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. 2018 Oct 19;9:1503.
doi: 10.3389/fpls.2018.01503. eCollection 2018.

WHIRLY1 Occupancy Affects Histone Lysine Modification and WRKY53 Transcription in Arabidopsis Developmental Manner

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Free PMC article

WHIRLY1 Occupancy Affects Histone Lysine Modification and WRKY53 Transcription in Arabidopsis Developmental Manner

Dongmei Huang et al. Front Plant Sci. .
Free PMC article

Abstract

Single-stranded DNA-binding proteins (SSBs) are assumed to involve in DNA replication, DNA repairmen, and gene transcription. Here, we provide the direct evidence on the functionality of an Arabidopsis SSB, WHIRLY1, by using loss- or gain-of-function lines. We show that WHIRLY1 binding to the promoter of WRKY53 represses the enrichment of H3K4me3, but enhances the enrichment of H3K9ac at the region contained WHIRLY1-binding sequences and TATA box or the translation start region of WRKY53, coincided with a recruitment of RNAPII. In vitro ChIP assays confirm that WHIRLY1 inhibits H3K4me3 enrichment at the preinitiation complex formation stage, while promotes H3K9ac enrichment and RNAPII recruitment at the elongation stage, consequently affecting the transcription of WRKY53. These results further explore the molecular actions underlying SSB-mediated gene transcription through epigenetic regulation in plant senescence.

Keywords: Arabidopsis; histone modification; leaf senescence; ssDNA binding protein; transcription.

