Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape.

The cistrome is the complete set of transcription factor (TF) binding sites (cis-elements) in an organism, while an epicistrome incorporates tissue-specific DNA chemical modifications and TF-specific chemical sensitivities into these binding profiles. Robust methods to construct comprehensive cistrome and epicistrome maps are critical for elucidating complex transcriptional networks that underlie growth, behavior, and disease. Here, we describe DNA affinity purification sequencing (DAP-seq), a high-throughput TF binding site discovery method that interrogates genomic DNA with in-vitro-expressed TFs. Using DAP-seq, we defined the Arabidopsis cistrome by resolving motifs and peaks for 529 TFs. Because genomic DNA used in DAP-seq retains 5-methylcytosines, we determined that >75% (248/327) of Arabidopsis TFs surveyed were methylation sensitive, a property that strongly impacts the epicistrome landscape. DAP-seq datasets also yielded insight into the biology and binding site architecture of numerous TFs, demonstrating the value of DAP-seq for cost-effective cistromic and epicistromic annotation in any organism.

Determination and inference of eukaryotic transcription factor sequence specificity.

Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ∼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ∼34% of the ∼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif "library" can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

Transcriptional corepressors in maize maintain meristem development.

The formation of the plant body proceeds in a sequential post-embryonic manner through the action of meristems. Tightly coordinated meristem regulation is required for development and reproductive success, eventually determining yield in crop species. In maize (Zea mays), the RAMOSA1 ENHANCER LOCUS2 (REL2) family of transcriptional corepressors includes four members, REL2, RELK1 (REL2-LIKE1), RELK2, and RELK3. In a screen for rel2 enhancers, we identified shorter double mutants with enlarged ear inflorescence meristems (IMs) carrying mutations in RELK1. Expression and genetic analysis indicated that REL2 and RELK1 cooperatively regulate ear IM development by controlling genes involved in redox balance, hormone homeostasis, and differentiation, ultimately tipping the meristem toward an environment favorable to expanded expression of the ZmWUSCHEL1 gene, which encodes a key stem-cell promoting transcription factor. We further demonstrated that RELK genes have partially redundant yet diverse functions in the maintenance of various meristem types during development. By exploiting subtle increases in ear IM size in rel2 heterozygous plants, we also showed that extra rows of kernels are formed across a diverse set of F1 hybrids. Our findings reveal that the REL2 family maintains development from embryonic initiation to reproductive growth and can potentially be harnessed for increasing seed yield in a major crop species.

Necrotic upper tips1 mimics heat and drought stress and encodes a protoxylem-specific transcription factor in maize.

Maintaining sufficient water transport during flowering is essential for proper organ growth, fertilization, and yield. Water deficits that coincide with flowering result in leaf wilting, necrosis, tassel browning, and sterility, a stress condition known as "tassel blasting." We identified a mutant, necrotic upper tips1 (nut1), that mimics tassel blasting and drought stress and reveals the genetic mechanisms underlying these processes. The nut1 phenotype is evident only after the floral transition, and the mutants have difficulty moving water as shown by dye uptake and movement assays. These defects are correlated with reduced protoxylem vessel thickness that indirectly affects metaxylem cell wall integrity and function in the mutant. nut1 is caused by an Ac transposon insertion into the coding region of a unique NAC transcription factor within the VND clade of Arabidopsis NUT1 localizes to the developing protoxylem of root, stem, and leaf sheath, but not metaxylem, and its expression is induced by flowering. NUT1 downstream target genes function in cell wall biosynthesis, apoptosis, and maintenance of xylem cell wall thickness and strength. These results show that maintaining protoxylem vessel integrity during periods of high water movement requires the expression of specialized, dynamically regulated transcription factors within the vasculature.

NEEDLE1 encodes a mitochondria localized ATP-dependent metalloprotease required for thermotolerant maize growth.

