WITHDRAWN: NMR structure verifies the eponymous zinc finger domain of transcription factor ZNF750.

This article was initially published in the Journal of Structural Biology, instead of the Journal of Structural Biology: X, due to a publisher error. We regret the inconvenience. The link to the article published in Journal of Structural Biology: X is presented below: https://www.sciencedirect.com/science/article/pii/S2590152423000090. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.

Transcription factor-binding k-mer analysis clarifies the cell type dependency of binding specificities and cis-regulatory SNPs in humans.

BACKGROUND: Transcription factors (TFs) exhibit heterogeneous DNA-binding specificities in individual cells and whole organisms under natural conditions, and de novo motif discovery usually provides multiple motifs, even from a single chromatin immunoprecipitation-sequencing (ChIP-seq) sample. Despite the accumulation of ChIP-seq data and ChIP-seq-derived motifs, the diversity of DNA-binding specificities across different TFs and cell types remains largely unexplored. RESULTS: Here, we applied MOCCS2, our k-mer-based motif discovery method, to a collection of human TF ChIP-seq samples across diverse TFs and cell types, and systematically computed profiles of TF-binding specificity scores for all k-mers. After quality control, we compiled a set of TF-binding specificity score profiles for 2,976 high-quality ChIP-seq samples, comprising 473 TFs and 398 cell types. Using these high-quality samples, we confirmed that the k-mer-based TF-binding specificity profiles reflected TF- or TF-family dependent DNA-binding specificities. We then compared the binding specificity scores of ChIP-seq samples with the same TFs but with different cell type classes and found that half of the analyzed TFs exhibited differences in DNA-binding specificities across cell type classes. Additionally, we devised a method to detect differentially bound k-mers between two ChIP-seq samples and detected k-mers exhibiting statistically significant differences in binding specificity scores. Moreover, we demonstrated that differences in the binding specificity scores between k-mers on the reference and alternative alleles could be used to predict the effect of variants on TF binding, as validated by in vitro and in vivo assay datasets. Finally, we demonstrated that binding specificity score differences can be used to interpret disease-associated non-coding single-nucleotide polymorphisms (SNPs) as TF-affecting SNPs and provide candidates responsible for TFs and cell types. CONCLUSIONS: Our study provides a basis for investigating the regulation of gene expression in a TF-, TF family-, or cell-type-dependent manner. Furthermore, our differential analysis of binding-specificity scores highlights noncoding disease-associated variants in humans.

Atypical aplasia cutis in association with Xia Gibbs syndrome.

Xia Gibbs syndrome is a genetic disorder first defined in 2014 characterized by hypotonia, intellectual disability, global developmental delay, and dysmorphic facial features. While many additional features may be present, there are few reports of dermatologic findings. We report a case of atypical aplasia cutis in a female infant who was found to have Xia Gibbs syndrome. This case highlights consideration of cutaneous manifestations of Xia Gibbs syndrome which may aid in diagnosis.

Structures of the activator of K. pneumonia biofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding.

The pathogenesis of Klebsiella pneumonia is linked to the bacteria's ability to form biofilms. Mannose-resistant Klebsiella-like (Mrk) hemagglutinins are critical for K pneumonia biofilm development, and the expression of the genes encoding these proteins is activated by a 3',5'-cyclic diguanylic acid (c-di-GMP)-regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architecture consisting of a PilZ C-domain connected to an N domain that unexpectedly also harbors a PilZ-like fold. Comparison of apo- and c-di-GMP-bound MrkH structures reveals a large 138° interdomain rotation that is induced by binding an intercalated c-di-GMP dimer. c-di-GMP interacts with PilZ C-domain motifs 1 and 2 (RxxxR and D/NxSxxG) and a newly described c-di-GMP-binding motif in the MrkH N domain. Strikingly, these c-di-GMP-binding motifs also stabilize an open state conformation in apo MrkH via contacts from the PilZ motif 1 to residues in the C-domain motif 2 and the c-di-GMP-binding N-domain motif. Use of the same regions in apo structure stabilization and c-di-GMP interaction allows distinction between the states. Indeed, domain reorientation by c-di-GMP complexation with MrkH, which leads to a highly compacted structure, suggests a mechanism by which the protein is activated to bind DNA. To our knowledge, MrkH represents the first instance of specific DNA binding mediated by PilZ domains. The MrkH structures also pave the way for the rational design of inhibitors that target K pneumonia biofilm formation.

The Arabidopsis WRINKLED1 transcription factor affects auxin homeostasis in roots.

