Nicolas Buisine

Arabidopsis TFL2/LHP1 Specifically Associates with Genes Marked by Trimethylation of Histone H3 Lysine 27

TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) is the only Arabidopsis protein with overall sequence similarity to the HETEROCHROMATIN PROTEIN 1 (HP1) family of metazoans and S. pombe. TFL2/LHP1 represses transcription of numerous genes, including the flowering-time genes FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC), as well as the floral organ identity genes AGAMOUS (AG) and APETALA 3 (AP3). These genes are also regulated by proteins of the Polycomb repressive complex 2 (PRC2), and it has been proposed that TFL2/LHP1 represents a potential stabilizing factor of PRC2 activity. Here we show by chromatin immunoprecipitation and hybridization to an Arabidopsis Chromosome 4 tiling array (ChIP-chip) that TFL2/LHP1 associates with hundreds of small domains, almost all of which correspond to genes located within euchromatin. We investigated the chromatin marks to which TFL2/LHP1 binds and show that, in vitro, TFL2/LHP1 binds to histone H3 di- or tri-methylated at lysine 9 (H3K9me2 or H3K9me3), the marks recognized by HP1, and to histone H3 trimethylated at lysine 27 (H3K27me3), the mark deposited by PRC2. However, in vivo TFL2/LHP1 association with chromatin occurs almost exclusively and co-extensively with domains marked by H3K27me3, but not H3K9me2 or -3. Moreover, the distribution of H3K27me3 is unaffected in lhp1 mutant plants, indicating that unlike PRC2 components, TFL2/LHP1 is not involved in the deposition of this mark. Rather, our data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2.

Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells

Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

Improved detection and annotation of transposable elements in sequenced genomes using multiple reference sequence sets.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes that impact many aspects of genome function. TE detection in genomic sequences is typically performed using similarity searches against a set of reference sequences built from previously identified TEs. Here, we demonstrate that this process can be improved by designing reference sets that incorporate key aspects of the structure and evolution of TEs and by combining these sets with Repbase Update (RU), which is composed mainly of consensus sequences. Using the Arabidopsis genome as a test case, our approach leads to the detection of an extra 12.4% of TE sequences. These correspond to novel TE fragments as well as to the extension of TE fragments already detected by RU. Significantly, we find that TE detection could be readily optimized using only two reference sets, one containing true consensus sequences and the other mosaic sequences that capture the structural diversity of TE copies within a family.

Specific Histone Lysine 4 Methylation Patterns Define TR-Binding Capacity and Differentiate Direct T3 Responses

The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.

Mechanisms of thyroid hormone receptor action during development: lessons from amphibian studies.

BACKGROUND Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. However, the in vivo mechanism of TR action remains poorly explored. SCOPE OF REVIEW Frog metamorphosis is controlled by TH and mimics the postembryonic period in mammals when high levels of TH are also required. We review here some of the findings on the developmental functions of TH and TR and the associated mechanisms obtained from this model system. MAJOR CONCLUSION A dual function model for TR in Anuran development was proposed over a decade ago. That is, unliganded TR recruits corepressors to TH response genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes. Subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model. Specifically, it has been shown that unliganded TR recruits histone deacetylase containing corepressor complexes during larval stages to control metamorphic timing, while liganded TR recruits multiple histone modifying and chromatin remodeling coactivator complexes during metamorphosis. These complexes can alter chromatin structure via nucleosome position alterations or eviction and histone modifications to contribute to the recruitment of transcriptional machinery and gene activation. GENERAL SIGNIFICANCE The molecular mechanisms of TR action in vivo as revealed from studies on amphibian metamorphosis are very likely applicable to mammalian development as well. These findings provide a new perspective for understanding the diverse effects of TH in normal physiology and diseases caused by TH dysfunction. This article is part of a Special Issue entitled Thyroid hormone signalling.

The thyroid hormone receptor β induces DNA damage and premature senescence

Thyroid hormone and its receptor act in concert with NRF1 to increase cellular respiration and reactive oxygen species production, leading to DNA damage and premature senescence in susceptible cells.

The transposon-like correia elements encode numerous strong promoters and provide a potential new mechanism for phase variation in the meningococcus

Neisseria meningitidis is the primary causative agent of bacterial meningitis. The genome is rich in repetitive DNA and almost 2% is occupied by a diminutive transposon called the Correia element. Here we report a bioinformatic analysis defining eight subtypes of the element with four distinct types of ends. Transcriptional analysis, using PCR and a lacZ reporter system, revealed that two ends in particular encode strong promoters. The activity of the strongest promoter is dictated by a recurrent polymorphism (Y128) at the right end of the element. We highlight examples of elements that appear to drive transcription of adjacent genes and others that may express small non-coding RNAs. Pair-wise comparisons between three meningococcal genomes revealed that no more than two-thirds of Correia elements maintain their subtype at any particular locus. This is due to recombinational class switching between elements in a single strain. Upon switching subtype, a new allele is available to spread through the population by natural transformation. This process may represent a hitherto unrecognized mechanism for phase variation in the meningococcus. We conclude that the strain-to-strain variability of the Correia elements, and the large number of strong promoters encoded by them, allows for potentially widespread effects within the population as a whole. By defining the strength of the promoters encoded by the eight subtypes of Correia ends, we provide a resource that allows the transcriptional effects of a particular subtype at a given locus to be predicted.

