the rest of the world. The complex life cycle of P. falciparum (Pf) between the vertebrate human host and the anopheline vector results in differential expression of genes within and between hosts. An in-depth understanding of Pf interaction with various human genes through regulatory elements will pave way for identification of newer tools in the arsenal for malaria control. Therefore, the regulatory elements (REs) involved in the over- or under-expression of various host immune genes hold the key to elucidating alternative control measures that can be applied for disease surveillance, prompt diagnosis and treatment. We carried out an RNAseq analysis to identify differentially expressed genes and network elucidation of non-coding RNAs and target genes associated with immune response in individuals with different clinical outcomes. Raw RNAseq datasets, retrieved for analyses include individuals with severe (Gambia—20), symptomatic (Burkina Faso—15), asymptomatic (Mali—16) malaria as well as uninfected controls (Tanzania—20; Mali—36). Of the total 107 datasets retrieved, we identified 5534 differentially expressed genes (DEGs) among disease and control groups. A peculiar pattern of DEGs was observed, with individuals presenting with severe/symptomatic malaria having the highest and most diverse upregulated genes, while a reverse phenomenon was recorded among asymptomatic and uninfected individuals. In addition, we identified 141 differentially expressed micro RNA (miRNA), of which 78 and 63 were upregulated and downregulated respectively. Interactome analysis revealed a moderate interaction between DEGs and miRNAs. Of all identified miRNA, five were unique (hsa-mir-32, hsa-mir-25, hsa-mir-221, hsa-mir-29 and hsa-mir-148) because of their connectivity to several genes, including hsa-mir-221 connected to 16 genes. Six-hundred and eight differentially expressed long non coding RNA (lncRNA) were also identified, including SLC7A11, LINC01524 among the upregulated ones. Our study provides important insight into host immune genes undergoing differential expression under different malaria conditions. It also identified unique miRNAs and lncRNAs that modify and/or regulate the expression of various immune genes. These regulatory elements we surmise, have the potential to serve a diagnostic purpose in discriminating between individuals with severe/symptomatic malaria and those with asymptomatic infection or uninfected, following further clinical validation from field isolates.

Improving crop traits requires genetic diversity, which allows breeders to select advantageous alleles of key genes. In species or loci that lack sufficient genetic diversity, synthetic directed evolution (SDE) can supplement natural variation, thus expanding the possibilities for trait engineering. In this review, we explore recent advances and applications of SDE for crop improvement, highlighting potential targets (coding sequences and cis-regulatory elements) and computational tools to enhance crop resilience and performance across diverse environments. Recent advancements in SDE approaches have streamlined the generation of variants and the selection processes; by leveraging these advanced technologies and principles, we can minimize concerns about host fitness and unintended effects, thus opening promising avenues for effectively enhancing crop traits. [ABSTRACT FROM AUTHOR]

