Putative Cis-regulatory Elements Research Articles

Bioinformatics and Functional Analysis of OsASMT1 Gene in Response to Abiotic Stress.

The study aimed to elucidate the functional characteristics of OsASMT1 gene under copper (Cu) or sodium chloride (NaCl) stress. Bioinformatics scrutiny unveiled that OsASMT1 is situated on chromosome 9. Its protein architecture, comprising dimerization and methyltransferase domains, showed significant similarities to OsASMT2 and OsASMT3. High expression in roots and panicles, along with abiotic stress putative cis-regulatory elements in the promoter, indicated potential stress responsiveness. Real-time quantitative PCR confirmed OsASMT1 induction under Cu and NaCl stress in rice. Surprisingly, yeast expressing OsASMT1 did not exhibit enhanced resistance to abiotic stresses. The results of subcellular localization analysis indicated that OsASMT1 plays a role in the cytoplasm. While OsASMT1 responded to Cu and NaCl stress in rice, its heterologous expression in yeast failed to confer abiotic stress resistance, highlighting the need for further investigation of its functional implications.

The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Shade responses and resistant mechanisms in Spatholobus suberectus

The medicinal plant Spatholobus suberectus Dunn is easily exposed to shade stress during growth, but its shade responses and shade stress resistant mechanisms have not been clarified. In this study, shade treatments including four attenuated sunlight intensities (100%, 60%, 40%, and 10%) and three shade durations (30 d, 45 d, and 60 d) were applied to S. suberectus. The shade-induced morphological indicators, phytohormonal regulations, metabolic flavonoids contents, transcriptomic flavonoid pathway gene expressions, and stress physiological changes of S. suberectus were analyzed. The putative promoter cis-regulatory elements (CREs) of 18 flavonoid biosynthetic pathway genes were identified. Results showed the stem growth indicators of S. suberectus were better at 40% light intensity. Phytohormones were involved in the shade-induced responses. Short-term shade (30 d) increased total flavonoids, gallated catechins and especially epigallocatechin gallate contents and favored for boosting medicinal value. Long-term shade (45 d, 60 d) tended to decrease flavonoids. The shade-induced flavonoids changes were attributed to their corresponding biosynthesizing genes expression variations. The high antioxidant capacity and the presence of phytohormone-, stress-, and development-related CREs provided the basis for stress resistance. In conclusion, the multiple responses under shade and the CREs analysis elucidated S. suberectus’ shade tolerance.

Computational analysis of regulation and expression of plant DNA repair genes under various abiotic stresses reveals a stochastic correlation

Biotic/abiotic stresses are known to induce genome instability in all organisms. Plants being sessile, are more vulnerable to stresses. Thus, they have evolved mechanisms to protect their genome from the unpredictably changing environment. DNA repair genes play a critical role in preserving genome integrity. Plants have about 15 different DNA repair pathways comprising more than 100 genes. To gain a better understanding of how plant DNA repair genes perceive stress and regulate their expression, we performed a computational analysis of the putative binding sites of different abiotic stress-responsive transcription factors (ASRTFs) on their promoters, in the model plant Arabidopsis thaliana. Of the 12 different ASRTFs that we analysed, MYC2 had the highest number of 1876 putative binding sites. Diversity study in 135 DNA repair gene promoters revealed that these had about 6 to 12 types of putative binding sites for the ASRTFs. While RAD51 promoter had the highest number of 207 putative binding sites, RECQ4B had only 11. Comparison of the Arabidopsis eFP browser-based temporal/spatial expression profiles of these genes under nine different abiotic stresses revealed that all of these genes had altered expression under various stresses. Some genes exhibited similar expression profiles. To identify the reasons for their similar expression pattern, we compared the promoter sequences and putative cis-regulatory elements such as binding sites for ASRTFs and methylation-prone nucleotide repeats. Our analysis indicated that the gene expression and gene regulation mediated by ASRTFs, and DNA methylation have a stochastic correlation

Altered binding affinity of SIX1-Q177R correlates with enhanced WNT5A and WNT pathway effector expression in Wilms tumor.

