Structure Of Transcription Factor Research Articles

Structure-based learning to predict and model protein-DNA interactions and transcription-factor co-operativity in cis-regulatory elements.

Transcription factor (TF) binding is a key component of genomic regulation. There are numerous high-throughput experimental methods to characterize TF-DNA binding specificities. Their application, however, is both laborious and expensive, which makes profiling all TFs challenging. For instance, the binding preferences of ∼25% human TFs remain unknown; they neither have been determined experimentally nor inferred computationally. We introduce a structure-based learning approach to predict the binding preferences of TFs and the automated modelling of TF regulatory complexes. We show the advantage of using our approach over the classical nearest-neighbor prediction in the limits of remote homology. Starting from a TF sequence or structure, we predict binding preferences in the form of motifs that are then used to scan a DNA sequence for occurrences. The best matches are either profiled with a binding score or collected for their subsequent modeling into a higher-order regulatory complex with DNA. Co-operativity is modelled by: (i) the co-localization of TFs and (ii) the structural modeling of protein-protein interactions between TFs and with co-factors. We have applied our approach to automatically model the interferon-β enhanceosome and the pioneering complexes of OCT4, SOX2 (or SOX11) and KLF4 with a nucleosome, which are compared with the experimentally known structures.

Cloning, Expression, and Functional Analysis of the MYB Transcription Factor SlMYB86-like in Tomato.

MYB transcription factors (TFs) have been shown to play a key role in plant growth and development and are in response to various types of biotic and abiotic stress. Here, we clarified the structure, expression patterns, and function of a MYB TF, SlMYB86-like (Solyc06g071690) in tomato using an inbred tomato line exhibiting high resistance to bacterial wilt (Hm 2-2 (R)) and one susceptible line (BY 1-2 (S)). The full-length cDNA sequence of this gene was 1226 bp, and the open reading frame was 966 bp, which encoded 321 amino acids; its relative molecular weight was 37.05055 kDa; its theoretical isoelectric point was 7.22; it was a hydrophilic nonsecreted protein; and it had no transmembrane structures. The protein also contains a highly conserved MYB DNA-binding domain and was predicted to be localized to the nucleus. Phylogenetic analysis revealed that SlMYB86-like is closely related to SpMYB86-like in Solanum pennellii and clustered with other members of the family Solanaceae. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of the SlMYB86-like gene was tissue specific and could be induced by Ralstonia solanacearum, salicylic acid, and jasmonic acid. The results of virus-induced gene silencing (VIGS) revealed that SlMYB86-like silencing decreased the resistance of tomato plants to bacterial wilt, suggesting that it positively regulates the resistance of tomatoes to bacterial wilt. Overall, these findings indicate that SlMYB86-like plays a key role in regulating the resistance of tomatoes to bacterial wilt.

Identification and characterization of the Cyamopsis tetragonoloba transcription factor MYC (CtMYC) under drought stress

The MYC transcription factor (TF) has a variety of roles in abiotic stress responses of plants. In the present work, MYC TF named CtMYC (Cymopsis tetragonoloba) from guar plant, which is induced by drought stress, was identified. The mature leaves of guar were employed to detect the full-length CtMYC TF on the 8th day of drought stress. The CtMYC gene showed tissue-specific expression and up regulated under drought stress conditions as compared to the control and maximum expression was observed in mature leaves. Additionally, CtMYC TF was cloned and expressed in E. coli Rosetta cells and CtMYC protein was purified. The circular dichroism (CD) analysis revealed the presence of helical content and beta sheets and in the presence of genomic DNA the conformational changes were observed in secondary structure, which showed DNA binding potential of CtMYC. These results were analyzed by CD and fluorescence studies. In silico studies reveal the presence of conserved bHLH domain and DNA-binding amino acid residues His, Glu and Arg in CtMYC. This is first report on CtMYC TF with DNA binding potential that is responsive to drought. This study provides the structure and characterization of CtMYC TF and DNA binding ability in drought tolerance mechanism in guar.

Rice WRKY13 TF protein binds to motifs in the promoter region to regulate downstream disease resistance-related genes.

In plants, pathogen resistance is brought about by the binding of certain transcription factor (TF) proteins to the cis-elements of certain target genes. These cis-elements are present upstream in the motif of the promoters of each gene. This ensures the binding of a specific TF to a specific promoter, therefore regulating the expression of that gene. Therefore, the study of each promoter sequence of all the rice genes would help identify the target genes of a specific TF. Rice 1kb upstream promoter sequences of 55,986 annotated genes were analyzed using the Perl program algorithm to detect WRKY13 binding motifs (bm). The resulting genes were grouped using Gene Ontology and gene set enrichment analysis. A gene with more than 4 TF bm in their promoter was selected. Ten genes reported to have a role in rice disease resistance were selected for further analysis. Cis-acting regulatory element analysis was carried out to find the cis-elements and confirm the presence of the corresponding motifs in the promoter sequences of these genes. The 3D structure of WRKY13 TF and the corresponding ten genes were built, and the interacting residues were determined. The binding capacity of WRKY13 to the promoter of these selected genes was analyzed using docking studies. WRKY13 was considered for docking analysis based on the prior reports of autoregulation. Molecular dynamic simulations provided more details regarding the interactions. Expression data revealed the expression of the genes that helped provide the mechanism of interaction. Further co-expression network helped to characterize the interaction of these selected disease resistance-related genes with the WRKY13 TF protein. This study suggests downstream target genes that are regulated by the WRKY13 TF. The molecular mechanism involving the gene network regulated by WRKY13 TF in disease resistance against rice fungal pathogens is explored.

Genome-Wide Identification, Evolution and Expression Profile Analysis of NAC Transcription Factor in Simmondsia chinensis.

NAC transcription factors (TFs) are one of the largest plant-specific gene families and play important roles in plant growth, development, and the biotic and abiotic stress response. Although the sequencing of Jojoba (Simmondsia chinensis) has been completed, the genome-wide identification and analysis of its NAC TFs has not been reported. In this study, a total of 57 genes were identified in Jojoba, which were divided into eight groups based on phylogenetic analysis. The genes clustered in the same groups have a similar gene structure and motif distribution. Based on the analysis of cis-elements in NAC TFs, nine cis-acting elements were identified in the promoter region that involved in light response, hormonal response, and stress response. Synteny analysis showed a greater collinearity between Jojoba and V. vinifera than Arabidopsis thaliana. The 24 genes in the Jojoba NAC TFs are derived from fragment replication, which may be the main source of NAC amplification. Gene expression analysis identified seven genes that were highly expressed in seeds. The differential expression analysis of NAC TFs in cotyledon and embryonic axis tissues showed that the expression of 10 genes was up-regulated and 1 gene was down-regulated. This study provides more information on the classification, gene structure, conserved motif, and evolution of NAC TFs in Jojoba, facilitating further exploration of their specific functional analysis in Jojoba seed development.

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF–promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF–promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF–promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF–promoter binding assay, which not only complements currently available approaches for DNA–protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria–host interface.

MYB gene family in the diatom Phaeodactylum tricornutum revealing their potential functions in the adaption to nitrogen deficiency and diurnal cycle.

The MYB transcription factor (TF) family is one of the largest and most important TF families, regulating the growth and response of microalgae to stress. However, the gene structure and characteristics of Phaeodactylum tricornutum MYB TFs, and their functions under nitrogen deficiency, have not been explored yet. To identify all P. tricornutum MYB (PtMYB) genes, the MYB gene family was analyzed at the genome-wide level in this study. A total ofm26 PtMYB genes were identified from the genome of P. tricornutum. These PtMYB genes were divided into 5 subfamilies: 5R-MYB, 4R-MYB, R2R3-MYB, R1R2R3-MYB, and MYB-related proteins. Phylogenetical motif and gene structure analyses of MYB genes indicated that the number and proportion of MYB TFs were species-specific, and MYB genes exhibited a lot of duplication events from microalgae to higher plants. Furthermore, the differentially expressed patterns of all 26 PtMYB TFs implied that PtMYB genes might have functional specificity under nitrogen deficiency. Homology analysis of MYB genes revealed that PtMYB3, PtMYB15, and PtMYB21 might play important roles in the regulation of the diurnal cycle and response to nitrogen stress in P. tricornutum. PtMYB3, PtMYB15, and PtMYB21 genes might be used as potential candidate genes for further studying the regulatory mechanisms of P. tricornutum under nitrogen deficiency. This work provides an important foundation for the future research of the potential functions of PtMYB genes and its diurnal regulatory mechanisms under nitrogen deficiency.

WRKY Transcription Factor Response to High-Temperature Stress.

Plant growth and development are closely related to the environment, and high-temperature stress is an important environmental factor that affects these processes. WRKY transcription factors (TFs) play important roles in plant responses to high-temperature stress. WRKY TFs can bind to the W-box cis-acting elements of target gene promoters, thereby regulating the expression of multiple types of target genes and participating in multiple signaling pathways in plants. A number of studies have shown the important biological functions and working mechanisms of WRKY TFs in plant responses to high temperature. However, there are few reviews that summarize the research progress on this topic. To fully understand the role of WRKY TFs in the response to high temperature, this paper reviews the structure and regulatory mechanism of WRKY TFs, as well as the related signaling pathways that regulate plant growth under high-temperature stress, which have been described in recent years, and this paper provides references for the further exploration of the molecular mechanisms underlying plant tolerance to high temperature.