Malaria transmission and endemicity in Africa remains hugely disproportionate compared to the rest of the world. The complex life cycle of P. falciparum (Pf) between the vertebrate human host and the anopheline vector results in differential expression of genes within and between hosts. An in-depth understanding of Pf interaction with various human genes through regulatory elements will pave way for identification of newer tools in the arsenal for malaria control. Therefore, the regulatory elements (REs) involved in the over- or under-expression of various host immune genes hold the key to elucidating alternative control measures that can be applied for disease surveillance, prompt diagnosis and treatment. We carried out an RNAseq analysis to identify differentially expressed genes and network elucidation of non-coding RNAs and target genes associated with immune response in individuals with different clinical outcomes. Raw RNAseq datasets, retrieved for analyses include individuals with severe (Gambia—20), symptomatic (Burkina Faso—15), asymptomatic (Mali—16) malaria as well as uninfected controls (Tanzania—20; Mali—36). Of the total 107 datasets retrieved, we identified 5534 differentially expressed genes (DEGs) among disease and control groups. A peculiar pattern of DEGs was observed, with individuals presenting with severe/symptomatic malaria having the highest and most diverse upregulated genes, while a reverse phenomenon was recorded among asymptomatic and uninfected individuals. In addition, we identified 141 differentially expressed micro RNA (miRNA), of which 78 and 63 were upregulated and downregulated respectively. Interactome analysis revealed a moderate interaction between DEGs and miRNAs. Of all identified miRNA, five were unique (hsa-mir-32, hsa-mir-25, hsa-mir-221, hsa-mir-29 and hsa-mir-148) because of their connectivity to several genes, including hsa-mir-221 connected to 16 genes. Six-hundred and eight differentially expressed long non coding RNA (lncRNA) were also identified, including SLC7A11, LINC01524 among the upregulated ones. Our study provides important insight into host immune genes undergoing differential expression under different malaria conditions. It also identified unique miRNAs and lncRNAs that modify and/or regulate the expression of various immune genes. These regulatory elements we surmise, have the potential to serve a diagnostic purpose in discriminating between individuals with severe/symptomatic malaria and those with asymptomatic infection or uninfected, following further clinical validation from field isolates. [ABSTRACT FROM AUTHOR]

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The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.

The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements. [ABSTRACT FROM AUTHOR]

Flavor is essential to consumer preference of foods and is an increasing focus of plant breeding programs. In fruit crops, identifying genes underlying volatile organic compounds has great promise to accelerate flavor improvement, but polyploidy and heterozygosity in many species have slowed progress. Here we use octoploid cultivated strawberry to demonstrate how genomic heterozygosity, transcriptomic intricacy and fruit metabolomic diversity can be treated as strengths and leveraged to uncover fruit flavor genes and their regulatory elements. Multi-omics datasets were generated including an expression quantitative trait loci map with 196 diverse breeding lines, haplotype-phased genomes of a highly-flavored breeding selection, a genome-wide structural variant map using five haplotypes, and volatile genome-wide association study (GWAS) with > 300 individuals. Overlaying regulatory elements, structural variants and GWAS-linked allele-specific expression of numerous genes to variation in volatile compounds important to flavor. In one example, the functional role of anthranilate synthase alpha subunit 1 in methyl anthranilate biosynthesis was supported via fruit transient gene expression assays. These results demonstrate a framework for flavor gene discovery in fruit crops and a pathway to molecular breeding of cultivars with complex and desirable flavor.

Abstract The majority of disease-associated variants identified through genome-wide association studies are located outside of protein-coding regions. Prioritizing candidate regulatory variants and gene targets to identify potential biological mechanisms for further functional experiments can be challenging. To address this challenge, we developed FORGEdb ( https://forgedb.cancer.gov/ ; https://forge2.altiusinstitute.org/files/forgedb.html ; and https://doi.org/10.5281/zenodo.10067458 ), a standalone and web-based tool that integrates multiple datasets, delivering information on associated regulatory elements, transcription factor binding sites, and target genes for over 37 million variants. FORGEdb scores provide researchers with a quantitative assessment of the relative importance of each variant for targeted functional experiments.

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology.

Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications. [Display omitted] • Novel method for tuning cancer receptor surface density using regulatory element constructs. • Swift co-culture protocol allows combination of tuned cancer receptor cells and leukocytes. • Presented platform for co-culturing and receptor tuning allows for evaluation of bispecifics. [ABSTRACT FROM AUTHOR]

