Long-read genome assemblies reveal a cis-regulatory landscape associated with phenotypic divergence in two sister Siniperca fish species

Due to the difficulty in accurately identifying structural variants(SVs) across genomes,their impact on cisregulato ry diverge n ce of closely related species,especially fish,remains to be explored.Recently identified broad H3K4me3 domains are essential for the regulation of genes involved in several biological processes.However,the role of broad H3K4me3 domains in phenotypic divergence remains poorly understood.Siniperca chuatsi and S.scherzeri are closely related but divergent in several phenotypic traits,making them an ideal model to study cis-regulatory evolution in sister species.Here,we generated chromosome-level genomes of S.chuatsi and S.scherzeri,with assembled genome sizes of 716.35 and740.54 Mb,respectively.The evolutionary histories of S.chuatsi and S.scherzeri were studied by inferring dynamic changes in ancestral population sizes.To explore the genetic basis of adaptation in S.chuatsi and S.scherzeri,we performed gene family expansion and contraction analysis and identified positively selected genes(PSGs).To investigate the role of SVs in cis-regulatory divergence of closely related fish species,we identified high-quality SVs as well as divergent H3K27ac and H3K4me3 domains in the genomes of S.chuatsi and S.scherzeri.Integrated analysis revealed that cis-regulatory divergence caused by SVs played an essential role in phenotypic divergence between S.chuatsi and S.scherzeri.Additionally,divergent broad H3K4me3 domains were mostly associated with cancer-related genes in S.chuatsi and S.scherzeri and contributed to their phenotypic divergence.

Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation

Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon(-1000 to-1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules(CCRMs), CCRM1(-300 to-101), CCRM2(-650 to-400), and CCRM3(-950 to-750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qP CR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter(-300 to-1), spanning CCRM1 and core region(-100 to-1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering(DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2(-300 to-209) containing Complete HMW Enhancer(-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1(-208 to-101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.

Drought-responsive genes expressed predominantly in root tissues are enriched with homotypic cis-regulatory clusters in promoters of major cereal crops

The root appears to be the most relevant organ for breeding drought stress tolerance.However, our knowledge about temporal and spatial regulation of drought-associated genes in the root remains fragmented, especially in crop plants. We performed a meta-analysis of expression divergence of essential drought-inducible genes and analyzed their association with cis-elements in model crops and major cereal crops. Our analysis of42 selected drought-inducible genes revealed that these are expressed primarily in roots,followed by shoot, leaf, and inflorescence tissues, especially in wheat. Quantitative real-time RT-PCR analysis confirmed higher expression of TaDREB2 and TaAQP7 in roots,correlated with extensive rooting and drought-stress tolerance in wheat. A promoter scan up to 2 kb upstream of the translation start site using phylogenetic footprinting revealed708 transcription factor binding sites, including drought response elements(DREs), auxin response elements(Aux REs), MYCREs/MYBREs, ABAREs, and ERD1 in 19 selected genes.Interestingly, these elements were organized into clusters of overlapping transcription factor binding sites known as homotypic clusters(HCTs), which modulate drought physiology in plants. Taken together, these results revealed the expression preeminence of major drought-inducible genes in the root, suggesting its crucial role in drought adaptation. The occurrence of HCTs in drought-inducible genes highlights the putative evolutionary modifications of crop plants in developing drought adaptation. We propose that these DNA motifs can be used as molecular markers for breeding drought-resilient cultivars, particularly in the cereal crops.

Generating a reporter mouse line marking medium spiny neurons in the developing striatum driven by Arpp21 cis-regulatory elements

The striatum, as the primary input nucleus in the basal ganglion,plays an important role in neural circuits crucial for the control of critical motivation, motor planning and procedural learning(Kreitzer and Malenka, 2008). Most cells in the striatum are GABAergic, including a large population (90%-95%) of medium spiny neurons (MSNs) and a small population of interneurons.

Dynamic chromatin architectures provide insights into the genetics of cattle myogenesis

Background Sharply increased beef consumption is propelling the genetic improvement projects of beef cattle in China.Three-dimensional genome structure is confirmed to be an important layer of transcription regulation.Although genome-wide interaction data of several livestock species have already been produced,the genome structure states and its regulatory rules in cattle muscle are still limited.Results Here we present the first 3D genome data in Longissimus dorsi muscle of fetal and adult cattle(Bos taurus).We showed that compartments,topologically associating domains(TADs),and loop undergo re-organization and the structure dynamics were consistent with transcriptomic divergence during muscle development.Furthermore,we annotated cis-regulatory elements in cattle genome during myogenesis and demonstrated the enrichments of promoter and enhancer in selection sweeps.We further validated the regulatory function of one HMGA2 intronic enhancer near a strong sweep region on primary bovine myoblast proliferation.Conclusions Our data provide key insights of the regulatory function of high order chromatin structure and cattle myogenic biology,which will benefit the progress of genetic improvement of beef cattle.