TF-PROTACs Enable Targeted Degradation of Transcription Factors.

Transcription factors (TFs) represent a major class of therapeutic targets for the treatment of human diseases including cancer. Although the biological functions and even crystal structures of many TFs have been clearly elucidated, there is still no viable approach to target the majority of TFs, thus rendering them undruggable for decades. PROTACs (proteolysis targeting chimeras) emerge as a powerful class of therapeutic modalities, which rely on induced protein-protein interactions between the proteins of interest (POIs) and E3 ubiquitin ligases to aid the degradation of POIs by the ubiquitin-proteasome system (UPS). Here, we report the development of a platform termed TF-PROTAC, which links an DNA oligonucleotide to an E3 ligase ligand via a click reaction, to selectively degrade the TF of interest. The selectivity of these TF-PROTACs depends on the DNA oligonucleotides utilized that can be specific to the TFs of interest. We have developed two series of VHL-based TF-PROTACs, NF-κB-PROTAC (dNF-κB) and E2F-PROTAC (dE2F), which effectively degrade endogenous p65 and E2F1 proteins in cells, respectively, and subsequently display superior antiproliferative effects in cells. Collectively, our results suggest that TF-PROTACs provide a generalizable platform to achieve selective degradation of TFs and a universal strategy for targeting most "undruggable" TFs.

NAC Transcription Factors as Positive or Negative Regulators during Ongoing Battle between Pathogens and Our Food Crops.

The NAC (NAM, ATAF1/2, and CUC2) family of proteins is one of the largest plant-specific transcription factor (TF) families and its members play varied roles in plant growth, development, and stress responses. In recent years, NAC TFs have been demonstrated to participate in crop-pathogen interactions, as positive or negative regulators of the downstream defense-related genes. NAC TFs link signaling pathways between plant hormones, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA), or other signals, such as reactive oxygen species (ROS), to regulate the resistance against pathogens. Remarkably, NAC TFs can also contribute to hypersensitive response and stomatal immunity or can be hijacked as virulence targets of pathogen effectors. Here, we review recent progress in understanding the structure, biological functions and signaling networks of NAC TFs in response to pathogens in several main food crops, such as rice, wheat, barley, and tomato, and explore the directions needed to further elucidate the function and mechanisms of these key signaling molecules.

Genome-Wide Identification of WRKY Genes in Artemisia annua: Characterization of a Putative Ortholog of AtWRKY40.

Artemisia annua L. is well-known as the plant source of artemisinin, a sesquiterpene lactone with effective antimalarial activity. Here, a putative ortholog of the Arabidopsis thaliana WRKY40 transcription factor (TF) was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends in A. annua and named AaWRKY40. A putative nuclear localization domain was identified in silico and experimentally confirmed by using protoplasts of A. annua transiently transformed with AaWRKY40-GFP. A genome-wide analysis identified 122 WRKY genes in A. annua, and a manually curated database was obtained. The deduced proteins were categorized into the major WRKY groups, with group IIa containing eight WRKY members including AaWRKY40. Protein motifs, gene structure, and promoter regions of group IIa WRKY TFs of A. annua were characterized. The promoter region of AaWRKY group IIa genes contained several abiotic stress cis-acting regulatory elements, among which a highly conserved W-box motif was identified. Expression analysis of AaWRKY40 compared to AaWRKY1 in A. annua cell cultures treated with methyl jasmonate known to enhance artemisinin production, suggested a possible involvement of AaWRKY40 in terpenoid metabolism. Further investigation is necessary to study the role of AaWRKY40 and possible interactions with other TFs in A. annua.

Transcription Factor Binding Affinities and DNA Shape Readout.

SummaryAn essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. Here, we use a binding assay we recently established to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, we find that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates our approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.

