Homeotic Gene Regulation Research Articles

Plant BBR/BPC transcription factors: unlocking multilayered regulation in development, stress and immunity.

This review provides a detailed structural and functional understanding of BBR/BPC TF, their conservation across the plant lineage, and their comparative study with animal GAFs. Plant-specific Barley B Recombinant/Basic PentaCysteine (BBR/BPC) transcription factor (TF) family binds to "GA" repeats similar to animal GAGA Factors (GAFs). These GAGA binding proteins are among the few TFs that regulate the genes at multiple steps by modulating the chromatin structure. The hallmark of the BBR/BPC TF family is the presence of a conserved C-terminal region with five cysteine residues. In this review, we present: first, the structural distinct yet functional similar relation of plant BBR/BPC TF with animal GAFs, second, the conservation of BBR/BPC across the plant lineage, third, their role in planta, fourth, their potential interacting partners and structural insights. We conclude that BBR/BPC TFs have multifaceted roles in plants. Besides the earliest identified function in homeotic gene regulation and developmental processes, presently BBR/BPCTFs were identified in hormone signaling, stress, circadian oscillation, and sex determination processes. Understanding how plants' development and stress processes are coordinated is central to divulging the growth-immunity trade-off regulation. The BBR/BPC TFs may hold keys to divulge the interactions between development and immunity. Moreover, the conservation of BBR/BPC across plant lineage makes it an evolutionary vital gene family. Consequently, BBR/BPCs are prospective to attract the increasing attention of the scientific communities as they are probably at the crossroads of diverse fundamental processes.

Characterizations of a Class-I BASIC PENTACYSTEINE Gene Reveal Conserved Roles in the Transcriptional Repression of Genes Involved in Seed Development.

The developmental regulation of flower organs involves the spatio-temporal regulation of floral homeotic genes. BASIC PENTACYSTEINE genes are plant-specific transcription factors that is involved in many aspects of plant development through gene transcriptional regulation. Although studies have shown that the BPC genes are involved in the developmental regulation of flower organs, little is known about their role in the formation of double-flower due. Here we characterized a Class I BPC gene (CjBPC1) from an ornamental flower—Camellia japonica. We showed that CjBPC1 is highly expressed in the central whorls of flowers in both single and doubled varieties. Overexpression of CjBPC1 in Arabidopsis thaliana caused severe defects in siliques and seeds. We found that genes involved in ovule and seed development, including SEEDSTICK, LEAFY COTYLEDON2, ABSCISIC ACID INSENSITIVE 3 and FUSCA3, were significantly down-regulated in transgenic lines. We showed that the histone 3 lysine 27 methylation levels of these downstream genes were enhanced in the transgenic plants, indicating conserved roles of CjBPC1 in recruiting the Polycomb Repression Complex for gene suppression.

Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, it was found that null mutations in the dCTCF gene died as pharate adults, which failed to eclose from their pupal case, or shortly after hatching of adults. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears is limited mainly to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not important for the functional activity of dCTCF in vivo.

Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing.

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.

The regulatory function of dIno80 correlates with its DNA binding activity

The INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. The Drosophila homologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.

Evolution: Remodelling Animal Body Plans, Gene by Gene

Changes in Homeotic (Hox) gene regulation have long been thought to drive the evolution of animal body plans. Direct genetic evidence of their evolutionary role has, however, remained limited. A new study reveals how several mutations distributed across a gene network mask the phenotypic effects of a Hox gene's evolution.

Phylogenetic Analyses and GAGA-Motif Binding Studies of BBR/BPC Proteins Lend to Clues in GAGA-Motif Recognition and a Regulatory Role in Brassinosteroid Signaling.

