Jose M. Muiño

Orchestration of Floral Initiation by APETALA1

Flower Power The transcription factor APETALA1 (AP1) controls the transition from vegetative growth to flower production in the plant Arabidopsis. A handful of factors that control AP1 have been identified, as well as some targets that AP1 controls. Kaufmann et al. (p. 85) now apply genome-wide microarray analysis to identify over a thousand genes whose transcription is regulated by AP1. By proximity to AP1 binding sites, over two thousand genes are implicated as putative AP1 targets. Analysis of this network of interactions indicates that AP1 functions first to repress vegetative identity, then to help establish floral primordia, and finally to shape the differentiation of floral parts. The master transcription factor APETALA1 dynamically regulates a complex genetic network to guide flower development. The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. Although AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning, and hormonal pathways.

Target Genes of the MADS Transcription Factor SEPALLATA3: Integration of Developmental and Hormonal Pathways in the Arabidopsis Flower

The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals, and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs.

Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development

Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the “floral quartet” model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.

Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP)

Chromatin immunoprecipitation (ChIP) is a powerful technique to study interactions between transcription factors (TFs) and DNA in vivo. For genome-wide de novo discovery of TF-binding sites, the DNA that is obtained in ChIP experiments needs to be processed for sequence identification. The sequences can be identified by direct sequencing (ChIP-SEQ) or hybridization to microarrays (ChIP-CHIP). Given the small amounts of DNA that are usually obtained in ChIP experiments, successful and reproducible sample processing is challenging. Here we provide a detailed procedure for ChIP of plant TFs, as well as protocols for sample preparation for ChIP-SEQ and for ChIP-CHIP. Our ChIP procedure is optimized for high signal-to-noise ratio starting with tissue fixation, followed by nuclei isolation, immunoprecipitation, DNA amplification and purification. We also provide a guide for primary data analysis of ChIP-SEQ data. The complete protocol for ChIP-SEQ/ChIP-CHIP sample preparation starting from plant harvest takes ∼7 d.

Arabidopsis Class I and Class II TCP Transcription Factors Regulate Jasmonic Acid Metabolism and Leaf Development Antagonistically.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) transcription factors control developmental processes in plants. The 24 TCP transcription factors encoded in the Arabidopsis (Arabidopsis thaliana) genome are divided into two classes, class I and class II TCPs, which are proposed to act antagonistically. We performed a detailed phenotypic analysis of the class I tcp20 mutant, showing an increase in leaf pavement cell sizes in 10-d-old seedlings. Subsequently, a glucocorticoid receptor induction assay was performed, aiming to identify potential target genes of the TCP20 protein during leaf development. The LIPOXYGENASE2 (LOX2) and class I TCP9 genes were identified as TCP20 targets, and binding of TCP20 to their regulatory sequences could be confirmed by chromatin immunoprecipitation analyses. LOX2 encodes for a jasmonate biosynthesis gene, which is also targeted by class II TCP proteins that are under the control of the microRNA JAGGED AND WAVY (JAW), although in an antagonistic manner. Mutation of TCP9, the second identified TCP20 target, resulted in increased pavement cell sizes during early leaf developmental stages. Analysis of senescence in the single tcp9 and tcp20 mutants and the tcp9tcp20 double mutants showed an earlier onset of this process in comparison with wild-type control plants in the double mutant only. Both the cell size and senescence phenotypes are opposite to the known class II TCP mutant phenotype in JAW plants. Altogether, these results point to an antagonistic function of class I and class II TCP proteins in the control of leaf development via the jasmonate signaling pathway.

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

BackgroundDevelopment of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.ResultsWe characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes.ConclusionsOur findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility.