First report for availability of HRT-like genes in Eragrostis tef and in silico analysis for elucidating their potential functions

Tef [Eragrostis tef (Zucc.) Troetter] is called ‘super grain’ because of its healthy and nutritious seeds along with compatibility to suboptimal environment. Plant Transcription factors (TFs) are key regulators of gene expression which affect transcription through interaction with proteins and DNA binding. Hordeum Repressor Transcription (HRT) are one of TF gene families with too scanty of information on their abundance, distribution, motifs, phylogenetic relationship among plants, promoter and association network analysis on cereals including tef. CoGeBlast, MEME, MEGA X and PLANTCARE were employed accordingly. Two HRT-like TFs (ET) were identified residing on chromosome 5A and 5B, the proteins exhibit CH residue at the C- terminal end and GIY-YIG domain in the N-terminal. The phylogenetic tree obtained depicted orthology in HRT genes in S. bicolor and Z. mays; O. sativa and O. glaberrima, S. italica and S. virdis; and H. vulgare and T. aestivum and homeologous pairs in E. tef. From the promoter analysis of EtHRTs, CRE elements like ABRE, MYBs, G-box, GC-motif, TC-rich repeats, O2-site were identified known to involve in the control of hormones, sugars and abiotic stresses (ABA, GA, zein and α-amylase) confirming the involvement/role of HRT-like TFs in growth, development and stress response of tef. The study contributes for the understanding of structure and function of HRT-like TFs among plants.

Genome-wide analysis of NAC transcription factors and their response to abiotic stress in celery (Apium graveolens L.)

In plants, NAC (NAM, ATAF, CUC) is a class of transcription factors (TFs) involved in growth regulation and is associated with abiotic stress, morphogenesis, and metabolism. Celery (Apium graveolens L.) is an important leafy vegetable and its yield and quality are considerably influenced by environmental factors. Currently, the characterization of NAC genes in celery is still limited. In this study, a total of 111 putative NAC TFs were determined based on the celery transcriptome and genome database. They were divided into 18 subfamilies on the basis of their NAC domain. NAC TFs in celery account for a moderate number compared with other species, similar to that in carrot. Real-time quantitative PCR (RT-qPCR) showed that some AgNAC genes were differentially expressed under adverse conditions (heat, cold, drought, and salt). AgNAC63 (ortholog of ANAC072/RD26) was highly induced by heat, cold, and salt conditions. The expression levels of AgNAC47 in leaf blades were 105.25- and 123.14-fold those of petioles and roots, respectively. AgNAC63 and AgNAC47 showed significant tissue specificity, high expression in leaves, and varying degrees of response under the four treatments. This study provides a basis for the improved investigation of the structure and function of AgNAC TFs and celery stress resistance.

An R2R3-MYB transcription factor, OjMYB1, functions in anthocyanin biosynthesis in Oenanthe javanica.

This study showed that an R2R3-MYB transcription factor, OjMYB1, is involved in anthocyanin biosynthesis and accumulation in Oenanthe javanica. Anthocyanins can be used as safe natural food colorants, obtained from many plants. R2R3-MYB transcription factors (TFs) play important roles in anthocyanins biosynthesis during plant development. Oenanthe javanica is a popular vegetable with high nutritional values and numerous medical functions. O. javanica has purple petioles that are mainly due to anthocyanins accumulation. In the present study, the gene encoding an R2R3-MYB TF, OjMYB1, was isolated from purple O. javanica. Sequencing results showed that OjMYB1 contained a 912-bp open reading frame encoding 303 amino acids. Sequence alignments revealed that OjMYB1 contained bHLH-interaction motif ([DE]Lx2[RK]x3Lx6Lx3R) and ANDV motif ([A/G]NDV). Phylogenetic analysis indicated that the OjMYB1 classified into the anthocyanins biosynthesis clade. Subcellular localization assay showed that OjMYB1 was a nuclear protein in vivo. The heterologous expression of OjMYB1 in Arabidopsis could enhance the anthocyanins content and up-regulate the expression levels of the structural genes-related anthocyanins biosynthesis. Yeast two-hybrid assay indicated that OjMYB1 could interact with AtTT8 and AtEGL3 proteins. Enzymatic analysis revealed that overexpression of OjMYB1 gene up-regulated the enzyme activity of 3-O-glycosyltransferase encoded by AtUGT78D2 in transgenic Arabidopsis. Our results provided a comprehensive understanding of the structure and function of OjMYB1 TF in O. javanica.

