Vernalization-Repression of Arabidopsis FLC Requires Promoter Sequences but Not Antisense Transcripts

The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts

Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium

Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity

Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb Repressive Complex 2 Components

Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis

Mutually exclusive sense–antisense transcription at FLC facilitates environmentally induced gene repression

Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns

Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat

The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley

Small RNA-Directed Epigenetic Natural Variation in Arabidopsis thaliana

Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of ∼24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of ∼24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes

Brahma Is Required for Proper Expression of the Floral Repressor FLC in Arabidopsis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development. [Methodology/Principal Findings]: Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable. [Conclusions/Significance]: BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.This work was supported by Ministerio de Educacin y Ciencia (BFU2008-00238, CSD2006-00049), and by Junta de Andaluca (P06-CVI-01400) to J.C.R. and by the National Institutes of Health (grant no. 1R01GM079525), and the National Science Foundation (grant no. 0446440) to R.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe