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.

Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans.

Gene regulatory divergence between species can result from cis-acting local changes to regulatory element DNA sequences or global trans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifying trans-acting changes. We present a comprehensive approach to directly identify cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using assay for transposase-accessible chromatin coupled to self-transcribing active regulatory region (ATAC-STARR) sequencing. In addition to thousands of cis changes, we discover an unexpected number (∼10,000) of trans changes and show that cis and trans elements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie ∼37% of trans differences and trace how cis changes can produce cascades of trans changes. Overall, we find that most divergent elements (67%) experienced changes in both cis and trans, revealing a substantial role for trans divergence-alone and together with cis changes-in regulatory differences between species.

Yeast heterochromatin stably silences only weak regulatory elements by altering burst duration.

Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.

The immunoglobulin heavy chain super enhancer controls class switch recombination in developing B cells.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

Extreme restructuring of cis-regulatory regions controlling a deeply conserved plant stem cell regulator.

A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent cis-regulatory sequences. It remains unclear how such drastic cis-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how cis-regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ~125 million years. Using in-vivo genome editing in two distantly related models, Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene CLAVATA3 (CLV3) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato CLV3 are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis CLV3 function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis CLV3 caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional cis-regulatory sequences. Our results demonstrate remarkable malleability in cis-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of cis-regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of cis-regulation to effectively engineer trait variation from conserved productivity genes in crops.

BestCRM: An Exhaustive Search for Optimal Cis-Regulatory Modules in Promoters Accelerated by the Multidimensional Hash Function.

The concept of cis-regulatory modules located in gene promoters represents today's vision of the organization of gene transcriptional regulation. Such modules are a combination of two or more single, short DNA motifs. The bioinformatic identification of such modules belongs to so-called NP-hard problems with extreme computational complexity, and therefore, simplifications, assumptions, and heuristics are usually deployed to tackle the problem. In practice, this requires, first, many parameters to be set before the search, and second, it leads to the identification of locally optimal results. Here, a novel method is presented, aimed at identifying the cis-regulatory elements in gene promoters based on an exhaustive search of all the feasible modules' configurations. All required parameters are automatically estimated using positive and negative datasets. To be computationally efficient, the search is accelerated using a multidimensional hash function, allowing the search to complete in a few hours on a regular laptop (for example, a CPU Intel i7, 3.2 GH, 32 Gb RAM). Tests on an established benchmark and real data show better performance of BestCRM compared to the available methods according to several metrics like specificity, sensitivity, AUC, etc. A great practical advantage of the method is its minimum number of input parameters-apart from positive and negative promoters, only a desired level of module presence in promoters is required.

Multi-omics analysis in human retina uncovers ultraconserved cis-regulatory elements at rare eye disease loci.

Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.

Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

Regulatory features aid interpretation of 3'UTR variants.

Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.

MAE-seq refines regulatory elements across the genome.

Proper cell fate determination relies on precise spatial and temporal genome-wide cooperation between regulatory elements (REs) and their targeted genes. However, the lengths of REs defined using different methods vary, which indicates that there is sequence redundancy and that the context of the genome may be unintelligible. We developed a method called MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional REs at a 25-bp scale. In this study, MAE-seq was used to identify 626879, 541617 and 554826 25-bp enhancers in mouse embryonic stem cells (mESCs), C2C12 and HEK 293T, respectively. Using ∼1.6 trillion 25 bp DNA fragments and screening 12 billion cells, we identified 626879 as active enhancers in mESCs as an example. Comparative analysis revealed that most of the histone modification datasets were annotated by MAE-Seq loci. Furthermore, 33.85% (212195) of the identified enhancers were identified as de novo ones with no epigenetic modification. Intriguingly, distinct chromatin states dictate the requirement for dissimilar cofactors in governing novel and known enhancers. Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previously defined larger elements, Enhanced resolution facilitated the identification of numerous cell-specific enhancers and their accurate annotation as super enhancers. Moreover, we characterized novel elements capable of augmenting gene activity. By integrating with high-resolution Hi-C data, over 55.64% of novel elements may have a distal association with different targeted genes. For example, we found that the Cdh1 gene interacts with one novel and two known REs in mESCs. The biological effects of these interactions were investigated using CRISPR-Cas9, revealing their role in coordinating Cdh1 gene expression and mESC proliferation. Our study presents an experimental approach to refine the REs at 25-bp resolution, advancing the precision of genome annotation and unveiling the underlying genome context. This novel approach not only advances our understanding of gene regulation but also opens avenues for comprehensive exploration of the genomic landscape.

Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Hold out the genome: a roadmap to solving the cis-regulatory code.

Gene expression is regulated by transcription factors that work together to read cis-regulatory DNA sequences. The 'cis-regulatory code' - how cells interpret DNA sequences to determine when, where and how much genes should be expressed - has proven to be exceedingly complex. Recently, advances in the scale and resolution of functional genomics assays and machine learning have enabled substantial progress towards deciphering this code. However, the cis-regulatory code will probably never be solved if models are trained only on genomic sequences; regions of homology can easily lead to overestimation of predictive performance, and our genome is too short and has insufficient sequence diversity to learn all relevant parameters. Fortunately, randomly synthesized DNA sequences enable testing a far larger sequence space than exists in our genomes, and designed DNA sequences enable targeted queries to maximally improve the models. As the same biochemical principles are used to interpret DNA regardless of its source, models trained on these synthetic data can predict genomic activity, often better than genome-trained models. Here we provide an outlook on the field, and propose a roadmap towards solving the cis-regulatory code by a combination of machine learning and massively parallel assays using synthetic DNA.