Allele-specific expression and alternative splicing in horse×donkey and cattle×yak hybrids

Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.

Analysis of Wheat GBSS1 Promoter:Tissue Specificity and DNA Methylation

The cis-regulatory elements of promoters regulate temporal and spatial expression of genes. DNA inethylation, histone methylation and histone acetylation are the main types of epigenetic modifications, which play important roles in plant growth and development. DNA methylation could seilenco transposons, affect gene imprinting and gene expression. In this study, we found that granule bound starch synthase 1 （GBSSI） gene is expressed specifically in wheat endosperm rath- er than in the embryo. We also analyzed the cis-elements within this promoter region and found some seed-specific elements. In order to confirm the tissue specifici- ty, we cloned 4k bp sequences upstream of GBSS1 gene to link to vector with GUS and this construct was transferred to tobacco by Agrobacterium mediated transfor- marion. The results showed that wheat GBSS1 promoter mediated the seed-specific expression of GUS gene, hut not mediated expression in embryo. In addition, we found that GBSSI promoter is methylated in wheat embryo and de-methylated in wheat endosperm. Our study might provide the molecular basis for specific expres- sion of GBSSI gene.

Genetic map construction and functional characterization of genes within the segregation distortion regions(SDRs)in the F_(2:3) populations derived from wild cotton species of the D genome

Background:Segregation distortion(SD)is a common phenomenon among stable or segregating populations,and the principle behind it still puzzles many researchers.The F2:3 progenies developed from the wild cotton species of the D genomes were used to investigate the possible plant transcription factors within the segregation distortion regions(SDRs).A consensus map was developed between two maps from the four D genomes,map A derived from F2:3 progenies of Gossypium klotzschianum and G.davidsonii while Map B from G.thurberi and G.trilobum F2:3 generations.In each map,188 individual plants were used.Results:The consensus linkage map had 1492 markers across the 13 linkage groups with a map size of 1467.445 cM and an average marker distance of 1.0370 cM.Chromosome D502 had the highest percentage of SD with 58.6%,followed by Chromosome D507 with 47.9%.Six thousand and thirty-eight genes were mined within the SDRs on chromosome D502 and D507 of the consensus map.Within chromosome D502 and D507,2308 and 3730 genes were mined,respectively,and were found to belong to 1117 gourp out of which 622 groups were common across the two chromosomes.Moreover,genes within the top 9 groups related to plant resistance genes(R genes),whereas 188 genes encoding protein kinase domain(PF00069)comprised the largest group.Further analysis of the dominant gene group revealed that 287 miRNAs were found to target various genes,such as the gra-miR398,gramiR5207,miR164a,miR164b,miR164c among others,which have been found to target top-ranked stress-responsive transcription factors such as NAC genes.Moreover,some of the stress-responsive cis-regulatory elements were also detected.Furthermore,RNA profiling of the genes from the dominant family showed that higher numbers of genes were highly upregulated under salt and osmotic stress conditions,and also they were highly expressed at different stages of fiber development.Conclusion:The results indicated the critical role of the SDRs in the evolution of the key regulatory genes in plants.

Low-affinity SPL binding sites contribute to subgenome expression divergence in allohexaploid wheat

Expression divergence caused by genetic variation and crosstalks among subgenomes of the allohexaploid bread wheat(Triticum aestivum.L.,BBAADD)is hypothesized to increase its adaptability and/or plasticity.However,the molecular basis of expression divergence remains unclear.Squamosa promoter-binding protein-like(SPL)transcription factors are critical for a wide array of biological processes.In this study,we constructed expression regulatory networks by combining DAP-seq for 40 SPLs,ATACseq,and RNA-seq.Our findings indicate that a group of low-affinity SPL binding regions(SBRs)were targeted by diverse SPLs and caused different sequence preferences around the core GTAC motif.The SBRs including the low-affinity ones are evolutionarily conserved,enriched GWAS signals related to important agricultural traits.However,those SBRs are highly diversified among the cis-regulatory regions(CREs)of syntenic genes,with less than 8%SBRs coexisting in triad genes,suggesting that CRE variations are critical for subgenome differentiations.Knocking out of Ta SPL7A/B/D and Ta SPL15A/B/D subfamily further proved that both high-and low-affinity SBRs played critical roles in the differential expression of genes regulating tiller number and spike sizes.Our results have provided baseline data for downstream networks of SPLs and wheat improvements and revealed that CRE variations are critical sources for subgenome divergence in the allohexaploid wheat.

