Nucleosomes are the basic repeating units of eukaryotic chromatin. They play important roles in chromatin compaction and gene regulation. Therefore, it is important to profile the in vivo locations ...of nucleosomes in the genome. Here we illustrate how to profile nucleosome occupancy at genome-wide scale using micrococcal nuclease (MNase) digestion combined with high throughput Illumina sequencing (MNase-seq). Nucleosome-associated DNA is relatively insensitive to digestion by micrococcal nuclease (MNase). Upon mild MNase treatment, the undigested nucleosomal DNA can be purified and sequenced allowing a precise localization of in vivo nucleosomes at a genome-wide level.
Floral organs are specified by the combinatorial action of MADSdomain transcription factors, yet the mechanisms by which MADSdomain 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 MADSdomain 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.
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.
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.
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 approximately 7 d.
Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is ...still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation.
SUMMARY
The development of photosynthetically competent seedlings requires both light and retrograde biogenic signaling pathways. The transcription factor GLK1 functions at the interface between ...these pathways and receives input from the biogenic signal integrator GUN1. BBX14 was previously identified, together with GLK1, in a core module that mediates the response to high light (HL) levels and biogenic signals, which was studied by using inhibitors of chloroplast development. Our chromatin immunoprecipitation‐Seq experiments revealed that BBX14 is a direct target of GLK1, and RNA‐Seq analysis suggests that BBX14 may function as a regulator of the circadian clock. In addition, BBX14 plays a role in chlorophyll biosynthesis during early onset of light. Knockout of BBX14 results in a long hypocotyl phenotype dependent on a retrograde signal. Furthermore, the expression of BBX14 and BBX15 during biogenic signaling requires GUN1. Investigation of the role of BBX14 and BBX15 in GUN‐type biogenic (gun) signaling showed that the overexpression of BBX14 or BBX15 caused de‐repression of CA1 mRNA levels, when seedlings were grown on norflurazon. Notably, transcripts of the LHCB1.2 marker are not de‐repressed. Furthermore, BBX14 is required to acclimate plants to HL stress. We propose that BBX14 is an integrator of biogenic signals and that BBX14 is a nuclear target of retrograde signals downstream of the GUN1/GLK1 module. However, we do not classify BBX14 or BBX15 overexpressors as gun mutants based on a critical evaluation of our results and those reported in the literature. Finally, we discuss a classification system necessary for the declaration of new gun mutants.
Significance Statement
A picture is emerging in which BBX proteins have multifaceted functions in plant development, acting as receivers of chloroplast signals as well as playing a role in responses to acute stress.
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 embryogenie growth. Embryogenie 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.
MADS-domain transcription factors are well known for their roles in plant development and regulate sets of downstream genes that have been uncovered by high-throughput analyses. A considerable number ...of these targets are predicted to function in hormone responses or responses to environmental stimuli, suggesting that there is a close link between developmental and environmental regulators of plant growth and development. Here, we show that the Arabidopsis MADS-domain factor FRUITFULL (FUL) executes several functions in addition to its noted role in fruit development. Among the direct targets of FUL, we identified SMALL AUXIN UPREGULATED RNA 10 (SAUR10), a growth regulator that is highly induced by a combination of auxin and brassinosteroids and in response to reduced R:FR light. Interestingly, we discovered that SAUR10 is repressed by FUL in stems and inflorescence branches. SAUR10 is specifically expressed at the abaxial side of these branches and this localized activity is influenced by hormones, light conditions and by FUL, which has an effect on branch angle. Furthermore, we identified a number of other genes involved in hormone pathways and light signalling as direct targets of FUL in the stem, demonstrating a connection between developmentally and environmentally regulated growth programs.
Development 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.
We 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.
Our 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.
PDF