Meristems are highly regulated structures ultimately responsible for the formation of branches, lateral organs, and stems, and thus directly affect plant architecture and crop yield. In meristems, genetic networks, hormones, and signaling molecules are tightly integrated to establish robust systems that can adapt growth to continuous inputs from the environment. Here we characterized needle1 (ndl1), a temperature-sensitive maize mutant that displays severe reproductive defects and strong genetic interactions with known mutants affected in the regulation of the plant hormone auxin. NDL1 encodes a mitochondria-localized ATP-dependent metalloprotease belonging to the FILAMENTATION TEMPERATURE-SENSITIVE H (FTSH) family. Together with the hyperaccumulation of reactive oxygen species (ROS), ndl1 inflorescences show up-regulation of a plethora of stress-response genes. We provide evidence that these conditions alter endogenous auxin levels and disrupt primordia initiation in meristems. These findings connect meristem redox status and auxin in the control of maize growth.

VviNAC33 promotes organ de-greening and represses vegetative growth during the vegetative-to-mature phase transition in grapevine.

Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green-to-mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood. We investigated the function of the transcriptional regulator VviNAC33 by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP-seq) approach combined with transcriptomic data. We demonstrated that VviNAC33 induces leaf de-greening, inhibits organ growth and directly activates the expression of STAY-GREEN PROTEIN 1 (SGR1), which is involved in Chl and photosystem degradation, and AUTOPHAGY 8f (ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibits AUXIN EFFLUX FACILITATOR PIN1, RopGEF1 and ATP SYNTHASE GAMMA CHAIN 1T (ATPC1), which are involved in photosystem II integrity and activity. Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative-to-mature phase transition.

OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem.

In rice, the florigens Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), OsFD-like basic leucine zipper (bZIP) transcription factors, and Gf14 proteins assemble into florigen activation/repressor complexes (FACs/FRCs), which regulate transition to flowering in leaves and apical meristem. Only OsFD1 has been described as part of complexes promoting flowering at the meristem, and little is known about the role of other bZIP transcription factors, the combinatorial complexity of FAC formation, and their DNA-binding properties. Here, we used mutant analysis, protein-protein interaction assays and DNA affinity purification (DAP) sequencing coupled to in silico prediction of binding syntaxes to study several bZIP proteins that assemble into FACs or FRCs. We identified OsFD4 as a component of a FAC promoting flowering at the shoot apical meristem, downstream of OsFD1. The osfd4 mutants are late flowering and delay expression of genes promoting inflorescence development. Protein-protein interactions indicate an extensive network of contacts between several bZIPs and Gf14 proteins. Finally, we identified genomic regions bound by bZIPs with promotive and repressive effects on flowering. We conclude that distinct bZIPs orchestrate floral induction at the meristem and that FAC formation is largely combinatorial. While binding to the same consensus motif, their DNA-binding syntax is different, suggesting discriminatory functions.

The FUSED LEAVES1-ADHERENT1 regulatory module is required for maize cuticle development and organ separation.

All aerial epidermal cells in land plants are covered by the cuticle, an extracellular hydrophobic layer that provides protection against abiotic and biotic stresses and prevents organ fusion during development. Genetic and morphological analysis of the classic maize adherent1 (ad1) mutant was combined with genome-wide binding analysis of the maize MYB transcription factor FUSED LEAVES1 (FDL1), coupled with transcriptional profiling of fdl1 mutants. We show that AD1 encodes an epidermally-expressed 3-KETOACYL-CoA SYNTHASE (KCS) belonging to a functionally uncharacterized clade of KCS enzymes involved in cuticular wax biosynthesis. Wax analysis in ad1 mutants indicates that AD1 functions in the formation of very-long-chain wax components. We demonstrate that FDL1 directly binds to CCAACC core motifs present in AD1 regulatory regions to activate its expression. Over 2000 additional target genes of FDL1, including many involved in cuticle formation, drought response and cell wall organization, were also identified. Our results identify a regulatory module of cuticle biosynthesis in maize that is conserved across monocots and eudicots, and highlight previously undescribed factors in lipid metabolism, transport and signaling that coordinate organ development and cuticle formation.

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

RAMOSA1 ENHANCER LOCUS2-Mediated Transcriptional Repression Regulates Vegetative and Reproductive Architecture.