Plant GAGA-motif binding factors are encoded by the BARLEY B RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family, which fulfill indispensable functions in growth and development. BBR/BPC proteins control flower development, size of the stem cell niche and seed development through transcriptional regulation of homeotic transcription factor genes. They are responsible for the context dependent recruitment of Polycomb repressive complexes (PRC) or other repressive proteins to GAGA-motifs, which are contained in Polycomb repressive DNA-elements (PREs). Hallmark of the protein family is the highly conserved BPC domain, which is required for DNA binding. Here we study the evolution and diversification of the BBR/BPC family and its DNA-binding domain. Our analyses supports a further division of the family into four main groups (I–IV) and several subgroups, to resolve a strict monophyletic descent of the BPC domain. We prove a polyphyletic origin for group III proteins, which evolved from group I and II members through extensive loss of domains in the N-terminus. Conserved motif searches lend to the identification of a WAR/KHGTN consensus and a TIR/K motif at the very C-terminus of the BPC-domain. We could show by DPI-ELISA that this signature is required for DNA-binding in AtBPC1. Additional binding studies with AtBPC1, AtBPC6 and mutated oligonucleotides consolidated the binding to GAGA tetramers. To validate these findings, we used previously published ChIP-seq data from GFP-BPC6. We uncovered that many genes of the brassinosteroid signaling pathway are targeted by AtBPC6. Consistently, bpc6, bpc4 bpc6, and lhp1 bpc4 bpc4 mutants display brassinosteroid-dependent root growth phenotypes. Both, a function in brassinosteroid signaling and our phylogenetic data supports a link between BBR/BPC diversification in the land plant lineage and the complexity of flower and seed plant evolution.

Arabidopsis HUA ENHANCER 4 delays flowering by upregulating the MADS-box repressor genes FLC and MAF4

The adaptive success of flowering plants is largely due to their ability to align floral production with optimal conditions. In Arabidopsis thaliana, MADS-box repressors of the FLC/MAF-clade prevent flowering under non-inductive conditions, although the role of some members is not yet clearly defined. Using a genetic strategy, we identified the KH-domain gene HEN4, previously shown to be involved in MADS-box floral homeotic gene regulation, as a modulator of flowering time. Loss-of-function hen4 mutants are early-flowering, and their response to low growth-temperature (16 °C) and day-length is altered. Interestingly, hen4 plants showed dramatic reduction of FLC and MAF4 transcripts, whereas other flowering repressors of the same clade (FLM, MAF2, MAF3, MAF5) remained unaltered. We also determined that hen4, partly due to loss of FLC, accelerates the vegetative phase-change. This report provides insight into flowering time control and highlights the potential of versatile regulators such as HEN4 to coordinate the juvenile-to-adult transition and floral timing.

Isolation and characterisation of oil palm LEAFY transcripts

ABSTRACTLEAFY (LFY) is an important regulator of plant's reproductive system, regulating the transition from vegetative growth to flowering, and also acts as an upstream regulator of floral homeotic MADS-box genes in Arabidopsis. Owing to its importance in flower development, oil palm LFY transcripts were isolated and the expression pattern was characterised to elucidate the role of LFY in oil palm. In addition, the gene expression pattern was analysed to determine a possible correlation of LFY to a type of floral abnormality in clonal palms termed mantling. In this study, three LFY transcripts were isolated from oil palm inflorescence tissue. The isolated cDNAs, OpLFY1, OpLFY2 and its splice variant, OpLFY2v were predicted to encode proteins each consisting of 430, 451 and 181 amino acids, respectively. OpLFY1 and OpLFY2 share 90% and 94% amino acid sequence identity to two different LFYs from Phoenix dactylifera, respectively. OpLFY1 was localised in all floral organ primordia. Quantitative polymerase chain reaction (qPCR) analysis revealed that OpLFY genes are expressed in shoot apical meristem and inflorescence tissues with the highest expression level. The expression of the OpLFY1 gene was significantly lower in stage 1 of inflorescence development in mantled palms compared with that of normal inflorescences, suggesting possible association of the gene with this somaclonal variation.

Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc.

Interaction of the Chromatin Remodeling Protein hINO80 with DNA.

The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity in vitro. Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes.

Tinkering with transcription factor networks for developmental robustness of Ranunculales flowers.