Figures

FIGURE 1
FIGURE 1
Histone modification and RNAPII occupancy at the promoter regions and translation start region of WRKY53. (A) Schematic diagram of the genomic structure of the WRKY53 gene. The lines with number represent qPCR amplicons in different regions of WRKY53 gene. Gray box, black box, and blank box represent WHIRLY1 binding domain (gray), exon (black), and untranslated regions (blank). (B–E) ChIP analyses of changes in H3K4me2 (B), H3K4me3 (C), H3K9ac (D) levels, and RNAPII (E) occupancy at different regions of WRKY53 from 5th to 8th week. The antibodies used for ChIP were optimized (Supplementary Figure S2). The quantitative PCR was performed with primers (I–VI) around the predicted WHIRLY1 binding sites on the promoter of WRKY53 and primer (P) covered the 5′ untranslated region and translational start region of WRKY53 (Figure 2) (Ay et al., 2009). ACT7 (Actin-related gene 7, AT5G09810) and MULE (Mutator-like transposable element, AT2G15810) were analyzed as controls. The relative level was normalized to INPUT DNA. Three biological replicates and three technique replicates were used to analyze. Error bar show the SD (n = 3×3). Asterisk indicates significant differences (P < 0.05 and ∗∗P < 0.01) based on Student’s t-test. (F) Alteration of ratio of H3K9ac/H3K4me2-3 at different regions of WRKY53 from 5th to 7th week compared to occupancy of WHIRLY1 and transcripts level of WRKY53.
FIGURE 2
FIGURE 2
WHIRLY1 regulates the H3K4me3 enrichment at WRKY53 at senescence initiation stage. (A,B) ChIP experiments were performed to assess H3K4me2 (A) and H3K4me3 (B) levels at WHIRLY1 binding region (WRKY53II) and translation start region (WRKY53P) of WRKY53 using rosette leaves of why1, oenWHY1, wild-type (WT), and PWHY1 plants. why1, whirly1; oenWHY1, oenWHIRLY1; and PWHY1, Pwhirly1:WHIRLY1. The relative level (input %) was normalized to that of 5-week-old WT. Three biological replicates and three technique replicates were used to analyze. Error bar shows the SD (n = 3×3). Asterisk indicate significant differences (P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001) based on Student’s t-test. (C–E) The binding affinity of WHIRLY1 at promoter of WRKY53 (pWRKY53II) (C), H3K4me3 (D), and H3K9ac (E) levels at WRKY53II and WRKY53P regions of WRKY53 using 6-week-old rosette leaves of PWHY1 plants after 24 h induction with 1, 5, and 10 μM 3-Deazaneplanocin A, dimethylsulfoxide (Dznep) was used as a control in treatment. Actin7 was used as an inner control for ChIP–qPCR. ChIP–qPCR were carried out with antibody against HA, H3K4me3, and H3K9ac. Three biological replicates and three technique replicates were used to analyze. Asterisk indicate significant differences (P < 0.05 and ∗∗P < 0.01) based on Student’s t-test.
FIGURE 3
FIGURE 3
WHIRLY1 enhances H3K9 deacetylation and represses RNAPII recruitment at WRKY53 at leaf early senescence stage. (A,B) ChIP experiments were performed to assess H3K9ac (A) and RNAPII occupancy (B) at WHIRLY1 binding region (WRKY53II) and translation start region (WRKY53P) of WRKY53 using rosette leaves of why1, oenWHY1, wild-type (WT), and PWHY1 plants. The relative level (input %) was normalized to that of 5-week-old WT. Three biological replicates and three technique replicates were used to analyze. Error bar shows the SD (n = 3×3). Asterisk indicate significant differences (P < 0.05 and ∗∗P < 0.01) based on Student’s t-test. (C) Expression of senescence-associated genes in 7-week-old why1, oenWHY1, WT, and PWHY1 plants. The transcript level in each case was normalized to that of GAPC2 as a reference gene and the expression level of WT was set as 1. Three biological replicates and three technique replicates were used to analyze. Asterisk indicate significant differences (P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001) based on Student’s t-test. (D) Senescent leaf fraction and chlorophyll content in 7-week-old why1, oenWHY1, WT, and PWHY1 plants. Mean and SD of at least 12 independent measurements are shown. Error bars represent SE.
FIGURE 4
FIGURE 4
WHIRLY1 impacts H3K4me3, H3K9ac levels on WRKY53 promoter in vitro. (A) Schematic of the in vitro ChIP assays. (B) Schematic of the pG5ML template indicating the amplicons used for qRT-PCR. (C) In vitro ChIP was performed with assembled chromatin. Chromatin was incubated with or without recombinant WHIRLY1 protein expressed in E. coli (Miao et al., 2013) acetylated by p300 or methylated by S-adenosyl-l-methionine (SAM), digested with MNase, and immunoprecipitated with antibodies against WHIRLY1, H3, H3K4me3 or H3K9ac, or RNAPII (A). The ChIP-quantitative PCR was carried out using primer containing WHIRLY1 binding sites (WRKY53II) and TATA box. H3K4me3 and H3K9ac levels were relative to H3 levels. Three biological replicates and three technique replicates were used to analyze. Asterisk indicate significant differences (∗∗P < 0.01 and ∗∗∗P < 0.001) based on Student’s t-test. Error bars represent SE. (D) The report gene transcription by run-on assay. (E) The report gene transcription level by qRT-PCR. Three biological replicates and three technique replicates were used to analyze. Error bars represent SE.
FIGURE 5
FIGURE 5
WHIRLY1 affects H3K4me3 and H3K9ac at the preinitiation conformation stage. (A) Schematic of the in vitro transcription assay. (B) ChIP experiments were performed on chromatin assembled in vitro after transcription in presence or absence of 0.01% sarkosyl or 800 nM B2RNA with indicated antibodies. H3K4me3 and H3K9ac levels were relative to H3 levels. Three biological replicates and three technique replicates were used to analyze. Error bars represent SE. (C) The report gene transcription level by qRT-PCR. Three biological replicates and three technique replicates were used to analyze. Error bars represent SE.
FIGURE 6
FIGURE 6
WHIRLY1 affects the HDAs gene expression. The expression of histone modification-related genes in 6-week-old why1, oenWHY1, and wild-type (WT) plants by qRT-PCR. The transcript level in each case was normalized to that of GAPC2 as a reference gene, and the expression level at WT was set as 1. Three biological replicates and three technique replicates were used to analyze. Error bars represent SE. Asterisk indicate significant differences (P < 0.05 and ∗∗P < 0.01) based on Student’s t-test.
FIGURE 7
FIGURE 7
Working model of WHIRLY1 regulates WRKY53 transcription. WHIRLY1 binding on WRKY53 promoter represses enrichment of H3K4 methylation by ATX1 inhibiting the PIC formation in leaf senescence initiation and modulates the enrichment of H3K9ac by association with HDACs and high-level transcription at early senescence stage. In absence of WHIRLY1, other transcription activators recruit histone lysine methyltransferase and HDACs is dissociated from WRKY53 resulting in elevated H3K4me3, H3K9ac levels, and higher occupancy of RNAPII in WRKY53 loci, thus increasing the transcription of WRKY53. TF, transcription factor; HDACs, histone deacetylases; RNAPII, RNA polymerase II; HKMT, histone lysine methyltransferase; nWHY1, nuclear WHY1 protein.

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