Transcriptional repression in multicellular organisms orchestrates dynamic and precise gene expression changes that enable complex developmental patterns. Here, we present phenotypic and molecular characterization of the maize (Zea mays) transcriptional corepressor RAMOSA1 ENHANCER LOCUS2 (REL2), a unique member of the highly conserved TOPLESS (TPL) family. Analysis of single recessive mutations in rel2 revealed an array of vegetative and reproductive phenotypes, many related to defects in meristem initiation and maintenance. To better understand how REL2-mediated transcriptional complexes relate to rel2 phenotypes, we performed protein interaction assays and transcriptional profiling of mutant inflorescences, leading to the identification of different maize transcription factors and regulatory pathways that employ REL2 repression to control traits directly impacting maize yields. In addition, we used our REL2 interaction data to catalog conserved repression motifs present on REL2 interactors and showed that two of these, RLFGV- and DLN-type motifs, interact with the C-terminal WD40 domain of REL2 rather than the N terminus, which is known to bind LxLxL EAR motifs. These findings establish that the WD40 domain of TPL family proteins is an independent protein interaction surface that may work together with the N-terminal domain to allow the formation of large macromolecular complexes of functionally related transcription factors.

Expanding the Regulatory Network for Meristem Size in Plants.

The remarkable plasticity of post-embryonic plant development is due to groups of stem-cell-containing structures called meristems. In the shoot, meristems continuously produce organs such as leaves, flowers, and stems. Nearly two decades ago the WUSCHEL/CLAVATA (WUS/CLV) negative feedback loop was established as being essential for regulating the size of shoot meristems by maintaining a delicate balance between stem cell proliferation and cell recruitment for the differentiation of lateral primordia. Recent research in various model species (Arabidopsis, tomato, maize, and rice) has led to discoveries of additional components that further refine and improve the current model of meristem regulation, adding new complexity to a vital network for plant growth and productivity.

Mapping transcription factor interactome networks using HaloTag protein arrays.

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

Auxin signaling modules regulate maize inflorescence architecture.

In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.

The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility.

Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.

Diversification and conservation of DNA binding specificities of SPL family of transcription factors.

SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors play vital roles in plant development and stress responses. In this study, we report a comprehensive DNA Affinity Purification sequencing (DAP-seq) analysis for 14 of the 16 SPL transcription factors in Arabidopsis thaliana, providing valuable insights into their DNA-binding specificities. We performed Gene Ontology (GO) analysis of the target genes to reveal their convergent and diverse biological functions among SPL family proteins. Comparative analysis between the paralogs AtSPL9 and AtSPL15 revealed differences in their binding motifs, suggesting divergent regulatory functions. Additionally, we expanded our investigation to homologs of AtSPL9/15 in Zea mays (ZmSBP8/30) and Triticum aestivum (TaSPL7/13), identifying conserved and unique DNA-binding patterns across species. These findings provide key resources for understanding the molecular mechanisms of SPL transcription factors in regulating plant development and evolution across different species.

Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation.

Regulatory elements are important constituents of plant genomes that have shaped ancient and modern crops. Their identification, function, and diversity in crop genomes however are poorly characterized, thus limiting our ability to harness their power for further agricultural advances using induced or natural variation. Here, we use DNA affinity purification-sequencing (DAP-seq) to map transcription factor (TF) binding events for 200 maize TFs belonging to 30 distinct families and heterodimer pairs in two distinct inbred lines historically used for maize hybrid plant production, providing empirical binding site annotation for 5.3% of the maize genome. TF binding site comparison in B73 and Mo17 inbreds reveals widespread differences, driven largely by structural variation, that correlate with gene expression changes. TF binding site presence-absence variation helps clarify complex QTL such as vgt1, an important determinant of maize flowering time, and DICE, a distal enhancer involved in herbivore resistance. Modification of TF binding regions via CRISPR-Cas9 mediated editing alters target gene expression and phenotype. Our functional catalog of maize TF binding events enables collective and comparative TF binding analysis, and highlights its value for agricultural improvement.

Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors.

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis-elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.