Pluripotent hematopoietic stem cells undergo maturation to regenerate blood cells. T-cell and B-cell maturation from common lymphoid progenitors (CLPs) is regulated by distinct transcription factors and signaling pathways. Notch signaling, GATA3, and TCF1 drive T-cell fate, while EBF1, PU.1, E2A, Pax5, TCF1, and Foxo1 regulate B-cell differentiation. Alterations in these factors can cause lineage deviations. Gene expression profiling of the chick thymus and bursa reveals dynamic expression patterns of transcription factors, cytokines, and signal molecules. Notch1 and Dll1 expression that increase during later stages indicate the ongoing role of Notch signaling in T-cell lineage maintenance. The current study aimed to identify differentially expressed genes during the development of the avian immune organs, focusing on the thymus and bursa using 24 Ross 308 avian breed. The mRNA libraries from these organs were analyzed using quantitative Real-time PCR analysis at six time points spanning the embryonic ages (days 15 and 18) and posthatch age (days 3, 7, 14, and 28). The data for the gene expression indicated significant variations across different stages of immune organ development. Differential gene expression was observed between sorted T and B-cells, with GATA3, CD3e, CD4, and Ptprc showing higher expression in the T-cell population, and Pax5 and CD81 exhibiting higher expression in the B-cell population. Notably, ENO1 and IRF4 showed higher expression in T-cells at E15 and B-cells at E18. The study highlights the importance of regulatory factors and genes in maintaining cellular identity, furthers the understanding of avian immunology, and has the potential for improving poultry health and studying immune-related diseases in humans. These findings pave the way for further research on the role of biochemical components under important disease conditions in avian immunology and their potential applications for poultry health. [ABSTRACT FROM AUTHOR]

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PR domain-containing protein 9, a key determinant of recombination dynamics in most metazoans. Historical recombination maps in birds show an apparent stasis in positioning recombination events. This highly conserved recombination pattern over long timescales may constrain the evolution of recombination in birds. At the same time, extensive variation in recombination rate is observed across the genome and between different species of birds. Here, we characterize the fine-scale historical recombination map of an iconic migratory songbird, the Eurasian blackcap (Sylvia atricapilla), using a linkage disequilibrium–based approach that accounts for population demography. Our results reveal variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content and negatively with chromosome size. Recombination rates increased significantly at regulatory regions but not necessarily at gene bodies. CpG islands are associated strongly with recombination rates, though their specific position and local DNA methylation patterns likely influence this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of heterogeneous intrachromosomal conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features in shaping this variation. This study opens the possibility of further investigating the impact of recombination on specific population-genomic features. [ABSTRACT FROM AUTHOR]

Genetic predisposition to cardiac arrhythmias has been a field of intense investigation. Research initially focused on rare hereditary arrhythmias, but over the last two decades, the role of genetic variation (single nucleotide polymorphisms) in heart rate, rhythm, and arrhythmias has been taken into consideration as well. In particular, genome-wide association studies have identified hundreds of genomic loci associated with quantitative electrocardiographic traits, atrial fibrillation, and less common arrhythmias such as Brugada syndrome. A significant number of associated variants have been found to systematically localize in non-coding regulatory elements that control the tissue-specific and temporal transcription of genes encoding transcription factors, ion channels, and other proteins. However, the identification of causal variants and the mechanism underlying their impact on phenotype has proven difficult due to the complex tissue-specific, time-resolved, condition-dependent, and combinatorial function of regulatory elements, as well as their modest conservation across different model species. In this review, we discuss research efforts aimed at identifying and characterizing-trait-associated variant regulatory elements and the molecular mechanisms underlying their impact on heart rate or rhythm. [ABSTRACT FROM AUTHOR]

