Cis-acting Regulatory Elements Research Articles

Ectopic enhancer-enhancer interactions as causal forces driving RNA-directed DNA methylation in gene regulatory regions.

Cis-regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower-specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non-coding RNAs (ncRNAs), which were processed into 24-nt small interfering RNAs (siRNAs) that drove RNA-directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis-activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE-CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable.

Genome-wide identification of shaker K+ channel gene family in sugar beet (Beta vulgaris L.) and function of BvSKOR in response to salt and drought stresses

Potassium (K+) is the most abundant cation in plants, which is absorbed by roots and distributed throughout the plants and within plant cells, and is involved in various cellular processes. Shaker K+ channel plays crucial roles in the absorption and distribution of K+ and in the response to abiotic stress in plants. Herein, a total of six shaker K+ channel genes, BvKAT1, BvKAT3, BvAKT1, BvAKT2, BvAKT5, and BvSKOR, were identified in the genome of sugar beet (Beta vulgaris L.). The coding domain sequences (CDS) of these genes ranged from 2232 to 2739bp, and protein lengths were varied from 743 to 912 aa. The shaker K+ channel genes contained hormone-related and light responsiveness cis-acting regulatory elements. The phylogenetic analysis showed that BvSKOR was highly conserved and contained six transmembrane structures. The expression patterns of BvSKOR under salt and osmotic stress were analyzed by qRT-PCR, and found that the expression level of BvSKOR under low concentration salt and osmotic stress at short period of treatment were significantly higher than that of the control group. The function of BvSKOR was further verified in tobacco (Nicotiana tabacum), and the results showed that under salt and osmotic stress, the roots of transgenic plants were significantly stronger than those of wild type (WT) plants, and the relative water content (RWC), chlorophyll, proline, soluble sugar, soluble proteins contents and antioxidant enzyme activity were significantly higher than those of WT plants. These results indicated that overexpression of BvSKOR can significantly enhance the salt and drought tolerance in transgenic tobacco plants. This study could provide theoretical support and genetic resources for genetic improvement of crops stress resistance.

Genome-Wide Identification, Characterization and Expression Patterns of the DBB Transcription Factor Family Genes in Wheat.

Double B-box (DBB) proteins are plant-specific transcription factors (TFs) that play crucial roles in plant growth and stress responses. This study investigated the classification, structure, conserved motifs, chromosomal locations, cis-elements, duplication events, expression levels, and protein interaction network of the DBB TF family genes in common wheat (Triticum aestivum L.). In all, twenty-seven wheat DBB genes (TaDBBs) with two conserved B-box domains were identified and classified into six subgroups based on sequence features. A collinearity analysis of the DBB family genes among wheat, Arabidopsis, and rice revealed some duplicated gene pairs and highly conserved genes in wheat. An expression pattern analysis indicated that wheat TaDBBs were involved in plant growth, responses to drought stress, light/dark, and abscisic acid treatment. A large number of cis-acting regulatory elements related to light response are enriched in the predicted promoter regions of 27 TaDBBs. Furthermore, some of TaDBBs can interact with COP1 or HY5 based on the STRING database prediction and yeast two-hybrid (Y2H) assay, indicating the potential key roles of TaDBBs in the light signaling pathway. Conclusively, our study revealed the potential functions and regulatory mechanisms of TaDBBs in plant growth and development under drought stress, light, and abscisic acid.

Machine-guided design of cell-type-targeting cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body1-3. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs4-8. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.

A Soybean Pyrroline-5-Carboxylate Dehydrogenase GmP5CDH1 Modulates Plant Growth and Proline Sensitivity

Soybean [Glycine max (L.) Merr.], as a globally commercialized crop, is an important source of protein and oil for both humans and livestock. With more frequent extreme weather disasters, abiotic stress has become one of the critical factors restricting soybean production. Proline (Pro) is a well-known substance in plants that responds to abiotic stress. To identify potential effector genes involved in soybean resistance to abiotic stress, we focused on the pyrroline-5-carboxylate dehydrogenase (P5CDH) which is a key enzyme in the degradation process of Pro. Through homologous sequence alignment, phylogenetic tree, and predicted expression, we chose GmP5CDH1 (Glyma.05G029200) for further research. Tissue-specific expression assay showed that GmP5CDH1 had higher expression levels in soybean seed and cotyledon development. Subcellular localization assay revealed that GmP5CDH1 was a nuclear-membrane-localized protein. As the result of the predicted cis-acting regulatory element indicates, the expression level of GmP5CDH1 was induced by low temperature, drought, salt stress, and ABA in soybean. Next, we constructed transgenic Arabidopsis overexpressing GmP5CDH1. The results showed that GmP5CDH1 also strongly responded to exogenous Pro, and overcame the toxicity of abiotic stress on plants by regulating the endogenous concentration of Pro. The interaction between GmP5CDH1 and GmSAM1 was validated through yeast two-hybrid, LUC fluorescence complementary, and BIFC. In conclusion, overexpression of a soybean pyrroline-5-carboxylate dehydrogenase GmP5CDH1 regulates the development of Arabidopsis thaliana by altering proline content dynamically under salt stress, especially improving the growth of plants under exogenous Pro.

