Histone H2B deubiquitination is required for transcriptional activation of FLOWERING LOCUS C and for proper control of flowering in Arabidopsis

Abstract

The spectrum of histone modifications at a given locus is a critical determinant for the correct output of gene expression. In Arabidopsis (Arabidopsis thaliana), many studies have examined the relationship between histone methylation and gene expression, but few studies exist on the relationship between other covalent histone modifications and gene expression. In this work, we describe the role of histone H2B deubiquitination in the activation of gene expression and the consequence of a perturbation of histone H2B deubiquitination in the timing of the floral transition in Arabidopsis. A mutation in a H2B deubiquitinase, UBIQUITIN-SPECIFIC PROTEASE26 (UBP26), results in an early-flowering phenotype. In the ubp26 mutant, mRNA levels of the floral repressor FLOWERING LOCUS C (FLC) and other related family members is decreased. Furthermore, this mutant accumulates H2B monoubiquitination, and has decreased levels of H3K36 trimethylation and increased levels of H3K27 trimethylation at the FLC locus. Thus, UBP26 is required for transcriptional activation of FLC through H2B deubiquitination and is consistent with a model in which deubiquitination is necessary for the accumulation of H3K36 trimethylation and the proper level of transcriptional activation.

Figure 1.

ubp26-1 mutants display an early-flowering phenotype. A, A gene structure of UBP26 containing exons (black boxes) and introns (black lines). The T-DNA in ubp26-1 is located in the fifth exon. (Sequence analysis of the T-DNA junction revealed the site of the T-DNA insertion was different from the insertion site published by Sridhar et al. [2007].) B, RT-PCR analysis of UBP26 in C24 and in ubp26-1 (the short fragment indicated below the gene structure in A represents the RT-PCR product). C and D, A picture of wild-type C24 and ubp26-1 plants grown in long days (C) and short days (D). E, Leaf count data of C24 and ubp26-1 in long days and short days. Black portions represent rosette leaves and white portions indicate cauline leaves. Error bars equal one sd. F, RT-PCR analysis of the FLC clade in C24 and in ubp26-1. [See online article for color version of this figure.]

Figure 2.

Mutations in ubp26 frequently result in a high rate of seed abortion in the Col genetic background. A, A gene structure of UBP26 including the positions of the ubp26-4 and the ubp26-3 T-DNA insertions. B, Pictures of a subset of seeds from seed stocks of Col and hemizygotes for the ubp26-4 and ubp26-3 T-DNAs. Arrows indicate shriveled/aborted seeds. The numbers below the picture indicate the frequency of healthy seeds to shriveled/aborted seeds. C and D are pictures of cleared embryos from a single silique of a plant hemizygous for the ubp26-4 T-DNA. In this particular silique, a majority of the cleared embryos were in the heart stage of embryo development (as pictured in C), whereas a minority had arrested at random stages of development (one example pictured in D). E, A picture depicting an example of a seedling lethal ubp26-4 mutant. The numbers indicate the frequency of observed seedling lethality from seed stocks of Col and hemizygotes for the ubp26-4 T-DNA. [See online article for color version of this figure.]

Figure 3.

Analysis of global levels of histone modifications. Analysis of global levels of monoubiquitinated H2B and histone methylation. Each mutant is compared to its respective wild type. elf7-2, pie1-2, and efs-3 are mutants in the Col genetic background.

Figure 4.

ChIP analysis of H2B monoubiquitination and H3K4me3 at FLC. A, A gene diagram of FLC indicating the location of the primers used in the ChIP analysis (see Supplemental Table S1 for primer information). B and C, ChIP analysis of H2Bub1 (B) and H3K4me3 (C) in FLC chromatin. Black bars indicate wild-type C24 and white bars indicate ubp26-1. Error bars represent ses.

Figure 5.

ChIP analysis of H3K36 methylation at FLC chromatin. A to C, ChIP analysis of H3K36me1 (A), H3K36me2 (B), and H3K36me3 (C) at FLC chromatin. Black bars indicate wild-type C24 and white bars indicate ubp26-1. Error bars represent ses.

Figure 6.

ChIP analysis of H3K27me3 at FLC chromatin. Shown is ChIP analysis of H3K27me3 at FLC chromatin. H3K27me3 is highly enriched in the ubp26-1 mutant compared to the wild-type C24. Black bars indicate wild-type C24 and white bars indicate ubp26-1. Error bars represent ses.

Figure 7.

A hypothetical model of FLC transcriptional activation by cycling of histone H2B monoubiquitination. A and B, A proposed model for transcriptional activation of FLC through H2B ubiquitination (A) and H2B deubiquitination (B). All factors shown above are essential for proper transcriptional activation of FLC. H2B and H3 indicate histones. K4 and K36 indicate Lys-4 and Lys-36 residues of histone H3. Ub and M indicate ubiquitin and methyl groups on histones, respectively. Solid arrows indicate a positive interaction. Broken arrows predict a positive interaction in Arabidopsis proteins based on experimental evidence from yeast.