Mortality from vmelanoma is associated with metastatic disease, but the mechanisms leading to spreading of the cancer cells remain obscure. Spatial profiling revealed that melanoma is characterized by a high degree of heterogeneity, which is established by the ability of melanoma cells to switch between different phenotypical stages. This plasticity, likely a heritage from embryonic pathways, accounts for a relevant part of the metastatic potential of these lesions, and requires the rapid and efficient reorganization of the transcriptional landscape of melanoma cells. A large part of the non‐coding genome cooperates to control gene expression, specifically through the activity of enhancers (ENHs). In this study, we aimed to identify ex vivo the network of active ENHs and to outline their cooperative interactions in supporting transcriptional adaptation during melanoma metastatic progression. We conducted a genome‐wide analysis to map active ENHs distribution in a retrospective cohort of 39 melanoma patients, comparing the profiles obtained in primary (N = 19) and metastatic (N = 20) melanoma lesions. Unsupervised clustering showed that the profile for acetylated histone H3 at lysine 27 (H3K27ac) efficiently segregates lesions into three different clusters corresponding to progressive stages of the disease. We reconstructed the map of super‐ENHs (SEs) and cooperative ENHs that associate with metastatic progression in melanoma, which showed that cooperation among regulatory elements is a mandatory requirement for transcriptional plasticity. We also showed that these elements carry out specialized and non‐redundant functions, and indicated the existence of a hierarchical organization, with SEs on top as masterminds of the entire transcriptional program and classical ENHs as executors. By providing an innovative vision of how the chromatin landscape of melanoma works during metastatic spreading, our data also point out the need to integrate functional profiling in the analysis of cancer lesions to increase definition and improve interpretation of tumor heterogeneity.

Abstract Background Cellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia. Results After validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions. Conclusions Neurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.

Bacteria belonging to Streptomyces have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in Streptomyces, system-level information remains limited. In this study, we integrated four multi-omics datasets from Streptomyces griseus NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3'-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5'-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in Streptomyces.

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

Abstract Background To identify the disease-causing gene in a Chinese family affected with congenital aniridia. Methods Patients underwent systematic ophthalmic examinations such as anterior segment photography, fundus photography, optical coherence tomography, and fundus fluorescein angiography. The proband was screened for pathogenic variants by whole exome sequencing (WES) and copy number variant (CNV) analysis. Real-time quantitative PCR (RT-qPCR) was applied to confirm the CNV results. Breakpoints were identified by long-range PCR followed by Sanger sequencing. Results All seven members of this Chinese family, including four patients and three normal individuals, were recruited for this study. All patients showed bilateral congenital aniridia with nystagmus, except the son of the proband, who presented with bilateral partial coloboma of the iris. A novel heterozygous deletion (chr11:31,139,019–31,655,997) containing the 3’ regulatory enhancers of the PAX6 gene was detected in this family. We also reviewed the reported microdeletions downstream of PAX6 in patients with aniridia. Conclusions We identified a novel microdeletion, 517 kb in size located about 133 kb downstream of the PAX6 gene, responsible for congenital aniridia in this Chinese family, which expands the spectrum of aniridia-associated mutations in PAX6.

Abstract Background Modern genome sequencing leads to an ever-growing collection of genomic annotations. Combining these elements with a set of input regions (e.g. genes) would yield new insights in genomic associations, such as those involved in gene regulation. The required data are scattered across different databases making a manual approach tiresome, unpractical, and prone to error. Semi-automatic approaches require programming skills in data parsing, processing, overlap calculation, and visualization, which most biomedical researchers lack. Our aim was to develop an automated tool providing all necessary algorithms, benefiting both bioinformaticians and researchers without bioinformatic training. Results We developed overlapping annotated genomic regions (OGRE) as a comprehensive tool to associate and visualize input regions with genomic annotations. It does so by parsing regions of interest, mining publicly available annotations, and calculating possible overlaps between them. The user can thus identify location, type, and number of associated regulatory elements. Results are presented as easy to understand visualizations and result tables. We applied OGRE to recent studies and could show high reproducibility and potential new insights. To demonstrate OGRE’s performance in terms of running time and output, we have conducted a benchmark and compared its features with similar tools. Conclusions OGRE’s functions and built-in annotations can be applied as a downstream overlap association step, which is compatible with most genomic sequencing outputs, and can thus enrich pre-existing analyses pipelines. Compared to similar tools, OGRE shows competitive performance, offers additional features, and has been successfully applied to two recent studies. Overall, OGRE addresses the lack of tools for automatic analysis, local genomic overlap calculation, and visualization by providing an easy to use, end-to-end solution for both biologists and computational scientists.