WRINKLED1 (WRI1) is a key transcriptional regulator of fatty acid biosynthesis genes in diverse oil-containing tissues. Loss of function of Arabidopsis WRI1 leads to a reduction in the expression of genes for fatty acid biosynthesis and glycolysis, and concomitant strong reduction of seed oil content. The wri1-1 loss-of-function mutant shows reduced primary root growth and decreased acidification of the growth medium. The content of a conjugated form of the plant growth hormone auxin, indole-3-acetic acid (IAA)-Asp, was higher in wri1-1 plants compared with the wild-type. GH3.3, a gene encoding an enzyme involved in auxin degradation, displayed higher expression in the wri1-1 mutant. EMSAs demonstrated that AtWRI1 bound to the promoter of GH3.3. Specific AtWRI1-binding motifs were identified in the promoter of GH3.3. In addition, wri1-1 displayed decreased auxin transport. Expression of some PIN genes, which encode IAA carrier proteins, was reduced in wri1-1 plants as well. Correspondingly, AtWRI1 bound to the promoter regions of some PIN genes. It is well known that auxin exerts its maximum effects at a specific, optimal concentration in roots requiring a finely balanced auxin homeostasis. This process appears to be disrupted when the expression of WRI1 and in turn a subset of its target genes are misregulated, highlighting a role for WRI1 in root auxin homeostasis.

ChIP-chip data for identifying target genes and consensus binding sequences of mutant p53 in MDA-MB-468 breast cancer cells.

The tumor suppressor p53 exerts its role mainly as a transcription factor. The TP53 gene, which encodes the p53 protein, is the most commonly mutated gene in human cancers, particularly triple negative breast cancer (TNBC). Variations in the TP53 gene occur mainly in exons 5-8 and result in missense mutations in the DNA-binding domain of the p53 protein that alter DNA binding specificity. To identify the target genes of mutant p53, we performed chromatin immunoprecipitation followed by DNA microarray (ChIP-chip). Briefly, the TNBC cell line MDA-MB-468 containing the endogenous p53-R273H mutation (the arginine residue at position 273 is mutated to a histidine) was cross-linked with 1% formaldehyde and ultrasonically sheared to generate chromatin fragments in a range of 200∼1000 bp. An aliquot of the sheared chromatin was kept as input, and the other chromatin was precipitated with a p53 monoclonal antibody. DNA was purified from the precipitated chromatin and the unprecipitated chromatin (i.e., input), amplified, and labeled with Cy5 (ChIP DNA) or Cy3 (input DNA). Cy5- and Cy3-labeled DNA samples were cohybridized with the NimbleGen Human ChIP-chip 2.1 M Deluxe Promoter Array. The raw and analyzed data are described in this article. They are useful for identifying target genes and consensus binding motifs of the p53 R273H mutant and for further clarifying the molecular mechanism underlying the oncogenic activity of the p53 mutant.

Unraveling the Gene Regulatory Networks of the Global Regulators VeA and LaeA in Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, the velvet family protein VeA and the global regulator of secondary metabolism LaeA govern development and secondary metabolism mostly by acting as the VelB/VeA/LaeA heterotrimeric complex. While functions of these highly conserved controllers have been well studied, the genome-wide regulatory networks governing cellular and chemical development remain to be uncovered. Here, by integrating transcriptomic analyses, protein-DNA interactions, and the known A. nidulans gene/protein interaction data, we have unraveled the gene regulatory networks governed by VeA and LaeA. Within the networks, VeA and LaeA directly control the expression of numerous genes involved in asexual/sexual development and primary/secondary metabolism in A. nidulans. Totals of 3,190 and 1,834 potential direct target genes of VeA and LaeA were identified, respectively, including several important developmental and metabolic regulators such as flbA·B·C, velB·C, areA, mpkB, and hogA. Moreover, by analyzing over 8,800 ChIP-seq peaks, we have revealed the predicted common consensus sequences 5'-TGATTGGCTG-3' and 5'-TCACGTGAC-3' that VeA and LaeA might bind to interchangeably. These findings further expand the biochemical and genomic studies of the VelB/VeA/LaeA complex functionality in the gene regulation. In summary, this study unveils genes that are under the regulation of VeA and LaeA, proposes the VeA- and LaeA-mediated gene regulatory networks, and demonstrates their genome-wide developmental and metabolic regulations in A. nidulans. IMPORTANCE Fungal development and metabolism are genetically programmed events involving specialized cellular differentiation, cellular communication, and temporal and spatial regulation of gene expression. In genus Aspergillus, the global regulators VeA and LaeA govern developmental and metabolic processes by affecting the expression of downstream genes, including multiple transcription factors and signaling elements. Due to their vital roles in overall biology, functions of VeA and LaeA have been extensively studied, but there still has been a lack of knowledge about their genome-wide regulatory networks. In this study, employing the model fungus A. nidulans, we have identified direct targets of VeA and LaeA and their gene regulatory networks by integrating transcriptome, protein-DNA interaction, and protein-protein interaction analyses. Our results demonstrate the genome-wide regulatory mechanisms of these global regulators, thereby advancing the knowledge of fungal biology and genetics.