Wilms tumors present as an amalgam of varying proportions of tissues located within the developing kidney, one being the nephrogenic blastema comprising multipotent nephron progenitor cells (NPCs). The recurring missense mutation Q177R in NPC transcription factors SIX1 and SIX2 is most correlated with tumors of blastemal histology and is significantly associated with relapse. Yet, the transcriptional regulatory consequences of SIX1/2-Q177R that might promote tumor progression and recurrence have not been investigated extensively. Utilizing multiple Wilms tumor transcriptomic datasets, we identified upregulation of the gene encoding non-canonical WNT ligand WNT5A in addition to other WNT pathway effectors in SIX1/2-Q177R mutant tumors. SIX1 ChIP-seq datasets from Wilms tumors revealed shared binding sites for SIX1/SIX1-Q177R within a promoter of WNT5A and at putative distal cis-regulatory elements (CREs). We demonstrate colocalization of SIX1 and WNT5A in Wilms tumor tissue and utilize in vitro assays that support SIX1 and SIX1-Q177R activation of expression from the WNT5A CREs, as well as enhanced binding affinity within the WNT5A promoter that may promote the differential expression of WNT5A and other WNT pathway effectors associated with SIX1-Q177R tumors.

A predominant role of genotypic variation in both expression of sperm competition genes and paternity success in Drosophila melanogaster.

Sperm competition is a crucial aspect of male reproductive success in many species, including Drosophila melanogaster, and seminal fluid proteins (Sfps) can influence sperm competitiveness. However, the combined effect of environmental and genotypic variation on sperm competition gene expression remains poorly understood. Here, we used Drosophila Genetic Reference Panel (DGRP) inbred lines and manipulated developmental population density (i.e. larval density) to test the effects of genotype, environment and genotype-by-environment interactions (GEI) on the expression of the known sperm competition genes Sex Peptide, Acp36DE and CG9997. High larval density resulted in reduced adult body size, but expression of sperm competition genes remained unaffected. Furthermore, we found no significant GEI but genotypic effects in the expression of SP and Acp36DE. Our results also revealed GEI for relative competitive paternity success (second male paternity; P2), with genes' expression positively correlated with P2. Given the effect of genotype on the expression of genes, we conducted a genome-wide association study (GWAS) and identified polymorphisms in putative cis-regulatory elements as predominant factors regulating the expression of SP and Acp36DE. The association of genotypic variation with sperm competition outcomes, and the resilience of sperm competition genes' expression against environmental challenges, demonstrates the importance of genome variation background in reproductive fitness.

Regulatory and coding sequences of TRNP1 co-evolve with brain size and cortical folding in mammals.

Brain size and cortical folding have increased and decreased recurrently during mammalian evolution. Identifying genetic elements whose sequence or functional properties co-evolve with these traits can provide unique information on evolutionary and developmental mechanisms. A good candidate for such a comparative approach is TRNP1, as it controls proliferation of neural progenitors in mice and ferrets. Here, we investigate the contribution of both regulatory and coding sequences of TRNP1 to brain size and cortical folding in over 30 mammals. We find that the rate of TRNP1 protein evolution (ω) significantly correlates with brain size, slightly less with cortical folding and much less with body size. This brain correlation is stronger than for >95% of random control proteins. This co-evolution is likely affecting TRNP1 activity, as we find that TRNP1 from species with larger brains and more cortical folding induce higher proliferation rates in neural stem cells. Furthermore, we compare the activity of putative cis-regulatory elements (CREs) of TRNP1 in a massively parallel reporter assay and identify one CRE that likely co-evolves with cortical folding in Old World monkeys and apes. Our analyses indicate that coding and regulatory changes that increased TRNP1 activity were positively selected either as a cause or a consequence of increases in brain size and cortical folding. They also provide an example how phylogenetic approaches can inform biological mechanisms, especially when combined with molecular phenotypes across several species.

Dynamics of Chromatin Accessibility During Hematopoietic Stem Cell Differentiation Into Progressively Lineage-Committed Progeny.

