Relics of repeat-induced point mutation direct heterochromatin formation in Neurospora crassa

Abstract

Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at 100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere was found in an approximately 350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa. In contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery. These data show that A:T-rich RIP'd DNA efficiently directs methylation of H3K9, which in turn, directs methylation of associated cytosines.

Figure 1.

Chromatin modification profile of previously identified methylated regions. Data from five representative control regions are shown. A scale bar indicating 2 kb is shown at the top right of each plot. The base composition (%GC) and a composite RIP index (CRI) of each region, plotted as the moving average for 500 bp windows with 100 bp steps, are shown at the top of each plot. Enrichment values for MeDIP and ChIP-chip experiments are shown as log₂ values (y-axis) for immunoprecipitation experiments with antibodies to 5mC, H3K9me3, green fluorescent protein for HP1-GFP (HP1) and H3K4me2 for each region. The positions of predicted open reading frames (genes) and the previously identified methylated DNA clones, with their identification numbers, are shown at the bottom.

Figure 2.

Chromatin modification profile for N. crassa LGVII. Base composition of the 3.9 Mb LGVII is shown as the moving average of %GC and the CRI calculated for 500 bp windows with 100 bp steps at the top of the plot. Enrichment values for MeDIP and ChIP-chip experiments are shown as log₂ values indicated on the y-axis (right) for immunoprecipitation experiments with antibodies to 5mC, H3K9me3, green fluorescent protein for HP1-GFP (HP1) and H3K4me2. The positions of predicted open reading frames (predicted genes) and repeats are indicated below. The scale bar on the top left indicates 0.5 Mb.

Figure 3.

Chromatin modification profile for the LGVII centromere and telomeres. For (A) N. crassa LGVII centromere and (B) N. crassa chromosome ends, nucleotide composition is shown as the moving average of %GC and CRI calculated for 500 bp windows with 100 bp steps at the top of the plot. Enrichment values for MeDIP and ChIP-chip experiments are shown as log₂ values indicated on the y-axis (right) for immunoprecipitation experiments using antibodies to 5mC, H3K9me3, green fluorescent protein for HP1-GFP (HP1) and H3K4me2. The position of predicted open reading frames (genes) and repeats are indicated below. The scale bars on the top indicate 30 kb and 2 kb for (A) and (B), respectively. The black dots above each plot in (B) indicate the position of the telomere. The broken line above telVIIR indicates that sequences containing the telomere repeats for telVIIR are missing from the current LGVII sequence assembly.

Figure 4.

Complex localization patterns of HP1. Microarray data from immunoprecipitation experiments using antibodies to 5mC, H3K9me3, green fluorescent protein (for HP1-GFP; HP1), and H3K4me2 are shown as log₂ values for 5mC peaks 35, 33, 34, and 19 (Supplemental Table 1). The position of predicted open reading frames (genes) and repeats are indicated below. Conventional ChIP was performed for each region using antibodies to H3K4me2, H3K9me3, and FLAG (for HP1-FLAG) to validate the microarray data. PCR products obtained using whole cell extracts (WCE), or the indicated immunoprecipitate fraction as the template, were resolved by gel electrophoresis. The black bars labeled P1–P5 depict the position of the expected PCR products within each region. The coding region of histone H4 was used as an internal euchromatin control. The labels to the right of each autoradiograph identify specific PCR products. The enrichment of each PCR product relative to the PCR product for H4 to is shown below.

Figure 5.

Genes and repeats reside in distinct chromatin environments. Scatter plots depicting the relationship between RIP and chromatin modifications are shown for LGVII genes (gray dots) and repeats (black dots). The CRI value calculated for each predicted open reading frame and annotated repeat is plotted on the y-axis. The median enrichment value for (A) 5mC, (B) H3K9me3, and (C) H3K4me2 for each gene and annotated repeat is plotted on the x-axis.

Figure 6.

Distribution of H3K9 methylation in wild-type, hpo, and dim-2 strains. (A) The distribution of H3K9me3 across LGVII is shown for wild-type, dim-5, hpo, and dim-2 strains. (B) The distribution of H3K9me3 is plotted for wild-type, hpo, and dim-2 strains for LGVII 5mC peak 40, (C) the previously identified methylated region 8:B1, and (D) LGVII 5mC peak 33 (Supplemental Table 1 and Selker et al. 2003). Conventional ChIP was performed using antibodies to H3K9me3 for the indicated strains. For each strain, PCR products obtained using the immunoprecipitated fraction (H3K9me3, top) and whole cell extract (WCE, bottom) as a template were resolved by gel electrophoresis. The black bars numbered P2–P7 depict the position of the expected PCR products within each region. The coding region of histone H4 (hH4) was used as an internal euchromatic control. The labels to the right of each autoradiogram indicate specific PCR products. The relative enrichment of each PCR product is shown below.

Figure 7.

N. crassa performs efficient de novo methylation of heterochromatic regions. The distribution of 5mC is shown across LGVII for wild type (wt), the methylation deficient strains dim-2 and hpo, and wild-type daughter strains from two crosses between methylation deficient strains. Enrichment values for MeDIP experiments are shown as log₂ values on the y-axis. The wild-type strain N3668 was obtained by crossing two strains that lack 5mC but retain H3K9me3, dim-2, and hpo. The wild-type strain N3669 was obtained by crossing two strains that lack both H3K9me3 and 5mC, dim-5, and dim-8. Thus, all methylation in these strains is a result of de novo methylation.