Massively parallel characterization of insulator activity across the genome

A key question in regulatory genomics is whether cis-regulatory elements (CREs) are modular elements that can function anywhere in the genome, or whether they are adapted to certain genomic locations. To distinguish between these possibilities we develop MPIRE (Massively Parallel Integrated Regulatory Elements), a technology for recurrently assaying CREs at thousands of defined locations across the genome in parallel. MPIRE allows us to separate the intrinsic activity of CREs from the effects of their genomic environments. We apply MPIRE to assay three insulator sequences at thousands of genomic locations and find that each insulator functions in locations with distinguishable properties. All three insulators can block enhancers, but each insulator blocks specific enhancers at specific locations. However, only ALOXE3 appears to block heterochromatin silencing. We conclude that insulator function is highly context dependent and that MPIRE is a robust method for revealing the context dependencies of CREs.

Genome-wide investigation of caffeic acid O-methyltransferases and expression analysis under different abiotic stresses and anthracnose infection in pepper (Capsicum annuum L.)

ABSTRACT The caffeic acid O-methyl transferases (COMTs) facilitate the biosynthesis of lignin and play important roles in many processes, including growth, development and response to stresses. Pepper (Capsicum annuum L.) is a major horticultural crop with significant nutritional and medicinal values. Nevertheless, a comprehensive assessment of the COMT genes in pepper and their involvement in drought, salt tolerance and anthracnose resistance remains unestablished. In the present study, 80 pepper COMT genes were identified and classified into four major groups based on domain structure and phylogenetics. Additionally, the sequence analysis, gene structures, and duplication events were analysed. The genes were unevenly distributed across 10 chromosomes, and 10 pairs of tandem and 13 pairs of segmental duplicated genes were detected in the COMT gene family. Examination of cis-acting regulatory elements detected the presence of phytohormone-specific, stress-receptive and light responsive elements in the pepper COMT promoters. Moreover, qRT-PCR analysis revealed significant upregulated expression of CanCOMT48 to drought stress, CanCOMT14 to salinity stress and CanCOMT18 to anthracnose infection by Colletotrichum truncatum, suggesting their important functions in different stresses. Our results provide significant insights into the expression and probable function of selective COMTs which could be used as potential candidate genes for pepper tolerance breeding.

Enrichment of Cis-Acting Regulatory Elements in Differentially Methylated Regions Following Lipopolysaccharide Treatment of Bovine Endometrial Epithelial Cells.

Endometritis is an inflammatory disease that negatively influences fertility and is common in milk-producing cows. An in vitro model for bovine endometrial inflammation was used to identify enrichment of cis-acting regulatory elements in differentially methylated regions (DMRs) in the genome of in vitro-cultured primary bovine endometrial epithelial cells (bEECs) before and after treatment with lipopolysaccharide (LPS) from E. coli, a key player in the development of endometritis. The enriched regulatory elements contain binding sites for transcription factors with established roles in inflammation and hypoxia including NFKB and Hif-1α. We further showed co-localization of certain enriched cis-acting regulatory motifs including ARNT, Hif-1α, and NRF1. Our results show an intriguing interplay between increased mRNA levels in LPS-treated bEECs of the mRNAs encoding the key transcription factors such as AHR, EGR2, and STAT1, whose binding sites were enriched in the DMRs. Our results demonstrate an extraordinary cis-regulatory complexity in these DMRs having binding sites for both inflammatory and hypoxia-dependent transcription factors. Obtained data using this in vitro model for bacterial-induced endometrial inflammation have provided valuable information regarding key transcription factors relevant for clinical endometritis in both cattle and humans.