Novel frameshift mutation in the AHDC1 gene in a Chinese global developmental delay patient: A case report.

BACKGROUND: Xia-Gibbs syndrome (XGS, OMIM: 615829), caused by mutations within the AT-Hook DNA-binding motif-containing protein 1 (AHDC1) gene (OMIM: 615790), located on the short arm of chromosome 1 within the cytogenetic band 1p36.11, contains five noncoding 5 exons, a single 4.9-kb coding exon, and a noncoding 3 exon. CASE SUMMARY: In this case report, we diagnosed and treated a 6-mo-old girl with XGS. The primary clinical symptoms included global developmental delay, hypotonia, and mild dysmorphic features. Using high-throughput whole-exosome sequencing to sequence the patient and her parents, and the results showed a novel frameshift mutation of c.1155dupG (p.Arg386Alafs*3) in the AHDC1 gene. The paternal gene was wild type. CONCLUSION: This report extends the mutation spectrum of the AHDC1 gene to provide the diagnostic basis for genetic counseling in families with XGS.

BORIS-mediated generation of circular RNAs induces inflammation.

Circular RNAs (circRNAs), which are more stable than linear mRNAs and long non-coding RNAs (LncRNAs), are detected in body fluids such as plasma, serum, and exosomes. Disease-associated circRNAs have significant clinical roles due to their diagnostic and prognostic values. Brother of regulator of imprinting site (BORIS) promotes cancer progression and is specifically highly expressed in the majority of carcinoma. However, the mechanism underlying the regulation of circRNAs by the oncoprotein BORIS and their role in regulating inflammation and immunity remain to be further explored. Vaccines prepared from circRNAs extracted from cancer cells showed that circRNAs induced inflammation and prevented cancer progression. Serum from animals injected with cancer cell-derived circRNAs vigorously reacted with cells that expressed cancer-specific antigen BORIS or cancer extracted circRNAs. It has been implicated that cancer-related circRNAs could be used as antigens to activate immune responses to prevent cancers and stimulate NF-κB signaling pathway by up-regulating and inducing TLR3. In the study we also found that BORIS regulated the expression of circRNAs and interacted with RNA motifs and the CCCTC binding factor (CTCF) motif adjacent to circRNA splicing sites to enhance the formation of circRNAs. Thus, our study delineated the novel mechanism by which cancer-specific antigen BORIS regulated circRNAs and identified that circRNAs could serve as a vaccine for cancer prevention.

Transcription Factors in the Regulation of Leydig Cell Gene Expression and Function.

Cell differentiation and acquisition of specialized functions are inherent steps in events that lead to normal tissue development and function. These processes require accurate temporal, tissue, and cell-specific activation or repression of gene transcription. This is achieved by complex interactions between transcription factors that form a unique combinatorial code in each specialized cell type and in response to different physiological signals. Transcription factors typically act by binding to short, nucleotide-specific DNA sequences located in the promoter region of target genes. In males, Leydig cells play a crucial role in sex differentiation, health, and reproductive function from embryonic life to adulthood. To better understand the molecular mechanisms regulating Leydig cell differentiation and function, several transcription factors important to Leydig cells have been identified, including some previously unknown to this specialized cell type. This mini review summarizes the current knowledge on transcription factors in fetal and adult Leydig cells, describing their roles and mechanisms of action.

Distinct p53 isoforms code for opposing transcriptional outcomes.

p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. Here, we investigate synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control in vivo. We show that transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

Mapping the DNA-Binding Motif of Scabin Toxin, a Guanine Modifying Enzyme from Streptomyces scabies.

Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rß1-RLa-NLc-STTß2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine and arginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family.

The master regulator MAT1-1-1 of fungal mating binds to its targets via a conserved motif in the human pathogen Aspergillus fumigatus.