Deciphering the Regulatory Logic of an Ancient, Ultraconserved Nuclear Receptor Enhancer Module

Cooperative, synergistic gene regulation by nuclear hormone receptors can increase sensitivity and amplify cellular responses to hormones. We investigated thyroid hormone (TH) and glucocorticoid (GC) synergy on the Krüppel-like factor 9 (Klf9) gene, which codes for a zinc finger transcription factor involved in development and homeostasis of diverse tissues. We identified regions of the Xenopus and mouse Klf9 genes 5-6 kb upstream of the transcription start sites that supported synergistic transactivation by TH plus GC. Within these regions, we found an orthologous sequence of approximately 180 bp that is highly conserved among tetrapods, but absent in other chordates, and possesses chromatin marks characteristic of an enhancer element. The Xenopus and mouse approximately 180-bp DNA element conferred synergistic transactivation by hormones in transient transfection assays, so we designate this the Klf9 synergy module (KSM). We identified binding sites within the mouse KSM for TH receptor, GC receptor, and nuclear factor κB. TH strongly increased recruitment of liganded GC receptor and serine 5 phosphorylated (initiating) RNA polymerase II to chromatin at the KSM, suggesting a mechanism for transcriptional synergy. The KSM is transcribed to generate long noncoding RNAs, which are also synergistically induced by combined hormone treatment, and the KSM interacts with the Klf9 promoter and a far upstream region through chromosomal looping. Our findings support that the KSM plays a central role in hormone regulation of vertebrate Klf9 genes, it evolved in the tetrapod lineage, and has been maintained by strong stabilizing selection.

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout.

In most of oviparous animals, vitellogenins (VTG) are the major egg yolk precursors. They are produced in the liver under the control of estrogens. In rainbow trout (Oncorhynchus mykiss), the vtg genes cluster contains an unusually large number of almost identical gene copies. In order to identify the regulatory elements in their promoters, we used a combination of reporter plasmids containing genomic sequences including putative estrogen response elements (EREs) and we performed transient transfection assays in MCF-7 and yeast cells. We found a functional ERE corresponding to the sequence GGGGCAnnnTAACCT (rtvtgERE), which differs from the consensus ERE (ERE(cs)) by three base pairs. This non-palindromic ERE is located in the env gene of a retrotransposon relic, 180 base pairs upstream of the transcriptional start site. Fluorescence anisotropy experiments confirmed that the purified human estrogen receptor alpha (hERalpha) can specifically bind to rtvtgERE. Furthermore, we observe that the stability of hERalpha-ERE(cs) and hERalpha-rtvtgERE complexes is similar with equilibrium dissociation constants of 3.0nM and 6.2nM respectively, under our experimental conditions. Additionally, this rtvtgERE sequence displays a high E2-responsiveness through ER activation in cellulo. In the rainbow trout, the functional ERE (rtvtgERE) lies within promoter sequences which are mostly composed of sequences derived from transposable elements (TEs), which therefore may have acted as an evolutionary buffer to secure the proper expression of these genes.

Two families of Xenopus tropicalis skeletal genes display well‐conserved expression patterns with mammals in spite of their highly divergent regulatory regions

SUMMARY The origin of bone and cartilage, and their subsequent diversification in specific vertebrate lineages, is intimately linked to the precise transcriptional control of genes involved in matrix mineralization. It is not yet clear, however, to which extent the osteoblasts, osteocytes, and chondrocytes of each of the major vertebrate groups express similar sets of genes. In this study we have focused on the evolution of two independent families of genes that code for extracellular matrix components of the skeleton and that include secreted protein, acidic, cysteine‐rich (SPARC), bone sialoprotein (BSP) and dentin matrix protein 1 (DMP1) paralogues, and the osteocalcin (OC) and matrix gla protein (MGP) paralogues. Analyzing developing Xenopus tropicalis skeletal elements, we show that the expression patterns of these genes are well conserved with mammals. The fact that only a few osteoblasts express DMP1, while only some osteocytes express SPARC and BSP, reveals a significant degree of molecular heterogeneity for these two populations of X. tropicalis cells, similarly to what has been described in mouse. Although the cis‐regulatory modules (CRM) of the mammalian OC, DMP1, and BSP orthologs have been functionally characterized, we found no evidence of sequence similarity between these regions and the X. tropicalis genome. Furthermore, these regulatory elements evolve rapidly, as they are only poorly conserved between human and rodents. Therefore, the SPARC/DMP1/BSP and the OC/MGP families provide a good paradigm to study how transcriptional output can be maintained in skeletal cells despite extensive sequence divergence of CRM.