Genome-wide identification and expression analysis of CmoADHs in Cucurbita moschata—Critical role of CmoADH9 in drought tolerance

In plants, alcohol dehydrogenases (ADHs) are involved in stress response, organ development, fruit ripening, and metabolite synthesis. However, little is known regarding ADH-encoding genes (ADHs) in Cucurbita moschata which is usually used as a rootstock for cucumber, melon, watermelon, and other cucurbit crops to resist soil-borne diseases and abiotic stresses. We identified 11 CmoADHs in the C. moschata genome that were unevenly distributed across seven chromosomes. These genes were predicted to encode stable cytoplasmic acidic proteins, sharing a low degree of identity with each other. The genes exhibited different intron–exon structures. Analysis of cis-acting regulatory elements showed that CmoADHs contain environmental stress-, hormone response-, light response-, and development/tissue specificity-related elements in their promoters. Expression pattern analysis revealed that CmoADH2, CmoADH3, CmoADH4, CmoADH9, CmoADH10, and CmoADH11 had the highest expression levels in the roots, which were significantly higher than those in the other tested tissues. These six genes may play important roles in the growth and development of roots, and in related abiotic stress responses. CmoADH1, CmoADH5, CmoADH6, CmoADH7, CmoADH8 had the highest expression in the apical region and could be involved in the differentiation of newly formed tissues. To study the role of CmoADHs in abiotic stress, salt, drought, low temperature, and ethephon treatments were performed. Under drought conditions, CmoADHs showed different expression trends. The expression levels of CmoADH1, CmoADH2, CmoADH3, and CmoADH9 increased significantly and peaked after 1 h of drought treatment, indicating that these four genes are more sensitive to drought stress. Under salt treatment, all CmoADHs showed a significant increase or decrease in expression within 6 h, except for CmoADH5 and CmoADH10, which were insensitive to salt treatment. The expression of most of the CmoADHs was significantly downregulated by low-temperature treatment. Ethephon treatment significantly induced the expression of all the CmoADHs, except CmoADH2, to different degrees within 12 h. CmoADH9 was found to be involved in root growth and drought stress resistance. Identification of these ADH genes can provide useful resources for conferring stress resistance in other economically important crops.

Genome-Wide Identification and Analysis of Maize DnaJ Family Genes in Response to Salt, Heat, and Cold at the Seedling Stage.

DnaJ proteins, also known as HSP40s, play a key role in plant growth and development, and response to environmental stress. However, little comprehensive research has been conducted on the DnaJ gene family in maize. Here, we identify 91 ZmDnaJ genes from maize, which are likely distributed in the chloroplast, nucleus, and cytoplasm. Our analysis revealed that ZmDnaJs were classified into three types, with conserved protein motifs and gene structures within the same type, particularly among members of the same subfamily. Gene duplication events have likely contributed to the expansion of the ZmDnaJ family in maize. Analysis of cis-regulatory elements in ZmDnaJ promoters suggested involvement in stress responses, growth and development, and phytohormone sensitivity in maize. Specifically, four cis-acting regulatory elements associated with stress responses and phytohormone regulation indicated a role in adaptation. RNA-seq analysis showed constitutive expression of most ZmDnaJ genes, some specifically in pollen and endosperm. More importantly, certain genes also responded to salt, heat, and cold stresses, indicating potential interaction between stress regulatory networks. Furthermore, early responses to heat stress varied among five inbred lines, with upregulation of almost tested ZmDnaJ genes in B73 and B104 after 6 h, and fewer genes upregulated in QB1314, MD108, and Zheng58. After 72 h, most ZmDnaJ genes in the heat-sensitive inbred lines (B73 and B104) returned to normal levels, while many genes, including ZmDnaJ55, 79, 88, 90, and 91, remained upregulated in the heat-tolerant inbred lines (QB1314, MD108, and Zheng58) suggesting a synergistic function for prolonged protection against heat stress. In conclusion, our study provides a comprehensive analysis of the ZmDnaJ family in maize and demonstrates a correlation between heat stress tolerance and the regulation of gene expression within this family. These offer a theoretical basis for future functional validation of these genes.

Genome-wide identification and characterization of FORMIN gene family in potato (Solanum tuberosum L.) and their expression profiles in response to drought stress condition.