ChIP-seq Analysis in R (CSAR): An R package for the statistical detection of protein-bound genomic regions

BackgroundIn vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally efficient manner. The generation of high copy numbers of DNA fragments as an artifact of the PCR step in ChIP-seq is an important source of bias of this methodology.ResultsWe present here an R package for the statistical analysis of ChIP-seq experiments. Taking the average size of DNA fragments subjected to sequencing into account, the software calculates single-nucleotide read-enrichment values. After normalization, sample and control are compared using a test based on the ratio test or the Poisson distribution. Test statistic thresholds to control the false discovery rate are obtained through random permutations. Computational efficiency is achieved by implementing the most time-consuming functions in C++ and integrating these in the R package. An analysis of simulated and experimental ChIP-seq data is presented to demonstrate the robustness of our method against PCR-artefacts and its adequate control of the error rate.ConclusionsThe software ChIP-seq Analysis in R (CSAR) enables fast and accurate detection of protein-bound genomic regions through the analysis of ChIP-seq experiments. Compared to existing methods, we found that our package shows greater robustness against PCR-artefacts and better control of the error rate.

Arabidopsis JAGGED links floral organ patterning to tissue growth by repressing Kip-related cell cycle inhibitors

Significance Plant organs, such as leaves, petals, or fruits, are shaped by the behavior of their constituent cells: cell growth, oriented extension of cell walls, and cell division. However, we know little about how these processes are coordinated by regulatory genes that shape plant organs, such as JAGGED (JAG) in Arabidopsis. By identifying the genes bound by JAG throughout the genome and the consequent changes in gene expression, we reveal that JAG functions as a direct mediator between genes that control the identity of organs and tissues and the cellular activities required for organ growth. In particular, we show that JAG sculpts floral organs by directly repressing genes that control entry into DNA replication. Plant morphogenesis requires coordinated cytoplasmic growth, oriented cell wall extension, and cell cycle progression, but it is debated which of these processes are primary drivers for tissue growth and directly targeted by developmental genes. Here, we used ChIP high-throughput sequencing combined with transcriptome analysis to identify global target genes of the Arabidopsis transcription factor JAGGED (JAG), which promotes growth of the distal region of floral organs. Consistent with the roles of JAG during organ initiation and subsequent distal organ growth, we found that JAG directly repressed genes involved in meristem development, such as CLAVATA1 and HANABA TARANU, and genes involved in the development of the basal region of shoot organs, such as BLADE ON PETIOLE 2 and the GROWTH REGULATORY FACTOR pathway. At the same time, JAG regulated genes involved in tissue polarity, cell wall modification, and cell cycle progression. In particular, JAG directly repressed KIP RELATED PROTEIN 4 (KRP4) and KRP2, which control the transition to the DNA synthesis phase (S-phase) of the cell cycle. The krp2 and krp4 mutations suppressed jag defects in organ growth and in the morphology of petal epidermal cells, showing that the interaction between JAG and KRP genes is functionally relevant. Our work reveals that JAG is a direct mediator between genetic pathways involved in organ patterning and cellular functions required for tissue growth, and it shows that a regulatory gene shapes plant organs by releasing a constraint on S-phase entry.

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.

The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male Gametophyte

Immature pollen of many species can be reprogrammed to form haploid embryos in vitro in response to a stress treatment. This work, in Brassica napus and Arabidopsis thaliana, showed that this stress-induced switch in developmental pathways relies on the inhibition of histone deacetylase activity. The haploid male gametophyte, the pollen grain, is a terminally differentiated structure whose function ends at fertilization. Plant breeding and propagation widely use haploid embryo production from in vitro–cultured male gametophytes, but this technique remains poorly understood at the mechanistic level. Here, we show that histone deacetylases (HDACs) regulate the switch to haploid embryogenesis. Blocking HDAC activity with trichostatin A (TSA) in cultured male gametophytes of Brassica napus leads to a large increase in the proportion of cells that switch from pollen to embryogenic growth. Embryogenic growth is enhanced by, but not dependent on, the high-temperature stress that is normally used to induce haploid embryogenesis in B. napus. The male gametophyte of Arabidopsis thaliana, which is recalcitrant to haploid embryo development in culture, also forms embryogenic cell clusters after TSA treatment. Genetic analysis suggests that the HDAC protein HDA17 plays a role in this process. TSA treatment of male gametophytes is associated with the hyperacetylation of histones H3 and H4. We propose that the totipotency of the male gametophyte is kept in check by an HDAC-dependent mechanism and that the stress treatments used to induce haploid embryo development in culture impinge on this HDAC-dependent pathway.