Elsevier

Gene

Volume 423, Issue 1, 15 October 2008, Pages 43-47
Gene

Dynamic and stable histone H3 methylation patterns at the Arabidopsis FLC and AP1 loci

https://doi.org/10.1016/j.gene.2008.06.022Get rights and content

Abstract

Mechanisms that chemically modify nucleosomes leading to inheritable activation or repression of pertinent genes are defined as epigenetic. H3K4me3 and H3K27me3 are interpreted as ‘activating’ and ‘silencing’ marks, respectively. Here, we demonstrate that even for related genes neither modification, alone, could serve as an indicator of expression status: despite being members of the same gene family selectively activated by ATX1, FLC and AP1 nucleosomes may be similarly decorated but, also, surprisingly different. ‘Activating’ H3K4me3 and ‘silencing’ H3K27me3 modifications co-exist at 5′-end nucleosomes of transcriptionally active FLC-gene, while highly transcribed AP1 displays neither of the two marks. The results suggest that distinct mechanisms ‘read’ and operate at each locus. In a remarkable contrast, H3K4me3–H3K27me3 profiles at downstream FLC and AP1 gene sequences remain unchanged and transmitted as stable marks throughout development. We propose that H3K4me3 and H3K27me3 produce a distinct bi-modular ‘syllable’ in the histone ‘code’ conveying different meaning on specific genes. Evidence that certain chromatin modifications might be common for active or non-active genome regions but, also, that the same histone signs might have gene-specific ‘meaning’, as reported here, might be critically important for large-scale genome analyses. ATX1 and CLF encode enzyme activities involved in establishing the H3K4me3 and H3K27me3 marks, respectively. The potential involvement of ATX1 and CLF in generating the dual H3K4me3 and H3K27me3 marks on FLC and AP1 nucleosomes was investigated.

Introduction

Plant development, differentiation, and environmental adaptation involve global reprogramming of the genome. Dynamic changes of chromatin structure are forces driving controlled shifts in genome activity. Specific chemical modifications of histone tails may trigger events leading to the establishment of transcriptionally active/silent chromatin domains. The Polycomb- and the Trithorax-Group (PcG/TrxG) complexes, known to regulate animal developmental genes, establish tri-methyl groups on lysine 27 of histone H3 (H3K27me3) and on lysine 4 (H3K4me3), respectively (Grimaud et al., 2006, Kouzarides, 2007). Similar to the metazoan counterparts, Arabidopsis PCR2 complexes deposit H3K27me3 through the biochemical activity of E(z) homologs (Goodrich et al., 1997, Chanvivattana et al., 2004, Schubert et al., 2005, Schubert et al., 2006), while Trithorax family members specifically tri-methylate histone H3K4 (Alvarez-Venegas et al., 2003, Pien and Grossniklaus, 2007). H3K27me3 and H3K4me3 marks are considered hallmarks of silent and actively transcribed genes, respectively. Emerging evidence, however, is suggesting more complex roles for histone modifications beyond simply ‘activating-silencing’ marks. Thus, histone deacetylase Rpd3 and histone deacetylation of the coding regions in actively transcribed genes are directly linked with methylated H3K36 labeling active genes (Kaplan et al., 2003, Keogh, 2005, Brown et al., 2006); simultaneously present K4me3 and K27me3 marks at promotor nucleosomes of embryonic stem cells establish a bivalent chromatin state for genes poised for expression later in development (Bernstein et al., 2006). In addition to pluripotent cells, K4me3–K27me3 co-localization is functional in more differentiated cells as well (rev. in Sharov and Ko, 2007). Here, we report that dual H3K27me3 and H3K4me3 marks co-exist on the nucleosomes of two related genes from the MADS-box family, FLC and AP1. We found that dual H3K27me3 and H3K4me3 marks at the 5′-transcription start site (TSS) have a different meaning from dual-marks labeling downstream gene (G)-nucleosomes. Distribution of H3K4me3 and H3K27me3 at TSS nucleosomes is dynamic and gene-specific. Erasure of both H3K27me3–H3K4me3 marks at the AP1-TSS region upon AP1 activation reflects removal of a nucleosome from the site. Changes in the methylation patterns of 5′-TSS nucleosomes of both FLC and AP1 reflect the genes' transcriptional activity, while dual marks on downstream nucleosomes remain unchanged. Thereby, preserved H3K27me3–H3K4me3 were passed on as stable marks independent of the FLC- and AP1 activity and/or developmental transitions. ATX1 modifies FLC, but not AP1, nucleosomes. However, CLF does not appear involved in modifying either the FLC or AP1 nucleosomes.

Section snippets

Plant material, growth conditions and chromatin isolation

Arabidopsis thaliana Ws seeds were sterilized and grown in either 40 ml of germination media or in pots, at 24 °C under long-day (16 h light/8 h darkness). The atx1 mutant line was described in Alvarez-Venegas et al. (2003). Seeds for the clf mutant line were kind gift by J. Goodrich. Tissues for analysis were harvested from 10-day old seedlings, from the rosette leaves of plants upon bolting and from buds and flowers of five-week old plants. Chromatins were isolated from wild type and from the

H3K4me3 and H3K27me3 patterns of FLC- and AP1-nucleosomes in transcribed and non-transcribed chromatins

The FLOWERING LOCUS C (FLC) indirectly represses the activity of APETALA1 (AP1) blocking the transition from vegetative to reproductive stages in Arabidopsis (Levy et al., 2002, Schonrock et al., 2006). After bolting, FLC transcripts were no longer detected in inflorescences or in rosette leaves while AP1-expression changed in reverse (Fig. 1a). To determine the nucleosomal methylation patterns of the two reciprocally expressed transcription factors, we analyzed chromatin H3K4me3 and H3K27me3

Discussion

Generally, H3K4me3 and H3K27me3 are interpreted as ‘activating’ and ‘silencing’ marks, respectively. Here, we provide strong evidence that neither modification, alone, could serve as an indicator of expression status even for related genes. The two antagonistic marks on downstream nucleosomes remained unchanged throughout developmental transitions, independently of the transcriptional activity of AP1 or FLC. In a stark contrast, the H3K4me3/H3K27me3 profile at 5′-TSS nucleosomes changed

Acknowledgements

I am grateful to Drs. S. Pien (U. of Zurich) and P. Crevillen (John Innes Center) for critically reading the manuscript and helpful suggestions. This work was partially supported by NSF-MCB-0343934 grant award ZA and by NSF grant EPS-0701892.

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