Epigenetic mechanisms regulate the multilineage differentiation capacity of hematopoietic stem cells (HSCs) into a variety of blood and immune cells. Mapping the chromatin dynamics of functionally defined cell populations will shed mechanistic insight into 2 major, unanswered questions in stem cell biology: how does epigenetic identity contribute to a cell type's lineage potential, and how do cascades of chromatin remodeling dictate ensuing fate decisions? Our recent work revealed evidence of multilineage gene priming in HSCs, where open cis-regulatory elements (CREs) exclusively shared between HSCs and unipotent lineage cells were enriched for DNA binding motifs of known lineage-specific transcription factors. Oligopotent progenitor populations operating between the HSCs and unipotent cells play essential roles in effecting hematopoietic homeostasis. To test the hypothesis that selective HSC-primed lineage-specific CREs remain accessible throughout differentiation, we used ATAC-seq to map the temporal dynamics of chromatin remodeling during progenitor differentiation. We observed epigenetic-driven clustering of oligopotent and unipotent progenitors into distinct erythromyeloid and lymphoid branches, with multipotent HSCs and MPPs associating with the erythromyeloid lineage. We mapped the dynamics of lineage-primed CREs throughout hematopoiesis and identified both unique and shared CREs as potential lineage reinforcement mechanisms at fate branch points. Additionally, quantification of genome-wide peak count and size revealed overall greater chromatin accessibility in HSCs, allowing us to identify HSC-unique peaks as putative regulators of self-renewal and multilineage potential. Finally, CRISPRi-mediated targeting of ATACseq-identified putative CREs in HSCs allowed us to demonstrate the functional role of selective CREs in lineage-specific gene expression. These findings provide insight into the regulation of stem cell multipotency and lineage commitment throughout hematopoiesis and serve as a resource to test functional drivers of hematopoietic lineage fate.

Pig fetal skeletal muscle development is associated with genome-wide DNA hypomethylation and corresponding alterations in transcript and microRNA expression.

Fetal myogenesis represents a critical period of porcine skeletal muscle development and requires coordinated expression of thousands of genes. Epigenetic mechanisms, including DNA methylation, drive transcriptional regulation during development; however, these processes are understudied in developing porcine tissues. We performed bisulfite sequencing to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-days gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45 739 differentially methylated regions (DMRs) between stages, and the majority (N=34 232) were hypomethylated at 70versus 41 dg. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and expression. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We show that developmental DMRs are enriched for GWAS SNPs for muscle- and meat-related traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics of porcine myogenesis and reveal putative cis-regulatory elements governed by epigenetic processes.

Identification of clade-wide putative cis-regulatory elements from conserved non-coding sequences in Cucurbitaceae genomes.

Cis-regulatory elements regulate gene expression and play an essential role in the development and physiology of organisms. Many conserved non-coding sequences (CNSs) function as cis-regulatory elements. They control the development of various lineages. However, predicting clade-wide cis-regulatory elements across several closely related species remains challenging. Based on the relationship between CNSs and cis-regulatory elements, we present a computational approach that predicts the clade-wide putative cis-regulatory elements in 12 Cucurbitaceae genomes. Using 12-way whole-genome alignment, we first obtained 632 112 CNSs in Cucurbitaceae. Next, we identified 16 552 Cucurbitaceae-wide cis-regulatory elements based on collinearity among all 12 Cucurbitaceae plants. Furthermore, we predicted 3271 potential regulatory pairs in the cucumber genome, of which 98 were verified using integrative RNA sequencing and ChIP sequencing datasets from samples collected during various fruit development stages. The CNSs, Cucurbitaceae-wide cis-regulatory elements, and their target genes are accessible at http://cmb.bnu.edu.cn/cisRCNEs_cucurbit/. These elements are valuable resources for functionally annotating CNSs and their regulatory roles in Cucurbitaceae genomes.

TF-Prioritizer: a Java pipeline to prioritize condition-specific transcription factors.