Purpose: The etiopathogenesis of coronal nonsyndromic craniosynostosis (cNCS), a congenital condition defined by premature fusion of 1 or both coronal sutures, remains largely unknown. Methods: We conducted the largest genome-wide association study of cNCS followed by replication, fine mapping, and functional validation of the most significant region using zebrafish animal model. Results: Genome-wide association study identified 6 independent genome-wide-significant risk alleles, 4 on chromosome 7q21.3 SEM1-DLX5-DLX6 locus, and their combination conferred over 7-fold increased risk of cNCS. The top variants were replicated in an independent cohort and showed pleiotropic effects on brain and facial morphology and bone mineral density. Fine mapping of 7q21.3 identified a craniofacial transcriptional enhancer (eDlx36) within the linkage region of the top variant (rs4727341; odds ratio [95% confidence interval], 0.48[0.39-0.59]; P = 1.2E−12) that was located in SEM1 intron and enriched in 4 rare risk variants. In zebrafish, the activity of the transfected human eDlx36 enhancer was observed in the frontonasal prominence and calvaria during skull development and was reduced when the 4 rare risk variants were introduced into the sequence. Conclusion: Our findings support a polygenic nature of cNCS risk and functional role of craniofacial enhancers in cNCS susceptibility with potential broader implications for bone health.

Mortality from vmelanoma is associated with metastatic disease, but the mechanisms leading to spreading of the cancer cells remain obscure. Spatial profiling revealed that melanoma is characterized by a high degree of heterogeneity, which is established by the ability of melanoma cells to switch between different phenotypical stages. This plasticity, likely a heritage from embryonic pathways, accounts for a relevant part of the metastatic potential of these lesions, and requires the rapid and efficient reorganization of the transcriptional landscape of melanoma cells. A large part of the non‐coding genome cooperates to control gene expression, specifically through the activity of enhancers (ENHs). In this study, we aimed to identify ex vivo the network of active ENHs and to outline their cooperative interactions in supporting transcriptional adaptation during melanoma metastatic progression. We conducted a genome‐wide analysis to map active ENHs distribution in a retrospective cohort of 39 melanoma patients, comparing the profiles obtained in primary (N = 19) and metastatic (N = 20) melanoma lesions. Unsupervised clustering showed that the profile for acetylated histone H3 at lysine 27 (H3K27ac) efficiently segregates lesions into three different clusters corresponding to progressive stages of the disease. We reconstructed the map of super‐ENHs (SEs) and cooperative ENHs that associate with metastatic progression in melanoma, which showed that cooperation among regulatory elements is a mandatory requirement for transcriptional plasticity. We also showed that these elements carry out specialized and non‐redundant functions, and indicated the existence of a hierarchical organization, with SEs on top as masterminds of the entire transcriptional program and classical ENHs as executors. By providing an innovative vision of how the chromatin landscape of melanoma works during metastatic spreading, our data also point out the need to integrate functional profiling in the analysis of cancer lesions to increase definition and improve interpretation of tumor heterogeneity. [ABSTRACT FROM AUTHOR]

Background: Cellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia. Results: After validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions. Conclusions: Neurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG. [ABSTRACT FROM AUTHOR]

This investigation discerns the expression profiles of genes within the leaf transcriptome of Arabidopsis thaliana subjected to salt stress (200 mM NaCl). Notably, the pivotal role of indole acetic acid emerged as a keystone orchestrating a multifaceted cascade of regulatory events aimed at enhancing the plant's adaptability under salt-induced stress. Cluster analysis elucidated upregulation of gene families with pivotal roles in supporting the availability of carbon dioxide, ameliorating photosynthetic processes and mitigating the deleterious effects of reactive oxygen species under salt stress. Analysis also unveiled the participation of several transcription factor families in the orchestration of a multitude of genes under salt stress. The investigation singled out a solitary TF, denominated as BH100, which was validated through RNA-Seq and qPCR, utilizing a VIGS line featuring the knockdown of the BH100 gene. This transcription factor was implicated in the upregulation of the FRO gene, thereby establishing a link between the synchronized expression of these two genes and their role in promoting iron acquisition under salt stress. In summation, our study unveiled the regulatory frameworks and salt-responsive genes underpinning the response of Arabidopsis to salt stress. We present compelling arguments for the potential applicability of this information in the realm of molecular breeding programs. [ABSTRACT FROM AUTHOR]