Developmental regulation of conserved non-coding element evolution provides insights into limb loss in squamates

Limb loss shows recurrent phenotypic evolution across squamate lineages.Here,based on three de novo-assembled genomes of limbless lizards from different lineages,we showed that divergence of conserved non-coding elements(CNEs)played an important role in limb development.These CNEs were associated with genes required for limb initiation and outgrowth,and with regulatory signals in the early stage of limb development.Importantly,we identified the extensive existence of insertions and deletions(In Dels)in the CNEs,with the numbers ranging from 111 to 756.Most of these CNEs with In Dels were lineagespecific in the limbless squamates.Nearby genes of these In Del CNEs were important to early limb formation,such as Tbx4,Fgf10,and Gli3.Based on functional experiments,we found that nucleotide mutations and In Dels both affected the regulatory function of the CNEs.Our study provides molecular evidence underlying limb loss in squamate reptiles from a developmental perspective and sheds light on the importance of regulatory element In Dels in phenotypic evolution.

Variation in cis-regulation of a NAC transcription factor contributes to drought tolerance in wheat

Drought is a major environmental factor limiting wheat production worldwide,and developing drought-tolerant cultivars is a central challenge for wheat breeders globally.Therefore,it is important to identify genetic components determining drought tolerance in wheat.In this study,we identified a wheat NAC gene(TaNAC071-A)that is tightly associated with drought tolerance by a genome-wide association study.Knockdown of TaNAC071-A in wheat attenuated plant drought tolerance,whereas its overexpression significantly enhanced drought tolerance through improved water-use efficiency and increased expression of stress-responsive genes.This heightened water-saving mechanism mitigated the yield loss caused by water deficit.Further candidate gene association analysis showed that a 108-bp insertion in the promoter of TaNAC071-A alters its expression level and contributes to variation in drought tolerance among wheat accessions.This insertion contains two MYB cis-regulatory elements(CREs)that can be directly bound by the MYB transcription activator,TaMYBL1,thereby leading to increased TaNAC071-A expression and plant drought tolerance.Importantly,introgression of this 108-bp insertion allele,TaNAC071-AIn-693,into drought-sensitive cultivars could improve their drought tolerance,demonstrating that it is a valuable genetic resource for wheat breeding.Taken together,our findings highlight a major breakthrough in determining the genetic basis underlying phenotypic variation in wheat drought tolerance and showcase the potential of exploiting CRE-containing indels for improving important agronomical traits.

Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit Development

Development and ripening of tomato fruit are precisely controlled by transcriptional regulation, which de- pends on the orchestrated accessibility of regulatory proteins to promoters and other c/s-regulatory DNA elements. This accessibility and its effect on gene expression play a major role in defining the develop- mental process. To understand the regulatory mechanism and functional elements modulating morpholog- ical and anatomical changes during fruit development, we generated genome-wide high-resolution maps of DNase I hypersensitive sites （DHSs） from the fruit tissues of the tomato cultivar ＂Moneymaker＂ at 20 days post anthesis as well as break stage. By exploring variation of DHSs across fruit development stages, we pinpointed the most likely hypersensitive sites related to development-specific genes. By detecting binding motifs on DHSs of these development-specific genes or genes in the ascorbic acid biosynthetic pathway, we revealed the common regulatory elements contributing to coordinating gene transcription of plant ripening and specialized metabolic pathways. Our results contribute to a better understanding of the regulatory dynamics of genes involved in tomato fruit development and ripening.

