Transcription‐dependence of histone H3 lysine 27 trimethylation at the Arabidopsis polycomb target gene FLC

Abstract

SUMMARYThe FLC gene encodes a MADS box repressor of flowering that is the main cause of the late-flowering phenotype of many Arabidopsis ecotypes. Expression of FLC is repressed by vernalization; maintenance of this repression is associated with the deposition of histone 3 K27 trimethylation (H3K27me3) at the FLC locus. However, whether this increased H3K27me3 is a consequence of reduced FLC transcription or the cause of transcriptional repression is not well defined. In this study we investigate the effect of changes in transcription rate on the abundance of H3K27me3 in the FLC gene body, a chromatin region that includes sequences required to maintain FLC repression following vernalization. We show that H3K27me3 is inversely correlated with transcription across the FLC gene body in a range of ecotypes and mutants with different flowering times. We demonstrate that the FLC gene body becomes marked with H3K27me3 in the absence of transcription. When transcription of the gene body is directed by an inducible promoter, H3K27me3 is removed following activation of transcription and H3K27me3 is added after transcription is decreased. The rate of addition of H3K27me3 to the FLC transgene following inactivation of transcription is similar to that observed in the FLC gene body following vernalization. Our data suggest that reduction of FLC transcription during vernalization leads to an increase of H3K27me3 levels in the FLC gene body that in turn maintains FLC repression.

Jumonji demethylases moderate precocious flowering at elevated temperature via regulation of FLC in Arabidopsis

“…10). A strong transcription activity driven by activators such as FRI may negatively affect the deposition of the repressive H3K27me3 marks at the FLC locus as previously suggested 54 , possibly masking the effect of JMJ30/ JMJ32. FRI-activated FLC is gradually silenced through vernalization with the accumulation of H3K27me3 (ref.…”

Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in <i>Brassica rapa</i> L.

“…Plant materials were harvested at the end of four weeks of cold treatment, or four-week vernalization, followed by seven or 12 days of normal growth conditions. Chromatin immunoprecipitation (ChIP) ChIP experiments were performed as described by Buzas et al (2011) Purified immunoprecipitated DNAs were amplified using a whole-genome amplification kit (Sigma, GenomePlex Kit, WGA2) and cloned into pGEM-T Easy vector (Promega). Individual clones were sequenced, and the location of sequenced fragments in the reference genome of B. rapa (Brassica genome release v1.5), Chiifu-401, was examined by BLAST search using the Brassica Database (http://brassicadb.org/brad/).…”

From the laboratory to the field: assaying histone methylation at <i>FLOWERING LOCUS C</i> in naturally growing <i>Arabidopsis halleri</i>

“…As reference genes for H3K27me3 ChIP, we selected orthologues of the following four genes: SHOOT MERISTEMLESS (STM) and AGAMOUS (AG), because these genes are silenced in tissues other than the shoot meristem (Yanofsky et al, 1990;Long et al, 1996); FUSCA3 (FUS3), silenced after germination (Gazzarrini et al, 2004); and F-box family protein-related gene (FBOX), which has been characterized as a low-expression gene (Czechowski et al, 2005). All of these genes are known to be enriched in H3K27me3 in A. thaliana (Zhang et al, 2007) and, moreover, some of them are already routinely used as internal controls (Schubert et al, 2006;Finnegan and Dennis, 2007;Buzas et al, 2011;Lafos et al, 2011). We determined the A. halleri sequences of these genes by homology search of A. thaliana genes against the draft genome of A. halleri (Supplementary Text S2), and designed the primers for ChIP-qPCR and RT-qPCR (Table 1, Supplementary Fig.…”

“…These results are consistent with the fact that the resolution of our ChIPs is 2-3 nucleosomes. To minimize the influence of histone density on the quantification of histone tail modifications, especially in the field, and for consistency with ChIP-qPCR normalization methods in other reports (Angel et al, 2011;Buzas et al, 2011), we used the H3 ChIP values for normalization in all subsequent calculations.…”