Inhibitor of differentiation 1 (ID1) protein is highly expressed in undifferentiated cells and associated with tumor progression and metastasis. Restricted regulation of the gene expression is necessary for cell maintenance. We performed the characterization of the response elements involved in the regulation of ID1 promoter activity using deletions and mutations. In silico analysis was used to predict regulatory binding sites. Mutants of the 5' upstream region of the ID1 promoter were constructed with nucleotide deletion or mutation, and the promoter activity was systematically analyzed. In silico analysis revealed several transcription binding sites presented in upstream region of the ID1 gene. Cluster of four Sp1 binding sites at position -1296 to -1078 contributed to the maximal expression activity of ID1 gene. The deletion and mutation of these Sp1 binding sites resulted in reduction of the expression level. Moreover, it is suggested that mutation of the Sp1 binding site at position -1078 could permit repressor proteins to suppress the gene expression. This study yields a new insight to a better understanding of the ID1 gene regulation, which pave the way to obtain the normal expression level of ID1 for cancer therapy development. [ABSTRACT FROM AUTHOR]

Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic-related and salt stress-inducible synthetic promoters for use in hybrid poplar. De novo motif-detecting algorithms yielded 30 water-deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water-deficit stress motifs (SD18, SD13 and SD9) found in 16 co-expressed gene promoters, and we discovered a well-conserved motif for salt response (SS16). We characterized several native poplar stress-inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five-to-eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18-1, SD9-2, SS16-1, SS16-2 and SS16-3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18-1 and SD9-2 were induced by water-deficit stress, whereas SS16-1, SS16-2 and SS16-3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.

DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (HBBA, IRF7, and PPARG) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.

Background: To identify the disease-causing gene in a Chinese family affected with congenital aniridia. Methods: Patients underwent systematic ophthalmic examinations such as anterior segment photography, fundus photography, optical coherence tomography, and fundus fluorescein angiography. The proband was screened for pathogenic variants by whole exome sequencing (WES) and copy number variant (CNV) analysis. Real-time quantitative PCR (RT-qPCR) was applied to confirm the CNV results. Breakpoints were identified by long-range PCR followed by Sanger sequencing. Results: All seven members of this Chinese family, including four patients and three normal individuals, were recruited for this study. All patients showed bilateral congenital aniridia with nystagmus, except the son of the proband, who presented with bilateral partial coloboma of the iris. A novel heterozygous deletion (chr11:31,139,019–31,655,997) containing the 3' regulatory enhancers of the PAX6 gene was detected in this family. We also reviewed the reported microdeletions downstream of PAX6 in patients with aniridia. Conclusions: We identified a novel microdeletion, 517 kb in size located about 133 kb downstream of the PAX6 gene, responsible for congenital aniridia in this Chinese family, which expands the spectrum of aniridia-associated mutations in PAX6. [ABSTRACT FROM AUTHOR]