The flowers of core eudicots and monocots are generally determined by the number of floral organs they produce, and their developmental set-up tolerates little change from the bauplan once the floral primordium is initiated. Many species outside the core eudicots and monocots are more plastic in the number of floral organs they produce. For example, the Nymphaeales (water lilies), within the basal angiosperms, arrange their floral organs spirally and show smooth transitions between floral organs, and many Ranunculales (buttercups) produce variable numbers of stamens by adjusting the number of stamen whorls generated from a specialized ring meristem. However, the interactions of regulatory genes governing those processes are unknown. This review provides an overview of the functional analyses of floral homeotic genes carried out in Ranunculales, summarizing knockdown and mutant phenotypes, and protein interactions to identify similarities and differences within the Ranunculales and in comparison with core eudicots. Floral gene regulatory networks in Ranunculales are identified showing intensive re-wiring amongst the floral homeotic genes to allow some degree of plasticity. The 'fading-border' model of floral organ identity evolution is extended by a hypothesis on how developmental plasticity can be achieved by interdependent regulation of floral homeotic genes. One aspect of floral plasticity may be achieved by regulation of the activity of a stamen-generating ring meristem and first ideas on its control are presented. While the amazing conservation of the major floral organ identity programme is being unravelled by analysing floral homeotic gene function and expression, we are only just beginning to understand the evolution of the gene network governing the organ identity genes, e.g. how plasticity can be achieved, and which aspects foster the robustness of the core eudicot floral bauplan.

Expansion of the polycomb system and evolution of complexity

Polycomb group (PcG) proteins regulate and maintain expression pattern of genes set early during development. Although originally isolated as regulators of homeotic genes, PcG members play a key role in epigenetic mechanisms that maintain the expression state of a large number of genes. All members of the two polycomb repressive complexes (PRC1 and PRC2) are conserved during evolution and while invertebrates generally have one gene for each of these, vertebrates have multiple homologues of them. It remains unclear, however, if different vertebrate PcG homologues have distinct or overlapping functions. We have identified and compared the sequence of PcG homologues in various organisms to analyze similarities and differences that shaped the evolutionary history of these proteins. Comparative analysis of the sequences led to the identification of several novel and signature motifs in the vertebrate homologues of these proteins, which can be directly used to pick respective homologues. Our analysis shows that PcG is an ancient gene group dating back to pre-bilaterian origin that has not only been conserved but also expanded during the evolution of complexity. The presence of unique motifs in each paralogue and its conservation for more than 500Ma indicates their functional relevance and probable unique role. Although this does not rule out completely any overlapping function, our finding that these homologues only minimally overlap in their nuclear localization suggests that each PcG homologue has distinct function. We further propose distinct complex formation by the PcG members. Taken together, our studies suggest non-redundant and specific role of multiple homologues of PcG proteins in vertebrates and indicate major expansion event preceded by emergence of vertebrates that contributed as enhanced epigenetic resource to the evolution of complexity.

A Targeted Genetic Modifier Screen Links the SWI2/SNF2 Protein Domino to Growth and Autophagy Genes inDrosophila melanogaster

Targeted genetic studies can facilitate phenotypic analyses and provide important insights into development and other complex processes. The SWI2/SNF2 DNA-dependent ATPase Domino (Dom) of Drosophila melanogaster, a component of the Tip60 acetyltransferase complex, has been associated with a wide spectrum of cellular processes at multiple developmental stages. These include hematopoiesis, cell proliferation, homeotic gene regulation, histone exchange during DNA repair, and Notch signaling. To explore the wider gene network associated with Dom action, we used RNAi directed against domino (dom) to mediate loss-of-function at the wing margin, a tissue that is readily scored for phenotypic changes. Dom RNAi driven through GAL4-UAS elicited dominant wing nicking that responded phenotypically to the dose of dom and other loci known to function with dom. We screened for phenotypic modifiers of this wing phenotype among 2500 transpositions of the EP P element and found both enhancers and suppressors. Several classes of modifier were obtained, including those encoding transcription factors, RNA regulatory proteins, and factors that regulate cell growth, proliferation and autophagy, a lysosomal degradation pathway that affects cell growth under conditions of starvation and stress. Our analysis is consistent with prior studies, suggesting that Dom acts pleiotropically as a positive effector of Notch signaling and a repressor of proliferation. This genetic system should facilitate screens for additional loci associated with Dom function, and complement biochemical approaches to their regulatory activity.