Eukaryotic gene expression is controlled by cis-regulatory elements (CREs), including promoters and enhancers, which are bound by transcription factors (TFs). Differential expression of TFs and their binding affinity at putative CREs determine tissue- and developmental-specific transcriptional activity. Consolidating genomic datasets can offer further insights into the accessibility of CREs, TF activity, and, thus, gene regulation. However, the integration and analysis of multimodal datasets are hampered by considerable technical challenges. While methods for highlighting differential TF activity from combined chromatin state data (e.g., chromatin immunoprecipitation [ChIP], ATAC, or DNase sequencing) and RNA sequencing data exist, they do not offer convenient usability, have limited support for large-scale data processing, and provide only minimal functionality for visually interpreting results. We developed TF-Prioritizer, an automated pipeline that prioritizes condition-specific TFs from multimodal data and generates an interactive web report. We demonstrated its potential by identifying known TFs along with their target genes, as well as previously unreported TFs active in lactating mouse mammary glands. Additionally, we studied a variety of ENCODE datasets for cell lines K562 and MCF-7, including 12 histone modification ChIP sequencing as well as ATAC and DNase sequencing datasets, where we observe and discuss assay-specific differences. TF-Prioritizer accepts ATAC, DNase, or ChIP sequencing and RNA sequencing data as input and identifies TFs with differential activity, thus offering an understanding of genome-wide gene regulation, potential pathogenesis, and therapeutic targets in biomedical research.

Dynamic DNA methylation reveals novel cis-regulatory elements in mouse hematopoiesis

Differentiation of hematopoietic stem and progenitor cells to terminally differentiated immune cells is accompanied by large-scale remodeling of the DNA methylation landscape. Although significant insights into the molecular mechanisms of hematopoietic tissue regeneration were derived from mouse models, profiling of DNA methylation has been hampered by high cost or low resolution using available methods. The recent development of the Infinium Mouse Methylation BeadChip (MMBC) array facilitates methylation profiling of the mouse genome at a single CpG resolution at affordable cost. We extended the RnBeads package to provide a computational framework for the analysis of MMBC data. This framework was applied to a newly generated reference map of mouse hematopoiesis encompassing nine different cell types. Analysis of dynamically regulated CpG sites showed progressive and unidirectional DNA methylation changes from hematopoietic stem and progenitor cells to differentiated hematopoietic cells and allowed the identification of lineage- and cell type-specific DNA methylation programs. Comparison with previously published catalogs of cis-regulatory elements (CREs) revealed 12,856 novel putative CREs that were dynamically regulated by DNA methylation (mdCREs). These mdCREs were predominantly associated with patterns of cell type-specific DNA hypomethylation and could be identified as epigenetic control regions regulating the expression of key hematopoietic genes during differentiation. In summary, we established an analysis pipeline for MMBC data sets and provide a DNA methylation atlas of mouse hematopoiesis. This resource allowed us to identify novel putative CREs involved in hematopoiesis and will serve as a platform to study epigenetic regulation of normal and malignant hematopoiesis.

Bioinformatics analyses of rice calmodulin-lysine N-methyltransferases gene (OsCaM KMT) and characterization of its promoter from Oryza sativa L. sub sp. indica through transgenic approach

• Cloning of OsCaM KMT gene and promoter region from “Swarna”, an indica rice variety. • Presence of OsCAM KMT transcript in meristematic zone of rice plants, with higher expression in root tips suggesting they might be involved in auxin responsiveness. • Promoter analysis of OsCaM KMT::GUS construct depicted strong expression of GUS reporter gene in root tip, lateral roots and young leaf tip region. • Sequencing result indicated single nucleotide difference between the OsCAM KMT CDS from indica and japonica rice variety, resulting in silent mutation. Calmodulin-lysine N-methyltransferases (CaM KMT) belong to a family of protein lysine methyltransferases that catalyzes methylation of non-histone proteins. Lysine present at 116 position in the protein sequence of calmodulin is apparently methylated by CaM KMT in most species, however not characterized in rice. In the present study rice calmodulin-lysine N-methyltransferase ( OsCaM KMT ) gene has been isolated from an indica rice variety ‘Swarna’. Derived amino acid sequences of CaM KMT gene from rice and other plants were used in constructing a phylogenetic tree to show the evolutionary relationship among those species. Motif analysis along with comparative protein modeling was done to find out structural relationships among rice, Arabidopsis, and human CaM KMT. Gene expression analysis revealed differential tissue specific expression of OsCaM KMT . The highest transcript expression was revealed in the root tip followed by flower and new leaf, while, the least expression was found in the old leaf. In silico analysis was performed considering the 2 kilobases (kb) upstream promoter region of OsCaM KMT gene to find out various transcription factor binding sites. Analyses of the putative cis-regulatory elements revealed their involvement in tissue specific and stress dependent responses. For further characterization, the promoter region (2 kb) of OsCaM KMT gene was isolated, cloned, and sequenced. Histochemical GUS detection of transgenic rice depicted strong OsCaM KMT promoter expression in midribs, stomata, and tip of leaf tissues, central cylinder as well as tip parts of the primary root and lateral roots, along with significant expression in pollen grains, ovary, and vascular bundles. OsCaM KMT gene expression was corroborated by the histological GUS detection in OsCaM KMT promoter:: GUS fusion construct. Although several putative microRNAs (miRNAs) has been identified targeting OsCaM KMT coding DNA sequence (CDS) and UTR region, further studies are required to elucidate their tissue-specific regulation.