The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot. The receptor binding domain (RBD) sequence from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein was used to replace the eGFP sequence in the expression vector and the RBD expression was detected by qRT-PCR and western blot. The results showed that protein expression can be regulated by optimizing the combination of cis-acting elements. The vector with the CMV enhancer, EF-1α core promoter, and TEF terminator was found to express approximately threefold higher eGFP than the unmodified vector in different animal cells, as well as 2.63-fold higher recombinant RBD protein than the original vector in HEK-293T cells. Moreover, we suggest that combinations of multiple regulatory elements capable of regulating gene expression do not necessarily exhibit synergistic effects to enhance expression further. Overall, our findings provide insights into biological applications that require the regulation of gene expression and will help to optimize expression vectors for biosynthesis and other fields. Additionally, we provide valuable insights into the production of RBD proteins, which may aid in developing reagents for diagnosis and treatment during the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

Background: Germline CDH1 pathogenic or likely pathogenic variants cause hereditary diffuse gastric cancer (HDGC). Once a genetic cause is identified, stomachs' and breasts' surveillance and/or prophylactic surgery is offered to asymptomatic CDH1 carriers, which is life-saving. Herein, we characterized an inherited mechanism responsible for extremely early-onset gastric cancer and atypical HDGC high penetrance. Methods: Whole-exome sequencing (WES) re-analysis was performed in an unsolved HDGC family. Accessible chromatin and CDH1 promoter interactors were evaluated in normal stomach by ATAC-seq and 4C-seq, and functional analysis was performed using CRISPR-Cas9, RNA-seq and pathway analysis. Results: We identified a germline heterozygous 23 Kb CDH1-TANGO6 deletion in a family with eight diffuse gastric cancers, six before age 30. Atypical HDGC high penetrance and young cancer-onset argued towards a role for the deleted region downstream of CDH1, which we proved to present accessible chromatin, and CDH1 promoter interactors in normal stomach. CRISPR-Cas9 edited cells mimicking the CDH1-TANGO6 deletion display the strongest CDH1 mRNA downregulation, more impacted adhesion-associated, type-I interferon immune-associated and oncogenic signalling pathways, compared to wild-type or CDH1-deleted cells. This finding solved an 18-year family odyssey and engaged carrier family members in a cancer prevention pathway of care. Conclusion: In this work, we demonstrated that regulatory elements lying down-stream of CDH1 are part of a chromatin network that control CDH1 expression and influence cell transcriptome and associated signalling pathways, likely explaining high disease penetrance and very young cancer-onset. This study highlights the importance of incorporating scientific–technological updates and clinical guidelines in routine diagnosis, given their impact in timely genetic diagnosis and disease prevention. [ABSTRACT FROM AUTHOR]

Oomycetes, a large group of fungus-like organisms, include some destructive plant pathogens causing enormous economic damage. Phylogenetically, oomycetes belong to the kingdom Straminipila and have diverse lifestyles, including saprotrophs and both general and specialized pathogens of various eukaryotic supergroups. A rapid increase in genomic studies and next-generation sequencing technologies have led to significant progress in understanding oomycete lifestyles. However, their genetics, including transcriptional regulation, have been studied to a much lesser extent. Here, we provide a cross-species analysis of oomycete promoter for providing a first step towards elucidating gene regulation networks related to pathogenicity and life cycle stages. The clustered DNA sequences of Plasmopara halstedii transcriptome time-series expression level dataset from a preliminary study have been used as a core reference for cross-species comparisons. Using a computational pipeline, 46 potential transcription factor binding site (TFBS) motifs in 25 clusters with functionally conserved downstream genes of downy mildew and two Phytophthora species, regardless of the gene expression levels of Phytophthora transcriptomes, were found. This can now be followed up by knock-out experiments in oomycete species amenable for genetic modification. [ABSTRACT FROM AUTHOR]