RNA-mediated symmetry breaking enables singular olfactory receptor choice.

Olfactory receptor (OR) choice provides an extreme example of allelic competition for transcriptional dominance, where every olfactory neuron stably transcribes one of approximately 2,000 or more OR alleles1,2. OR gene choice is mediated by a multichromosomal enhancer hub that activates transcription at a single OR3,4, followed by OR-translation-dependent feedback that stabilizes this choice5,6. Here, using single-cell genomics, we show formation of many competing hubs with variable enhancer composition, only one of which retains euchromatic features and transcriptional competence. Furthermore, we provide evidence that OR transcription recruits enhancers and reinforces enhancer hub activity locally, whereas OR RNA inhibits transcription of competing ORs over distance, promoting transition to transcriptional singularity. Whereas OR transcription is sufficient to break the symmetry between equipotent enhancer hubs, OR translation stabilizes transcription at the prevailing hub, indicating that there may be sequential non-coding and coding mechanisms that are implemented by OR alleles for transcriptional prevalence. We propose that coding OR mRNAs possess non-coding functions that influence nuclear architecture, enhance their own transcription and inhibit transcription from their competitors, with generalizable implications for probabilistic cell fate decisions.

Chromatin priming elements direct tissue-specific gene activity before hematopoietic specification.

Tissue-specific gene regulation during development involves the interplay between transcription factors and epigenetic regulators binding to enhancer and promoter elements. The pattern of active enhancers defines the cellular differentiation state. However, developmental gene activation involves a previous step called chromatin priming which is not fully understood. We recently developed a genome-wide functional assay that allowed us to functionally identify enhancer elements integrated in chromatin regulating five stages spanning the in vitro differentiation of embryonic stem cells to blood. We also measured global chromatin accessibility, histone modifications, and transcription factor binding. The integration of these data identified and characterised cis-regulatory elements which become activated before the onset of gene expression, some of which are primed in a signalling-dependent fashion. Deletion of such a priming element leads to a delay in the up-regulation of its associated gene in development. Our work uncovers the details of a complex network of regulatory interactions with the dynamics of early chromatin opening being at the heart of dynamic tissue-specific gene expression control.

NCAD v1.0: a database for non-coding variant annotation and interpretation.

The application of whole genome sequencing is expanding in clinical diagnostics across various genetic disorders, and the significance of non-coding variants in penetrant diseases is increasingly being demonstrated. Therefore, it is urgent to improve the diagnostic yield by exploring the pathogenic mechanisms of variants in non-coding regions. However, the interpretation of non-coding variants remains a significant challenge, due to the complex functional regulatory mechanisms of non-coding regions and the current limitations of available databases and tools. Hence, we develop the non-coding variant annotation database (NCAD, http://www.ncawdb.net/), encompassing comprehensive insights into 665,679,194 variants, regulatory elements, and element interaction details. Integrating data from 96 sources, spanning both GRCh37 and GRCh38 versions, NCAD v1.0 provides vital information to support the genetic diagnosis of non-coding variants, including allele frequencies of 12 diverse populations, with a particular focus on the population frequency information for 230,235,698 variants in 20,964 Chinese individuals. Moreover, it offers prediction scores for variant functionality, five categories of regulatory elements, and four types of non-coding RNAs. With its rich data and comprehensive coverage, NCAD serves as a valuable platform, empowering researchers and clinicians with profound insights into non-coding regulatory mechanisms while facilitating the interpretation of non-coding variants.

The ftz upstream element drives late ftz stripes but is not required for regulation of Ftz target genes.

The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. Here, we asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS7S homozygotes, ftz stripes appear in the same order as wildtype, and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz stripe expression, with all 7 stripes decaying earlier than wildtype in ftzΔUPS7S homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. We propose that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, our results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Ultrafine mapping of chromosome conformation at hundred basepair resolution reveals regulatory genome architecture.

The resolution limit of chromatin conformation capture methodologies (3Cs) has restrained their application in detection of fine-level chromatin structure mediated by cis-regulatory elements (CREs). Here, we report two 3C-derived methods, Tri-4C and Tri-HiC, which utilize multirestriction enzyme digestions for ultrafine mapping of targeted and genome-wide chromatin interaction, respectively, at up to one hundred basepair resolution. Tri-4C identified CRE loop interaction networks and quantitatively revealed their alterations underlying dynamic gene control. Tri-HiC uncovered global fine-gauge regulatory interaction networks, identifying >20-fold more enhancer:promoter (E:P) loops than in situ Hi-C. In addition to vastly improved identification of subkilobase-sized E:P loops, Tri-HiC also uncovered interaction stripes and contact domain insulation from promoters and enhancers, revealing their loop extrusion behaviors resembling the topologically associating domain boundaries. Tri-4C and Tri-HiC provide robust approaches to achieve the high-resolution interactome maps required for characterizing fine-gauge regulatory chromatin interactions in analysis of development, homeostasis, and disease.

Epigenetic regulation during cancer transitions across 11 tumour types.

Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis1-4. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of ABCC1 and VEGFA; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of FGF19, ASAP2 and EN1, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial-mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.