Mads floral integrators : Insights into molecular mechanisms of MADS domain proteins in the floral transition

The main aim of this thesis is understanding the molecular regulation of flowering time in Arabidopsis thaliana. More specifically, we focus on of key regulatory genes of flowering that integrate several internal and external flowering signals and examine in detail how they are regulated at the transcriptional and post-transcriptional level. Many of the key regulatory genes encode transcription factors (TFs), which are often functioning in larger protein complexes and are part of complex gene regulatory network. This thesis focuses on two important regulators that are MADS-domain TFs, SHORT VEGETATIVE PHASE (SVP) and SUPPERESSOR OF OVEREXPRESSION of CONSTANCE 1 (SOC1) and we studied the protein-protein interactions, chromosomal interactions and TF-DNA interactions, all connections that are part of the gene regulatory networks involved in flowering time control.</p

Identification of in planta protein–protein interactions using IP-MS

Gene regulation by transcription factors involves complex protein interaction networks, which include chromatin remodeling and modifying proteins as an integral part. Decoding these protein interactions is crucial for our understanding of chromatin-mediated gene regulation. Here, we describe a method for the immunoprecipitation of in planta nuclear protein complexes followed by mass spectrometry (IP-MS) to identify interactions between transcription factors and chromatin remodelers/modifiers in plants. In addition to a step-by-step bench protocol for immunoprecipitation and subsequent mass spectrometry, we provide guidelines and pointers on necessary controls and data analysis approaches

3C in maize and arabidopsis

With Chromosome Conformation Capture (3C), the relative interaction frequency of one chromosomal fragment with another can be determined. The technique is especially suited for unraveling the 3D organization of specific loci when focusing on aspects such as enhancer–promoter interactions or other topological conformations of the genome. 3C has been extensively used in animal systems, among others providing insight into gene regulation by distant cis-regulatory elements. In recent years, the 3C technique has been applied in plant research. However, the complexity of plant tissues prevents direct application of existing protocols from animals. Here, we describe an adapted protocol suitable for plant tissues, especially Arabidopsis thaliana and Zea mays

A cautionary note on the use of chromosome conformation capture in plants

Background: The chromosome conformation capture (3C) technique is a method to study chromatin interactions at specific genomic loci. Initially established for yeast the 3C technique has been adapted to plants in recent years in order to study chromatin interactions and their role in transcriptional gene regulation. As the plant scientific community continues to implement this technology, a discussion on critical controls, validations steps and interpretation of 3C data is essential to fully benefit from 3C in plants. Results: Here we assess the reliability and robustness of the 3C technique for the detection of chromatin interactions in Arabidopsis. As a case study, we applied this methodology to the genomic locus of a floral integrator gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and demonstrate the need of several controls and standard validation steps to allow a meaningful interpretation of 3C data. The intricacies of this promising but challenging technique are discussed in depth. Conclusions: The 3C technique offers an interesting opportunity to study chromatin interactions at a resolution infeasible by microscopy. However, for interpretation of 3C interaction data and identification of true interactions, 3C technology demands a stringent experimental setup and extreme caution

A molecular network for functional versatility of HECATE transcription factors

During the plant life cycle, diverse signaling inputs are continuously integrated and engage specific genetic programs depending on the cellular or developmental context. Consistent with an important role in this process, HECATE (HEC) basic helix–loop–helix transcription factors display diverse functions, from photomorphogenesis to the control of shoot meristem dynamics and gynoecium patterning. However, the molecular mechanisms underlying their functional versatility and the deployment of specific HEC subprograms remain elusive. To address this issue, we systematically identified proteins with the capacity to interact with HEC1, the best-characterized member of the family, and integrated this information with our data set of direct HEC1 target genes. The resulting core genetic modules were consistent with specific developmental functions of HEC1, including its described activities in light signaling, gynoecium development and auxin homeostasis. Importantly, we found that HEC genes also play a role in the modulation of flowering time, and uncovered that their role in gynoecium development may involve the direct transcriptional regulation of NGATHA1 (NGA1) and NGA2 genes. NGA factors were previously shown to contribute to fruit development, but our data now show that they also modulate stem cell homeostasis in the shoot apical meristem. Taken together, our results delineate a molecular network underlying the functional versatility of HEC transcription factors. Our analyses have not only allowed us to identify relevant target genes controlling shoot stem cell activity and a so far undescribed biological function of HEC1, but also provide a rich resource for the mechanistic elucidation of further context-dependent HEC activities