Structural modelling and molecular dynamics of a multi-stress responsive WRKY TF-DNA complex towards elucidating its role in stress signalling mechanisms in chickpea

Chickpea is a premier food legume crop with high nutritional quality and attains prime importance in the current era of 795 million people being undernourished worldwide. Chickpea production encounters setbacks due to various stresses and understanding the role of key transcription factors (TFs) involved in multiple stresses becomes inevitable. We have recently identified a multi-stress responsive WRKY TF in chickpea. The present study was conducted to predict the structure of WRKY TF to identify the DNA-interacting residues and decipher DNA-protein interactions. Comparative modelling approach produced 3D model of the WRKY TF with good stereochemistry, local/global quality and further revealed W19, R20, K21, and Y22 motifs within a vicinity of 5 Å to the DNA amongst R18, G23, Q24, K25, Y36, Y37, R38 and K47 and these positions were equivalent to the 2LEX WRKY domain of Arabidopsis. Molecular simulations analysis of reference protein -PDB ID 2LEX, along with Car-WRKY TF modelled structure with the DNA coordinates derived from PDB ID 2LEX and docked using HADDOCK were executed. Root Mean Square (RMS) Deviation and RMS Fluctuation values yielded consistently stable trajectories over 50 ns simulation. Strengthening the obtained results, neither radius of gyration, distance and total energy showed any signs of DNA-WRKY complex falling apart nor any significant dissociation event over 50 ns run. Therefore, the study provides first insights into the structural properties of multi-stress responsive WRKY TF-DNA complex in chickpea, enabling genome wide identification of TF binding sites and thereby deciphers their gene regulatory networks.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

NanoScript: A Versatile Nanoparticle-Based Synthetic Transcription Factor for Innovative Gene Manipulation.

Cellular reprogramming and stem cell-based therapies have shown tremendous potential in the field of regenerative medicine. To that end, developing tools to control stem cell fate is an attractive area of research for replacing damaged and diseased cells and reestablishing functional connections for tissue repair. Transcription factor (TFs) proteins are well known to regulate gene expression and direct stem cell fate. Inspired by natural TFs, NanoScript, a nanoparticle (NP)-based platform, mimics TFs to afford control over gene expression and stem cell fate for regenerative medicine. Here, we describe the construction of the NanoScript platform, which is designed with tunable properties to replicate the structure and function of TFs to bind to specific portions of the genome and regulate gene expression in a way that does not involve viral delivery.

Genome-wide analysis of PHD family transcription factors in carrot (Daucus carota L.) reveals evolution and response to abiotic stress

The plant homeodomain (PHD)-finger proteins are universally found in eukaryotes. In animals, the structure and function of PHD-finger proteins have been well studied. The PHD family is one of the important transcription factor (TF) families that have crucial roles in different biological processes in plants. In this study, 106 putative TFs were identified from carrot based on the carrot genomic and transcriptomic sequences. According to the phylogenetic comparisons in Arabidopsis and Populus trichocarpa, the PHD proteins were distinguished into 11 groups (A–J, L). The amino acid sequence, phylogenetic tree, physical characterizations, and conserved motifs of the PHD family TFs were predicted and analyzed. The analysis on the PHD family TFs in different species revealed that the number of PHD TFs in different species had a close relationship with the evolution of plant. The expression profiles of the four selected DcPHD genes were detected in leaf, root, and stem of carrot. Gene expression appeared to be tissue-specific in carrot. To analyze the response of DcPHD genes to abiotic stress, the expression profiles of the four DcPHD genes that were selected from the subgroup I were also detected through quantitative real time-PCR under abiotic stress treatments. In carrot, the expression levels of some genes were up-regulated, whereas the others were down-regulated, such as DcPHD-I-15 under heat stress treatment at 1 h. Results show that different DcPHD genes had different relative expression levels in the same condition. In this study, we mainly introduced the progress in the research on the structure and function of PHD TFs in carrot. This study may offer rich information for in depth research on PHD factors, as well as becoming a useful reference for PHD evolutionary relationship in plants. Results provide novel insights into the stress responses of DcPHD genes and promote a better understanding of the construction and function of this gene in carrot.

Evolving insights on how cytosine methylation affects protein-DNA binding.

Many anecdotal observations exist of a regulatory effect of DNA methylation on gene expression. However, in general, the underlying mechanisms of this effect are poorly understood. In this review, we summarize what is currently known about how this important, but mysterious, epigenetic mark impacts cellular functions. Cytosine methylation can abrogate or enhance interactions with DNA-binding proteins, or it may have no effect, depending on the context. Despite being only a small chemical change, the addition of a methyl group to cytosine can affect base readout via hydrophobic contacts in the major groove and shape readout via electrostatic contacts in the minor groove. We discuss the recent discovery that CpG methylation increases DNase I cleavage at adjacent positions by an order of magnitude through altering the local 3D DNA shape and the possible implications of this structural insight for understanding the methylation sensitivity of transcription factors (TFs). Additionally, 5-methylcytosines change the stability of nucleosomes and, thus, affect the local chromatin structure and access of TFs to genomic DNA. Given these complexities, it seems unlikely that the influence of DNA methylation on protein–DNA binding can be captured in a small set of general rules. Hence, data-driven approaches may be essential to gain a better understanding of these mechanisms.