Epigenetic regulation of transcription in intermediate heterochromatin

Abstract

Constitutive heterochromatin is a compact, transcriptionally inert structure formed in gene-poor and repeat- and transposon-rich regions. In Arabidopsis, constitutive heterochromatin is characterized by hypermethylated DNA and histone H3 dimethylated at lysine (K) 9 (H3K9me2) together with depletion of histone H3 dimethylated at lysine 4 (H3K4me2). Here, we describe loci with intermediate properties of heterochromatin in which transcription downregulation is inherited in a manner similar to constitutive heterochromatin, although the loci are associated with opposing histone marks--H3K4me2 and H3K9me2. In the ddm1 (decrease in DNA methylation 1) mutants, their transcriptional activation is accompanied by the expected shift in the H3 modifications--depletion of H3K9me2 and enrichment in H3K4me2. In mom1 (Morpheus' molecule 1) mutants, however, a marked increase in transcription is not accompanied by detectable changes in the levels of H3K4me2 and H3K9me2. Therefore, transcriptional regulation in the intermediate heterochromatin involves two distinct epigenetic mechanisms. Interestingly, silent transgenic inserts seem to acquire properties characteristic of the intermediate heterochromatin.

Figure 1

Accumulation of transcripts from MULE-F19G14–CyP40 and transposons in mom1 and ddm1. (A) RNA blot analysis of MULE-F19G14/CyP40 transcripts. Total RNAs extracted from Col-0 (wild type), ddm1-2 and mom1-2 were probed with CyP40 and MULE-F19G14 probes. Positions of the size markers are shown on the right in kilobases (kb). (B) A chromosomal region containing CyP40 and MULE-F19G14. Filled and open boxes represent exons and introns, respectively. Grey boxes represent terminal inverted repeats of MULE-F19G14. The direction and position of annotated genes are indicated by arrows. A deduced structure of the 7-kb transcript is shown by bars connected with dashed lines corresponding to introns. The positions of probes for RNA blots and ChIP are indicated at the bottom. The main transcription start site observed in mom1 and ddm1 is indicated by a hooked arrow. (C) Accumulation of the transcripts of AtMu1. Reverse transcription–PCR (RT–PCR) was carried out using RNA samples for Col-0 (wild type, wt), ddm1-2, and mom1-2 with (+) or without (−) RT. (D) Accumulation of transcripts of Tar17. RNA blot analysis was carried out using total RNA of Col-0 (wild type (wt)), ddm1-2 and mom1-2. ChIP, chromatin immunoprecipitation; CyP40, Cyclophilin 40; ddm1, decrease in DNA methylation 1; mom1, Morpheus' molecule 1; MULE, Mutator-like element.

Figure 2

DNA and histone methylation in MULE-F19G14/CyP40 and other representative genes. (A) Similarity in the DNA sequence of MULE-F19G14 with other MULEs. The structure and positions of annotated genes are indicated as in Fig 1B. Regions in MULE-F19G14 showing sequence similarities to other MULEs (expected value cutoff=3 in BLASTN) are shown as bars at the bottom, with the corresponding names of BACs carrying the MULEs. (B) DNA blot analysis of a region surrounding the transcription start site of the 7-kb transcript with methylation-sensitive restriction enzymes. Genomic DNAs were digested with SspI (methylation insensitive) followed by digestion with methylation-sensitive restriction enzymes. Recognition sites for the restriction enzymes around the main transcription start site (hooked arrow) of the 7-kb transcript are shown at the bottom. Av, AvaI; d, ddm1-2; Hc, HincII; Hp, HpaII; m, mom1-2; Nh, NheI; Sc, SacII; Sp, SspI; w, Col-0. (C) Bisulphite genomic sequencing of a region surrounding the main transcription start site of the 7-kb transcript. The percentage of methylated cytosine at a particular site is indicated in grey. Positions of cytosines in CpG and CpNpG are indicated by filled and open squares, respectively. Positions of primers, used for ChIP (D), are indicated by arrows. The transcription start site of the 7-kb transcript is indicated by the hooked arrow. (D) ChIP analysis of the methylation status of histone H3. ChIP analysis using antibodies recognizing H3K4me2, H3K9me2, H3K27me2 and H3K27me3 was carried out using chromatin extracts from wild-type plants (C, Col-0), mom1-2 (m) and ddm1-2 (d). Target loci that were analysed are listed on the left (supplementary Table III online); IP, input. (E) ChIP analysis of the methylation status of histones H3 associated with the transgenic HPT locus. A, line A (the wild-type parent of mom1-1; Mittelsten Scheid et al, 1998); IP, input; m, mom1-1. (F) ChIP analysis of the H3 methylation status of histones H3 associated with the AtSN1 transposon. ChIP, chromatin immunoprecipitation; ddm1, decrease in DNA methylation 1; H3K4me2, histone H3 dimethylated at lysine 4; H3K27me2, histone H3 dimethylated at lysine 27; H3K27me3, histone H3 trimethylated at lysine 27; HPT, hygromycin phosphotransferase; mom1, Morpheus' molecule 1; MULE, Mutator-like element. BACs, bacterial artificial chromosomes; CyP40, Cyclophilin 40.