Formin proteins, characterized by the FH2 domain, are critical in regulating actin-driven cellular processes and cytoskeletal dynamics during abiotic stress. However, no genome-wide analysis of the formin gene family has yet to be conducted in the economically significant plant potato (Solanum tuberosum L.). In this study, 26 formin genes were identified and characterized in the potato genome (named as StFH), each containing the typical FH2 domain and distributed across the ten chromosomes. The StFH was categorized into seven subgroups (A-G) and the gene structure and motif analysis demonstrated higher structural similarities within the subgroups. Besides, the StFH exhibited ancestry and functional similarities with Arabidopsis. The Ka/Ks ratio indicated that StFH gene pairs were evolving through purifying selection, with five gene pairs exhibiting segmental duplications and two pairs exhibiting tandem duplications. Subcellular localization analysis suggested that most of the StFH genes were located in the chloroplast and plasma membrane. Moreover, 54 cis-acting regulatory elements (CAREs) were identified in the promoter regions, some of which were associated with stress responses. According to gene ontology analysis, the majority of the StFH genes were involved in biological processes, with 63 out of 74 GO terms affecting actin polymerization. Six major transcription factor families, including bZIP, C2H2, ERF, GATA, LBD, NAC, and HSF, were identified that were involved in the regulation of StFH genes in various abiotic stresses, including drought. Further, the 60 unique microRNAs targeted 24 StFH by regulating gene expression in response to drought stress were identified. The expression of StFH genes in 14 different tissues, particularly in drought-responsive tissues such as root, stem, shoot apex, and leaf, underscores their significance in managing drought stress. RNA-seq analysis of the drought-resistant Qingshu No. 9 variety revealed the potential role of up-regulated genes, including StFH2, StFH10, StFH19, and StFH25, in alleviating drought stress. Overall, these findings provide crucial insights into the response to drought stress in potatoes and can be utilized in breeding programs to develop potato cultivars with enhanced drought-tolerant traits.

Systematic Analysis of the BrHAT Gene Family and Physiological Characteristics of Brassica rapa L. Treated with Histone Acetylase and Deacetylase Inhibitors under Low Temperature.

Brassica rapa L. is an important overwintering oilseed crop in Northwest China. Histone acetyltransferases (HATs) play an important role in epigenetic regulation, as well as the regulation of plant growth, development, and responses to abiotic stresses. To clarify the role of histone acetylation in the low-temperature response of B. rapa L., we identified 29 HAT genes in B. rapa L. using bioinformatics tools. We also conducted a comprehensive analysis of the physicochemical properties, gene structure, chromosomal localization, conserved structural domains and motifs, cis-acting regulatory elements, and evolutionary relationships of these genes. Using transcriptome data, we analyzed the expression patterns of BrHAT family members and predicted interactions between proteins; the results indicated that BrHATs play an important role in the low-temperature response of B. rapa L. HAT inhibitor (curcumin; CUR) and histone deacetylase inhibitor (Trichostatin A; TSA) were applied to four B. rapa L. varieties varying in cold resistance under the same low-temperature conditions, and changes in the physiological indexes of these four varieties were analyzed. The inhibitor treatment attenuated the effect of low temperature on seed germination, and curcumin treatment was most effective, indicating that the germination period was primarily regulated by histone acetylase. Both inhibitor treatments increased the activity of protective enzymes and the content of osmoregulatory substances in plants, suggesting that histone acetylation and deacetylation play a significant role in the response of B. rapa L. to low-temperature stress. The qRT-PCR analyses showed that the expression patterns of BrHATs were altered under different inhibitor treatments and low-temperature stress; meanwhile, we found three significantly differentially expressed genes. In sum, the process of histone acetylation is involved in the cold response and the BrHATs gene plays a role in the cold stress response.

Genome-Wide Analyses of CCHC Family Genes and Their Expression Profiles under Drought Stress in Rose (Rosa chinensis).

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.

Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq

Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.

Genome-wide identification of Ankyrin (ANK) repeat gene families in three Dendrobium species and the expression of ANK genes in D. officinale under gibberellin and abscisic acid treatments