The Arabidopsis thaliana SET-domain-containing protein ASHH1/SDG26 interacts with itself and with distinct histone lysine methyltransferases

Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have central roles in cell proliferation, growth and development. In animals, PcG and trxG proteins form higher order protein complexes that contain SET domain proteins with histone methyltransferase activity, and are responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. However, whether H3K4 methyltransferase complexes exist in Arabidopsis thaliana remains unknown. Here, we make use of the yeast two-hybrid system and the bimolecular fluorescence complementation assay to provide evidence for the self-association of the Arabidopsis thaliana SET-domain-containing protein SET DOMAIN GROUP 26 (SDG26), also known as ABSENT, SMALL, OR HOMEOTIC DISCS 1 HOMOLOG 1 (ASHH1). In addition, we show that the ASHH1 protein associates with SET-domain-containing sequences from two distinct histone lysine methyltransferases, the ARABIDOPSIS HOMOLOG OF TRITHORAX-1 (ATX1) and ASHH2 proteins. Furthermore, after screening a cDNA library we found that ASHH1 interacts with two proteins from the heat shock protein 40kDa (Hsp40/DnaJ) superfamily, thus connecting the epigenetic network with a system sensing external cues. Our findings suggest that trxG complexes in Arabidopsis thaliana could involve different sets of histone lysine methyltransferases, and that these complexes may be engaged in multiple developmental processes in Arabidopsis.

Unique morphological changes in plant pathogenic phytoplasma‐infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ‐specific manner

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.

Chromatin remodeling protein INO80 has a role in regulation of homeotic gene expression in Drosophila

The homologues of yeast INO80 are identified across phyla from Caenorhabditis elegans to human. In Drosophila it has been shown that dINO80 forms a complex with Pleiohomeotic but does not interact with Hox PRE (polycomb responsive element). As some proteins of the INO80 complex are implicated in homeotic gene regulation, we examined if dINO80 is involved in regulation of homeotic genes. We find that dINO80 null mutants generated by imprecise excision of P-element are late embryonic lethals and show homeotic transformation. We detect misexpression of homeotic genes like Sex-comb reduced, Antennapedia, Ultrabithorax and Abdominal-B in dIno80 mutant embryos by immunostaining which is further substantiated by quantitative PCR. Polycomb phenotype in dIno80-Pc is enhanced in double mutants. Concurrently, the localization of dINO80 to sequences upstream of misexpressed genes in vivo shows that dINO80 is involved in homeotic gene regulation and probably through its interactions with PcG-trxG complexes.

Rm62, a DEAD‐box RNA helicase, complexes with DSP1 in Drosophila embryos

Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB-like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD-box RNA helicase, Rm62, as partner of DSP1 in a 250-kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3- to 12-h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes.

Regulation by Polycomb and Trithorax Group Proteins in Arabidopsis

Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have crucial roles in cell proliferation, growth and development. PcG and trxG proteins form higher order protein complexes that contain SET domain proteins, with a histone methyltransferase (HMTase) activity, responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. In recent years, genetic studies along with biochemical and cell biological analyses in Arabidopsis have enabled researchers to begin to understand how PcG and trxG proteins are recruited to chromatin and how they regulate their target genes and to elucidate their functions. This review focuses on the advances in our understanding of the biological roles of PcG and trxG proteins, their molecular mechanisms of action and further examines the role of histone marks in PcG and trxG regulation in Arabidopsis.

RNA-directed DNA methylation induces transcriptional activation in plants

A class-C floral homeotic gene of Petunia, pMADS3, is specifically expressed in the stamen and carpels of developing flowers. We had previously reported the ect-pMADS3 phenomenon in which introduction of a part of the pMADS3 genomic sequence, including intron 2, induces ectopic expression of endogenous pMADS3. Unlike transcriptional or posttranscriptional gene silencing triggered by the introduction of homologous sequences, this observation is unique in that the gene expression is up-regulated. In this study, we demonstrated that the ect-pMADS3 phenomenon is due to transcriptional activation based on RNA-directed DNA methylation (RdDM) occurring in a particular CG in a putative cis-element in pMADS3 intron 2. The CG methylation was maintained over generations, along with pMADS3 ectopic expression, even in the absence of RNA triggers. These results demonstrate a previously undescribed transcriptional regulatory mechanism that could lead to the generation of a transcriptionally active epiallele, thereby contributing to plant evolution. Our results also reveal a putative negative cis-element for organ-specific transcriptional regulation of class-C floral homeotic genes, which could be difficult to identify by other approaches.