Characterization of the Tomato Prosystemin Promoter:Organ-specific Expression,Hormone Specificity and Methyl Jasmonate Responsiveness by Deletion Analysis in Transgenic Tobacco Plants

Tomato systemin is a bioactive peptide that regulates the systemic activation of wound-responsive genes. It is released from its 200 amino acid precursor called prosystemin. Initial tissue-localization and hormone-induced expression assays indicated that the tomato prosystemin gene （SIPS） accumulates mainly in floral tissues and in response to exogenous abscisic acid and methyl jasmonate （MeJA） treatments, respectively. Later, the promoter regions of the PS gene in tomato （Solanum lycopersicum L. cv. Castlemart）, pepper （Capsicum annuum） and potato （Solanum tuberosum） were isolated and an in silico analysis of the SIPS promoter revealed an over-representation of stress- and MeJA-responsive motifs. A subsequent 5＇ deletion analysis of the SIPS promoter fused to the/^-glucuronidase reporter （GUS） gene showed that the -221 to ＋40 bp proximal SIPS promoter region was sufficient to direct the stigma, vascular bundle-specific and MeJA-responsive expression of GUS in transgenic tobacco plants. Important vascular.tissue-specific, light- and MeJA-responsive cis-elements were also present in this region. These findings provide relevant information regarding the transcriptional regulation mechanisms of the SIPS promoter operating in transgenic tobacco plants. They also suggest that its Ussue-specificity and inducible nature could have wide applicability in plant biotechnology.

Distinct Role of Core Promoter Architecture in Regulation of Light-Mediated Responses in Plant Genes

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less （initiator-containing） promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr （Initiator） elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS （transcription start site）, and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

Inducible CRISPRa screen identifies putative enhancers

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates.However,the identification of enhancers in native cellular contexts still remains a challenge.Here,we develop an inducible CRISPR activation(CRISPRa)system by transgenic expression of doxycycline(Dox)-inducible dCas9-VPR in mouse embryonic stem cells(iVPR ESC).With this line,a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible,reversible,and Dox concentration-dependent manner.Taking advantage of this system,we induce tiled CRISPRa across genomic regions(105 kilobases)surrounding T(Brachyury),one of the key mesodermal development regulator genes.Moreover,we identify several CRISPRa-responsive elements with chromatin features of putative enhancers,including a region the homologous sequence in which humans harbors a body height risk variant.Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation.Therefore,our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.

The schizophrenia/bipolar disorder candidate gene GNB1L is regulated in human temporal cortex by a cis-acting element located within the 3'-region

22q 11.2 deletion syndrome（DS） is a complex developmental disorder with a high incidence of psychiatric illnesses,including schizophrenia and mood disorders.Recent studies have identified Guanine Nucleotide Binding Protein（G protein）Beta Polypeptide 1-Like（GNB1L）,located within the 1.5 Mbp 22q11.2 DS critical region,as a candidate liability gene for schizophrenia and bipolar disorder.In this study,we used mRNA expression measurements in Han Chinese postmortem temporal cortex and linkage disequilibrium（LD） analysis to show that GNB1 L is regulated by a cis-acting genetic variant within the 3＇-region of the gene.Significantly,this variant is located within an LD block that contains all of the common SNPs previously shown to associate with schizophrenia and bipolar disorder in Han Chinese and Caucasian populations.Contrary to our expectations,re-analysis of previously published case-control study data in light of our mRNA expression results implies that the GNB1 L highexpression allele is the risk allele for schizophrenia and bipolar disorder in the Han Chinese population.

A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana

The control of gene expression by transcriptional regulators and other types of functional y relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in al organisms. DNA transactions require the control ed interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions （NDRs） of the chromatin. Formaldehyde-assisted isolation of regulatory elements （FAIRE） has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successful y employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol wil be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.

Profiling Chromatin Accessibility at Single-cell Resolution

How distinct transcriptional programs are enacted to generate cellular heterogeneity and plasticity,and enable complex fate decisions are important open questions.One key regulator is the cell’s epigenome state that drives distinct transcriptional programs by regulating chromatin accessibility.Genome-wide chromatin accessibility measurements can impart insights into regulatory sequences(in)accessible to DNA-binding proteins at a single-cell resolution.This review outlines molecular methods and bioinformatic tools for capturing cell-to-cell chromatin variation using single-cell assay for transposase-accessible chromatin using sequencing(scATAC-seq)in a scalable fashion.It also covers joint profiling of chromatin with transcriptome/proteome measurements,computational strategies to integrate multi-omic measurements,and predictive bioinformatic tools to infer chromatin accessibility from single-cell transcriptomic datasets.Methodological refinements that increase power for cell discovery through robust chromatin coverage and integrate measurements from multiple modalities will further expand our understanding of gene regulation during homeostasis and disease.