Split Hand-Foot Malformation (SHFM) is a congenital limb defect characterized by a median cleft of the hands and/or feet due to the absence/hypoplasia of the central rays. It may occur as part of a syndromic condition or as an isolated malformation. The most common of the six genetic loci identified for this condition is correlated to SHFM1 and maps in the 7q21q22 region. SHFM1 is characterized by autosomal dominant transmission, incomplete penetrance and variable expressivity. Associated features often include hearing loss, intellectual disability/developmental delay and craniofacial abnormalities. Disruption of the DLX5/DLX6 genes, mapping within the SHFM1 locus, is now known to be responsible for the phenotype. Through SNP array, we analyzed a patient affected by SHFM1 associated with deafness and an abnormality of the inner ear (incomplete partition type I); we identified a deletion in 7q21, not involving the DLX5/6 genes, but including exons 15 and 17 of DYNC1I1, known to act as exonic enhancers (eExons) of the DLX5/6 genes. We further demonstrated the role of DYNC1I1 eExons in regulating DLX5/6 expression by means of showing a reduced expression of the DLX5/6 genes through RT-PCR in a patient-derived lymphoblastoid cell line. Furthermore, our data and a review of published cases do not support the hypothesis that DLX5/6 are imprinted in humans. This work is an example of how the disruption of regulatory elements can be responsible for congenital malformations. [ABSTRACT FROM AUTHOR]

Plants have evolved various mechanisms to adapt to adverse environmental stresses, such as the modulation of gene expression. Expression of stressresponsive genes is controlled by specific regulators, including transcription factors (TFs), that bind to sequence-specific binding sites, representing key components of cis-regulatory elements and regulatory networks. Our understanding of the underlying regulatory code remains, however, incomplete. Recent studies have shown that, by training machine learning (ML) algorithms on genomic sequence features, it is possible to predict which genes will transcriptionally respond to a specific stress. By identifying the most important features for gene expression prediction, these trained ML models allow, in theory, to further elucidate the regulatory code underlying the transcriptional response to abiotic stress. Here, we trained random forest ML models to predict gene expression in rice (Oryza sativa) in response to heat or drought stress. Apart from thoroughly assessing model performance and robustness across various input training data, the importance of promoter and gene body sequence features to train ML models was evaluated. The use of enriched promoter oligomers, complementing known TF binding sites, allowed us to gain novel insights in DNA motifs contributing to the stress regulatory code. By comparing genomic feature importance scores for drought and heat stress over time, general and stress-specific genomic features contributing to the performance of the learned models and their temporal variation were identified. This study provides a solid foundation to build and interpret ML models accurately predicting transcriptional responses and enables novel insights in biological sequence features that are important for abiotic stress responses. [ABSTRACT FROM AUTHOR]

Background: Modern genome sequencing leads to an ever-growing collection of genomic annotations. Combining these elements with a set of input regions (e.g. genes) would yield new insights in genomic associations, such as those involved in gene regulation. The required data are scattered across different databases making a manual approach tiresome, unpractical, and prone to error. Semi-automatic approaches require programming skills in data parsing, processing, overlap calculation, and visualization, which most biomedical researchers lack. Our aim was to develop an automated tool providing all necessary algorithms, benefiting both bioinformaticians and researchers without bioinformatic training. Results: We developed overlapping annotated genomic regions (OGRE) as a comprehensive tool to associate and visualize input regions with genomic annotations. It does so by parsing regions of interest, mining publicly available annotations, and calculating possible overlaps between them. The user can thus identify location, type, and number of associated regulatory elements. Results are presented as easy to understand visualizations and result tables. We applied OGRE to recent studies and could show high reproducibility and potential new insights. To demonstrate OGRE's performance in terms of running time and output, we have conducted a benchmark and compared its features with similar tools. Conclusions: OGRE's functions and built-in annotations can be applied as a downstream overlap association step, which is compatible with most genomic sequencing outputs, and can thus enrich pre-existing analyses pipelines. Compared to similar tools, OGRE shows competitive performance, offers additional features, and has been successfully applied to two recent studies. Overall, OGRE addresses the lack of tools for automatic analysis, local genomic overlap calculation, and visualization by providing an easy to use, end-to-end solution for both biologists and computational scientists. [ABSTRACT FROM AUTHOR]

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6+/- mouse model of fast-spiking CIN dysfunction.

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.