Genome-wide chromatin accessibility analysis unveils open chromatin convergent evolution during polyploidization in cotton

Allopolyploidization, resulting in divergent genomes in the same cell, is believed to trigger a "genome shock", leading to broad genetic and epigenetic changes. However, little is understood about chromatin and gene-expression dynamics as underlying driving forces during allopolyploidization. Here, we examined the genome-wide DNase I-hypersensitive site (DHS) and its variations in domesticated allotetraploid cotton (Gossypium hirsutum and Gossypium barbadense, AADD) and its extant AA (Gossypium arboreum) and DD (Gossypium raimondii) progenitors. We observed distinct DHS distributions between G. arboreum and G. raimondii. In contrast, the DHSs of the two subgenomes of G. hirsutum and G. barbadense showed a convergent distribution. This convergent distribution of DHS was also present in the wild allotetraploids Gossypium darwinii and G. hirsutum var. yucatanense, but absent from a resynthesized hybrid of G. arboreum and G. raimondii, suggesting that it may be a common feature in polyploids, and not a consequence of domestication after polyploidization. We revealed that putative cis-regulatory elements (CREs) derived from polyploidization-related DHSs were dominated by several families, including Dof, ERF48, and BPC1. Strikingly, 56.6% of polyploidization-related DHSs were derived from transposable elements (TEs). Moreover, we observed positive correlations between DHS accessibility and the histone marks H3K4me3, H3K27me3, H3K36me3, H3K27ac, and H3K9ac, indicating that coordinated interplay among histone modifications, TEs, and CREs drives the DHS landscape dynamics under polyploidization. Collectively, these findings advance our understanding of the regulatory architecture in plants and underscore the complexity of regulome evolution during polyploidization.

Temporal regulation of cold transcriptional response in switchgrass.

Switchgrass low-land ecotypes have significantly higher biomass but lower cold tolerance compared to up-land ecotypes. Understanding the molecular mechanisms underlying cold response, including the ones at transcriptional level, can contribute to improving tolerance of high-yield switchgrass under chilling and freezing environmental conditions. Here, by analyzing an existing switchgrass transcriptome dataset, the temporal cis-regulatory basis of switchgrass transcriptional response to cold is dissected computationally. We found that the number of cold-responsive genes and enriched Gene Ontology terms increased as duration of cold treatment increased from 30 min to 24 hours, suggesting an amplified response/cascading effect in cold-responsive gene expression. To identify genomic sequences likely important for regulating cold response, machine learning models predictive of cold response were established using k-mer sequences enriched in the genic and flanking regions of cold-responsive genes but not non-responsive genes. These k-mers, referred to as putative cis-regulatory elements (pCREs) are likely regulatory sequences of cold response in switchgrass. There are in total 655 pCREs where 54 are important in all cold treatment time points. Consistent with this, eight of 35 known cold-responsive CREs were similar to top-ranked pCREs in the models and only these eight were important for predicting temporal cold response. More importantly, most of the top-ranked pCREs were novel sequences in cold regulation. Our findings suggest additional sequence elements important for cold-responsive regulation previously not known that warrant further studies.