BackgroundDendrobium Sw. represents one of the most expansive genera within the Orchidaceae family, renowned for its species’ high medicinal and ornamental value. In higher plants, the ankyrin (ANK) repeat protein family is characterized by a unique ANK repeat domain, integral to a plethora of biological functions and biochemical activities. The ANK gene family plays a pivotal role in various plant physiological processes, including stress responses, hormone signaling, and growth. Hence, investigating the ANK gene family and identifying disease-resistance genes in Dendrobium is of paramount importance.ResultsThis research identified 78 ANK genes in Dendrobium officinale Kimura et Migo, 77 in Dendrobium nobile Lindl., and 58 in Dendrobium chrysotoxum Lindl. Subsequently, we conducted comprehensive bioinformatics analyses on these ANK gene families, encompassing gene classification, chromosomal localization, phylogenetic relationships, gene structure and motif characterization, cis-acting regulatory element identification, collinearity assessment, protein-protein interaction network construction, and gene expression profiling. Concurrently, three DoANK genes (DoANK14, DoANK19, and DoANK47) in D. officinale were discerned to indirectly activate the NPR1 transcription factor in the ETI system via SA, thereby modulating the expression of the antibacterial PR gene. Hormonal treatments with GA3 and ABA revealed that 17 and 8 genes were significantly up-regulated, while 4 and 8 genes were significantly down-regulated, respectively. DoANK32 was found to localize to the ArfGAP gene in the endocytosis pathway, impacting vesicle transport and the polar movement of auxin.ConclusionOur findings provide a robust framework for the taxonomic classification, evolutionary analysis, and functional prediction of Dendrobium ANK genes. The three highlighted ANK genes (DoANK14, DoANK19, and DoANK47) from D. officinale may prove valuable in disease resistance and stress response research. DoANK32 is implicated in the morphogenesis and development of D. officinale through its role in vesicular transport and auxin polarity, with subcellular localization studies confirming its presence in the nucleus and cell membrane. ANK genes displaying significant expression changes in response to hormonal treatments could play a crucial role in the hormonal response of D. officinale, potentially inhibiting its growth and development through the modulation of plant hormones such as GA3 and ABA.

Identification, Design, and Application of Noncoding Cis-Regulatory Elements.

Cis-regulatory elements (CREs) play a pivotal role in orchestrating interactions with trans-regulatory factors such as transcription factors, RNA-binding proteins, and noncoding RNAs. These interactions are fundamental to the molecular architecture underpinning complex and diverse biological functions in living organisms, facilitating a myriad of sophisticated and dynamic processes. The rapid advancement in the identification and characterization of these regulatory elements has been marked by initiatives such as the Encyclopedia of DNA Elements (ENCODE) project, which represents a significant milestone in the field. Concurrently, the development of CRE detection technologies, exemplified by massively parallel reporter assays, has progressed at an impressive pace, providing powerful tools for CRE discovery. The exponential growth of multimodal functional genomic data has necessitated the application of advanced analytical methods. Deep learning algorithms, particularly large language models, have emerged as invaluable tools for deconstructing the intricate nucleotide sequences governing CRE function. These advancements facilitate precise predictions of CRE activity and enable the de novo design of CREs. A deeper understanding of CRE operational dynamics is crucial for harnessing their versatile regulatory properties. Such insights are instrumental in refining gene therapy techniques, enhancing the efficacy of selective breeding programs, pushing the boundaries of genetic innovation, and opening new possibilities in microbial synthetic biology.

Genome mining of WOX-ARF gene linkage in Machilus pauhoi underpinned cambial activity associated with IAA induction.

As an upright tree with multifunctional economic application, Machilus pauhoi is an excellent choice in modern forestry from Lauraceae. The growth characteristics is of great significance for its molecular breeding and improvement. However, there still lack the information of WUSCHEL-related homeobox (WOX) and Auxin response factor (ARF) gene family, which were reported as specific transcription factors in plant growth as well as auxin signaling. Here, a total of sixteen MpWOX and twenty-one MpARF genes were identified from the genome of M. pauhoi. Though member of WOX conserved in the Lauraceae, MpWOX and MpARF genes were unevenly distributed on 12 chromosomes as a result of region duplication. These genes presented 45 and 142 miRNA editing sites, respectively, reflecting a potential post-transcriptional restrain. Overall, MpWOX4, MpWOX13a, MpWOX13b, MpARF6b, MpARF6c, and MpARF19a were highly co-expressed in the vascular cambium, forming a working mode as WOX-ARF complex. MpWOXs contains typical AuxRR-core and TGA-element cis-acting regulatory elements in this auxin signaling linkage. In addition, under IAA and NPA treatments, MpARF2a and MpWOX1a was highly sensitive to IAA response, showing significant changes after 6 hours of treatment. And MpWOX1a was significantly inhibited by NPA treatment. Through all these solid analysis, our findings provide a genetic foundation to growth mechanism analysis and further molecular designing breeding in Machilus pauhoi.

Symbolic recording of signalling and cis-regulatory element activity to DNA.

Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter1, we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.

Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana

Main conclusionTranscription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease.Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

Interspecies regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes.

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.