Mating-type transcription factors are master regulators of sexually related signal transduction pathways in fungi; however, their recognition of specific DNA sequences from target genes is widely undetermined. Here, we identified and characterized the DNA-binding sequence of the MAT1-1-1 alpha-box domain transcription factor from the human pathogen Aspergillus fumigatus. In order to explore MAT1-1-1 DNA-binding targets, we used the previously reported MAT1-1-1 binding motif from Penicillium chrysogenum, in a bioinformatics approach. We identified 18 A. fumigatus genes carrying the MAT1.1 sequence in their upstream region, among them genes for the α-pheromone precursor (PpgA), G-protein-coupled pheromone receptor (PreA), and for TomA, an unidentified protein. To validate our prediction further, quantification of transcript levels showed a decrease in expression of ppgA, tomA, and others in a MAT1-1 deletion strain. For a functional analysis of the binding sites, truncated variants of the A. fumigatus MAT1-1-1 gene were introduced into Escherichia coli for heterologous expression. The yield of recombinant protein was further optimized for the AfMAT1-1-178-235 variant that harbors an extended alpha-box domain. AfMAT1-1-178-235 bound to a subset of the most strongly upregulated genes: ppgA, preA, and tomA. The DNA-binding specificity was confirmed by testing mutated binding sequences, as well as performing competition experiments with specific and non-specific sequences. Finally, equilibrium dissociation constants of 1.83 ± 0.1 and 1.45 ± 0.26 µM were determined for AfMAT1-1-178-235 and fusion protein GST-AfMAT1-1-178-235. Collectively, these findings provide further insights into AfMAT1-1-1-mediated gene expression and imply that alpha-box domain regulators from other members of Eurotiales control fungal development in a conserved manner.

Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression.

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

RhizoBindingSites, a Database of DNA-Binding Motifs in Nitrogen-Fixing Bacteria Inferred Using a Footprint Discovery Approach.

Basic knowledge of transcriptional regulation is needed to understand the mechanisms governing biological processes, i.e., nitrogen fixation by Rhizobiales bacteria in symbiosis with leguminous plants. The RhizoBindingSites database is a computer-assisted framework providing motif-gene-associated conserved sequences potentially implicated in transcriptional regulation in nine symbiotic species. A dyad analysis algorithm was used to deduce motifs in the upstream regulatory region of orthologous genes, and only motifs also located in the gene seed promoter with a p-value of 1e-4 were accepted. A genomic scan analysis of the upstoream sequences with these motifs was performed. These predicted binding sites were categorized according to low, medium and high homology between the matrix and the upstream regulatory sequence. On average, 62.7% of the genes had a motif, accounting for 80.44% of the genes per genome, with 19613 matrices (a matrix is a representation of a motif). The RhizoBindingSites database provides motif and gene information, motif conservation in the order Rhizobiales, matrices, motif logos, regulatory networks constructed from theoretical or experimental data, a criterion for selecting motifs and a guide for users. The RhizoBindingSites database is freely available online at rhizobindingsites.ccg.unam.mx.

The C-terminal domain of HpDprA is a DNA-binding winged helix domain that does not bind double-stranded DNA.

DNA-processing protein A, a ubiquitous multidomain DNA-binding protein, plays a crucial role during natural transformation in bacteria. Here, we carried out the structural analysis of DprA from the human pathogen Helicobacter pylori by combining data issued from the 1.8-Å resolution X-ray structure of the Pfam02481 domain dimer (RF), the NMR structure of the carboxy terminal domain (CTD), and the low-resolution structure of the full-length DprA dimer obtained in solution by SAXS. In particular, we sought a molecular function for the CTD, a domain that we show here is essential for transformation in H. pylori. Albeit its structural homology to winged helix DNA-binding motifs, we confirmed that the isolated CTD does not interact with ssDNA nor with dsDNA. The key R52 and K137 residues of RF are crucial for these two interactions. Search for sequences harboring homology to either HpDprA or Rhodopseudomonas palustris DprA CTDs led to the identification of conserved patches in the two CTD. Our structural study revealed the similarity of the structures adopted by these residues in RpDprA CTD and HpDprA CTD. This argues for a conserved, but yet to be defined, CTD function, distinct from DNA binding.

A novel DNA-binding motif in prostate tumor overexpressed-1 (PTOV1) required for the expression of ALDH1A1 and CCNG2 in cancer cells.