Robust Genetic Analysis of the X-Linked Anophthalmic (Ie) Mouse.

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000–58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism.

Single-Molecule Mapping of Chromatin Accessibility Using NOMe-seq/dSMF.

The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments.

Evolutionary Changes in the Chromatin Landscape Contribute to Reorganization of a Developmental Gene Network During Rapid Life History Evolution in Sea Urchins.

Chromatin configuration is highly dynamic during embryonic development in animals, exerting an important point of control in transcriptional regulation. Yet there exists remarkably little information about the role of evolutionary changes in chromatin configuration to the evolution of gene expression and organismal traits. Genome-wide assays of chromatin configuration, coupled with whole-genome alignments, can help address this gap in knowledge in several ways. In this study we present a comparative analysis of regulatory element sequences and accessibility throughout embryogenesis in three sea urchin species with divergent life histories: a lecithotroph Heliocidaris erythrogramma, a closely related planktotroph H. tuberculata, and a distantly related planktotroph Lytechinus variegatus. We identified distinct epigenetic and mutational signatures of evolutionary modifications to the function of putative cis-regulatory elements in H. erythrogramma that have accumulated nonuniformly throughout the genome, suggesting selection, rather than drift, underlies many modifications associated with the derived life history. Specifically, regulatory elements composing the sea urchin developmental gene regulatory network are enriched for signatures of positive selection and accessibility changes which may function to alter binding affinity and access of developmental transcription factors to these sites. Furthermore, regulatory element changes often correlate with divergent expression patterns of genes involved in cell type specification, morphogenesis, and development of other derived traits, suggesting these evolutionary modifications have been consequential for phenotypic evolution in H. erythrogramma. Collectively, our results demonstrate that selective pressures imposed by changes in developmental life history rapidly reshape the cis-regulatory landscape of core developmental genes to generate novel traits and embryonic programs.

Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks.

Gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed, it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here, we developed an approach for systematic discovery of upstream regulators of critical immune factors-IL2RA, IL-2 and CTLA4-in primary human T cells. Then, we mapped the network of the target genes of these regulators and putative cis-regulatory elements using CRISPR perturbations, RNA-seq and ATAC-seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.

Machine Learning Prediction of Non-Coding Variant Impact in Human Retinal cis-Regulatory Elements.

PurposePrior studies have demonstrated the significance of specific cis-regulatory variants in retinal disease; however, determining the functional impact of regulatory variants remains a major challenge. In this study, we utilized a machine learning approach, trained on epigenomic data from the adult human retina, to systematically quantify the predicted impact of cis-regulatory variants.MethodsWe used human retinal DNA accessibility data (ATAC-seq) to determine a set of 18.9k high-confidence, putative cis-regulatory elements. Eighty percent of these elements were used to train a machine learning model utilizing a gapped k-mer support vector machine–based approach. In silico saturation mutagenesis and variant scoring was applied to predict the functional impact of all potential single nucleotide variants within cis-regulatory elements. Impact scores were tested in a 20% hold-out dataset and compared to allele population frequency, phylogenetic conservation, transcription factor (TF) binding motifs, and existing massively parallel reporter assay data.ResultsWe generated a model that distinguishes between human retinal regulatory elements and negative test sequences with 95% accuracy. Among a hold-out test set of 3.7k human retinal CREs, all possible single nucleotide variants were scored. Variants with negative impact scores correlated with higher phylogenetic conservation of the reference allele, disruption of predicted TF binding motifs, and massively parallel reporter expression.ConclusionsWe demonstrated the utility of human retinal epigenomic data to train a machine learning model for the purpose of predicting the impact of non-coding regulatory sequence variants. Our model accurately scored sequences and predicted putative transcription factor binding motifs. This approach has the potential to expedite the characterization of pathogenic non-coding sequence variants in the context of unexplained retinal disease.Translational RelevanceThis workflow and resulting dataset serve as a promising genomic tool to facilitate the clinical prioritization of functionally disruptive non-coding mutations in the retina.