A molecular network for functional versatility of HECATE transcription factors

During the plant life cycle, diverse signaling inputs are continuously integrated and engage specific genetic programs depending on the cellular or developmental context. Consistent with an important role in this process, HECATE (HEC) basic helix–loop–helix transcription factors display diverse functions, from photomorphogenesis to the control of shoot meristem dynamics and gynoecium patterning. However, the molecular mechanisms underlying their functional versatility and the deployment of specific HEC subprograms remain elusive. To address this issue, we systematically identified proteins with the capacity to interact with HEC1, the best-characterized member of the family, and integrated this information with our data set of direct HEC1 target genes. The resulting core genetic modules were consistent with specific developmental functions of HEC1, including its described activities in light signaling, gynoecium development and auxin homeostasis. Importantly, we found that HEC genes also play a role in the modulation of flowering time, and uncovered that their role in gynoecium development may involve the direct transcriptional regulation of NGATHA1 (NGA1) and NGA2 genes. NGA factors were previously shown to contribute to fruit development, but our data now show that they also modulate stem cell homeostasis in the shoot apical meristem. Taken together, our results delineate a molecular network underlying the functional versatility of HEC transcription factors. Our analyses have not only allowed us to identify relevant target genes controlling shoot stem cell activity and a so far undescribed biological function of HEC1, but also provide a rich resource for the mechanistic elucidation of further context-dependent HEC activities.</p

How a Retrotransposon Exploits the Plant's Heat Stress Response for Its Activation

<div><p>Retrotransposons are major components of plant and animal genomes. They amplify by reverse transcription and reintegration into the host genome but their activity is usually epigenetically silenced. In plants, genomic copies of retrotransposons are typically associated with repressive chromatin modifications installed and maintained by RNA-directed DNA methylation. To escape this tight control, retrotransposons employ various strategies to avoid epigenetic silencing. Here we describe the mechanism developed by <i>ONSEN</i>, an LTR-copia type retrotransposon in <i>Arabidopsis thaliana</i>. <i>ONSEN</i> has acquired a heat-responsive element recognized by plant-derived heat stress defense factors, resulting in transcription and production of full length extrachromosomal DNA under elevated temperatures. Further, the <i>ONSEN</i> promoter is free of CG and CHG sites, and the reduction of DNA methylation at the CHH sites is not sufficient to activate the element. Since dividing cells have a more pronounced heat response, the extrachromosomal <i>ONSEN</i> DNA, capable of reintegrating into the genome, accumulates preferentially in the meristematic tissue of the shoot. The recruitment of a major plant heat shock transcription factor in periods of heat stress exploits the plant's heat stress response to achieve the transposon's activation, making it impossible for the host to respond appropriately to stress without losing control over the invader.</p></div

Chromatin and epigenetics in all their states : Meeting report of the first conference on Epigenetic and Chromatin Regulation of Plant Traits - January 14 – 15, 2016 - Strasbourg, France

In January 2016, the first Epigenetic and Chromatin Regulation of Plant Traits conference was held in Strasbourg, France. An all-star lineup of speakers, a packed audience of 130 participants from over 20 countries, and a friendly scientific atmosphere contributed to make this conference a meeting to remember. In this article we summarize some of the new insights into chromatin, epigenetics, and epigenomics research and highlight nascent ideas and emerging concepts in this exciting area of research.</p

<i>ONSEN</i> is activated by heat stress.

<p>(<b>A</b>) Schematic representation of the experiments. White and black boxes represent light or darkness intervals; blue and red boxes represent periods of standard growth temperature or heat stress, arrows indicate sampling time points. (<b>B</b>) Relative amounts of <i>ONSEN</i> RNA determined by quantitative RT-PCR in three week-old wild type seedlings harvested as indicated in A. Bars represent the <i>ONSEN</i> transcripts in relation to that of AtSAND (equal level in all samples) and normalized to WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>C</b>) Southern blot analysis of undigested genomic DNA isolated from the same material as in B and hybridized to an <i>ONSEN</i>-specific probe. Upper and lower arrows indicate integrated and extrachromosomal <i>ONSEN</i> copies, respectively. The EtBr image indicates the loading of genomic DNA. (<b>D</b>) Quantification of <i>ONSEN</i> extrachromosomal DNA based on C. Bars represent the ratio between signal intensities of extrachromosomal and integrated copies determined by densitometry. Error bars correspond to the s.d. (n = 3).</p