PTOV1 is a transcription and translation regulator and a promoter of cancer progression. Its overexpression in prostate cancer induces transcription of drug resistance and self-renewal genes, and docetaxel resistance. Here we studied PTOV1 ability to directly activate the transcription of ALDH1A1 and CCNG2 by binding to specific promoter sequences. Chromatin immunoprecipitation and electrophoretic mobility shift assays identified a DNA-binding motif inside the PTOV-A domain with similarities to known AT-hooks that specifically interacts with ALDH1A1 and CCNG2 promoters. Mutation of this AT-hook-like sequence significantly decreased the expression of ALDH1A1 and CCNG2 promoted by PTOV1. Immunohistochemistry revealed the association of PTOV1 with mitotic chromosomes in high grade prostate, colon, bladder, and breast carcinomas. Overexpression of PTOV1, ALDH1A1, and CCNG2 significantly correlated with poor prognosis in prostate carcinomas and with shorter relapse-free survival in colon carcinoma. The previously described interaction with translation complexes and its direct binding to ALDH1A1 and CCNG2 promoters found here reveal the PTOV1 capacity to modulate the expression of critical genes at multiple levels in aggressive cancers. Remarkably, the AT-hook motifs in PTOV1 open possibilities for selective targeting its nuclear and/or cytoplasmic activities.

Conservation of DNA and ligand binding properties of retinoid X receptor from the placozoan Trichoplax adhaerens to human.

Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXRɑ. The binding sites for these two RXR proteins were broadly conserved (∼85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution.

Comparison of ChIP-Seq Data and a Reference Motif Set for Human KRAB C2H2 Zinc Finger Proteins.

KRAB C2H2 zinc finger proteins (KZNFs) are the largest and most diverse family of human transcription factors, likely due to diversifying selection driven by novel endogenous retroelements (EREs), but the vast majority lack binding motifs or functional data. Two recent studies analyzed a majority of the human KZNFs using either ChIP-seq (60 proteins) or ChIP-exo (221 proteins) in the same cell type (HEK293). The ChIP-exo paper did not describe binding motifs, however. Thirty-nine proteins are represented in both studies, enabling the systematic comparison of the data sets presented here. Typically, only a minority of peaks overlap, but the two studies nonetheless display significant similarity in ERE binding for 32/39, and yield highly similar DNA binding motifs for 23 and related motifs for 34 (MoSBAT similarity score >0.5 and >0.2, respectively). Thus, there is overall (albeit imperfect) agreement between the two studies. For the 242 proteins represented in at least one study, we selected a highest-confidence motif for each protein, utilizing several motif-derivation approaches, and evaluating motifs within and across data sets. Peaks for the majority (158) are enriched (96% with AUC >0.6 predicting peak vs. nonpeak) for a motif that is supported by the C2H2 "recognition code," consistent with intrinsic sequence specificity driving DNA binding in cells. An additional 63 yield motifs enriched in peaks, but not supported by the recognition code, which could reflect indirect binding. Altogether, these analyses validate both data sets, and provide a reference motif set with associated quality metrics.

Genome-wide identification of grain filling genes regulated by the OsSMF1 transcription factor in rice.

BACKGROUND: Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by gene promoters. Therefore, it is important to identify the binding motifs of transcription factors to better understand the networks associated with embryogenesis. RESULTS: Here, we used a protein-binding microarray (PBM) to identify the binding motifs of OsSMF1, which is a basic leucine zipper transcription factor involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1 or OsbZIP58) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage protein synthesis, and it functions as a key regulator of starch synthesis. Quadruple 9-mer-based PBM analysis and electrophoretic mobility shift assay revealed that OsSMF1 bound to the GCN4 (TGA(G/C)TCA), ACGT (CCACGT(C/G)), and ATGA (GGATGAC) motifs with three different affinities. We predicted 44 putative OsSMF1 target genes using data obtained from both the PBM and RiceArrayNet. Among these putative target genes, 18, 21, and 13 genes contained GCN4, ACGT, and ATGA motifs within their 1-kb promoter regions, respectively. Among them, six genes encoding major grain filling proteins and transcription factors were chosen to confirm the activation of their expression in vivo. OsSMF1 was shown to bind directly to the promoters of Os03g0168500 (GCN4 motif), patatin-like gene (GCN4 motif), α-globulin (ACGT motif), rice prolamin box-binding factor (RPBF) (ATGA motif), and ONAC024 (GCN4 and ACGT motifs) and to regulate their expression. CONCLUSIONS: The results of this study suggest that OsSMF1 is one of the key transcription factors that functions in a wide range of seed developmental processes with different specific binding affinities for the three DNA-binding motifs.