Arabidopsis ORC1 is a PHD-containing H3K4me3 effector that regulates transcription

Abstract

Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Downstream effector proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landmark of gene expression control in eukaryotes. Its recognition depends on the presence in the effector protein of a motif termed plant homeodomain (PHD) that specifically binds to H3K4me3. Here, we establish that Arabidopsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. Arabidopsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter region of ORC1 targets. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in Arabidopsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a novel class of H3K4me3 effector proteins characteristic of the plant kingdom.

Fig. 1.

The Arabidopsis ORC1 proteins contain a PHD motif that binds H3K4me3. (A) Schema of ORC1 showing the location of DNA replication motifs (I-VI) and alignment of the two Arabidopsis ORC1 PHD motifs with that of several PHD-containing proteins. The main PHD regions appear color-coded, as indicated. Asterisks indicate two key cysteine residues of one of the two Zn²⁺ fingers and a phenylalanine of the cage that were mutated in this study. (B) Three-dimensional modeling of Arabidopsis ORC1b PHD using the crystal structure information of CHD4 (PDB 1mm2) and ING2 (PDB 2g6q) PHDs. Colored residues refer to those in panel A. White spheres in CHD4 and ING2 indicate the position of Zn²⁺ ions. Red lines in ING2 indicate the position of a histone H3K4me3 peptide. (C) Pull-down assay of plant histone extracts with Arabidopsis His-ORC1b. Bound histones were detected by Western blot with anti-H3K4me3, anti-H3K9me3, and anti-H4K20me3 antibodies. (D) Binding assay of Arabidopsis His-ORC1b with biotinylated H3 peptides either unmodified, mono-, di-, or trymethylated at K4. Bound peptides we detected by Western blot with anti-biotin antibodies. (E) Pull-down assay of plant histone extracts with Arabidopsis His-ORC1b, His-ORC1b^PHD(C/A), and His-ORC1b^PHD(F/A). His-ORC1b^PHD(C/A) contains two point mutations that change C183 and C186 to A. His-ORC1b^PHD(F/A) contains a F190A mutation (asterisks in A).

Fig. 2.

Expression of Arabidopsis ORC1 activates transcription. (A) Determination of mRNA levels of the indicated DNA replication genes by real-time RT-PCR in control and transgenic plants expressing Myc-ORC1a, Myc-ORC1b, and Myc-ORC1b^PHD(C/A). Values represent mean ± SD (n = 3). (B) Determination of CDT1a (At2g31270) and APG9 (At2g31260) mRNA levels by real-time RT-PCR in control and transgenic plants (10-day-old) expressing Myc-ORC1b and Myc-ORC1b^PHD(C/A). Values represent mean ± SD (n = 3). (C) Effect of ORC1a protein on the spatial expression pattern of CDT1a detected in pCDT1a:GUS reporter plants (7–10-day-old). Bars, 200 μm.

Fig. 3.

Identification of ORC1 binding sites in vivo. (A) ChIP assays were carried out using anti-Myc antibodies with control plants (transformed with an empty vector) and transgenic plants (10-day-old) expressing Myc-ORC1b and Myc-ORC1b^PHD(C/A). The genomic location of the CDT1a and APG9 genes is shown together with their direction of transcription. Letters and small black bars refer to location in the map of the fragment amplified by PCR and its size. A representative example of a ChIP experiment is shown in Fig. S5. Enrichment was calculated as (ChIP/Input)/(ChIP control/Input control) using the band intensity values of the “Input” and “ChIP” lanes after substracting the corresponding value in the “no Ab” lanes. Data shown are representative of at least two independent assays. (B) ChIP assays carried out as in (A) show ORC1 binding to the promoters of other ORC1-responsive or nonresponsive genes used in this study. Black boxes indicate position and size of PCR-amplified fragment.

Fig. 4.

Histone acetylation status in response to ORC1 binding. (A) Determination of CDT1a mRNA levels by real-time RT-PCR in control and transgenic plants (10-day-old) expressing Myc-ORC1b with and without treatment with the histone deacetylase inhibitor trichostatin A (TSA; 1 μg/ml). Values represent mean of two independent experiments, made relative to the control without or with TSA. (B) Quantification of histone H4ac throughout the CDT1a and APG9 loci. Fragments amplified are indicated in the map. A representative example of a ChIP experiment is shown in Fig. S5. Values were calculated as described in Fig. 3A. Data shown are representative of at least two independent assays. (C) ChIP assays were carried out as in (B) to determine histone H4ac in the promoters of other ORC1-responsive genes used in this study. Black boxes indicate position and size of PCR-amplified fragment.

Fig. 5.

ORC1 binding and transcriptional activation is linked to enrichment in H4K20me3. (A) Quantification of histone H4K20me3 throughout the CDT1a and APG9 loci by ChIP analysis. Fragments amplified are indicated in the map. Representative example of a ChIP experiment is shown in Fig. S5. Values were calculated as described in Fig. 3A. Data shown are representative of at least two independent assays. (B) ChIP assays were carried out as in (A) to determine histone H4K20me3 in the promoters of other ORC1-responsive genes. Black boxes indicate position and size of PCR-amplified fragment. (C) Determination of mRNA levels by real-time RT-PCR of DNA replication genes in Arabidopsis MM2d-cultured cells arrested by sucrose deprivation for 24 hours and proliferating cells (2 hours after sucrose addition), during which sodium butyrate (10 mM) was added. (D) Histone H4K20me3 status at the promoter region of CDT1a, ORC3, MCM3 (test genes), and ACT2 (control) in Arabidopsis-cultured cells. ChIP assays were carried out in arrested and proliferating cells as described in (C). Fragments amplified correspond to fragment B of CDT1a (see A) and to an equivalent fragment in the promoters of ORC3 and MCM3. (E) Simplified model of Arabidopsis ORC1 function in transcriptional activation. We propose that Arabidopsis ORC1 is a H3K4me3 effector protein that binds to target promoters through its PHD motif. ORC binding is associated with an enrichment of H4 acetylation and H4K20me3, which are histone marks that determine transcriptional activation in Arabidopsis and, possibly, in the entire plant kingdom.