The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9

Abstract

Background: Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized.

Results: The selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1) mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased.

Conclusion: We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.

Figure 1

A) Phylogenetic tree of Sir2 homologues from N. crassa (NST-1 through NST-7) and humans (SIRT1 through SIRT7) based on a single CLUSTALW alignment of their putative Sir2 catalytic domains. B) Alignment of the Sir2 domain from S. cerevisiae Sir2p with the corresponding regions of Neurospora sirtuins NST-1, NST-2, NST-3, and NST-5. The most highly conserved regions are underlined in the Sir2p sequence.

Figure 2

Engineered subtelomeric markers. A-C) Top of each panel shows a cartoon map (not to scale) of inserted selectable markers at telomeres VR (contig 7.37, panel A), VIIL (contig 7.251, panel B) and IIR (contig 7.77, panel C). Vertical striped bars represent telomeric (TTAGGG)_n repeats and diagonal striped bars in A represent Pogo LTRs [29]. Gray triangles on Tel VIIL (B) represent CenVII repeat element (194–280 bp from end) and a second unnamed repeat element (280–1077 bp from end). Southern blots of DNA from strains 1 (N150; WT), 2 (N3120; Tel VR::hph), 3 (N3440; TelVIIL::bar), 4 (N3456; TelIIR::bar) digested with the enzyme named at the top of each autoradiogram. Probes used are indicated under each panel and shown as gray bars in the maps.

Figure 3

Telomeric silencing depends on dim-5, hpo and nst genes. Conidia from strains carrying the telomere VR hph allele were spotted at densities noted at the bottom of each panel to test sensitivity to moderate (A) or high (B) levels of Hygromycin. Strains used in A were: N2285 (nst⁺, "WT"); N2130 (nst-1^RIP1); N2635 (nst-3^RIP1). Strains used in B were: N3120 (nst⁺, "WT"); N2833 (dim-2); N2997 (dim-5); N3004 (hpo); N3125 (nst-1^RIP1); N2667 (nst-2^RIP1); N3126 (nst-3^RIP1); N3130 (nst-5^RIP1); N3132 (nst-1, nst-3, nst-5). C) Conidia from strains N3441 (nst⁺), N3447 (dim-2), N3449 (dim-5), N3443 (hpo), N3452 (nst-1^RIP1), N3453 (nst-2^RIP1), N3455 (nst-3^RIP1), and N3445 (nst-5^RIP1) were spotted on plates with or without Basta to assay expression of the bar transgene targeted to telomere VIIL. D) Conidia from strains N3457 (nst⁺) and N3456 (hpo) were spotted on plates with or without Basta to assay expression of the bar transgene targeted to telomere IIR.

Figure 4

Derepression of the subtelomeric transgenes by inhibitors of histone acetyltransferases and DNA methyltransferases. A) Nicotinamide (NAM) relieved silencing of telomeric hph (N2285) but not hph flanked by am^RIP (N2015) at a non-telomeric site. All plates contained 0.6 mg/ml Hygromycin. Plates with NAM contained 0.1 mg/ml (middle plate) or 1 mg/ml (right plate) of the drug. Each plate received approximately 100,000 conidia. B) Approximately 1000 conidia from an nst⁺ strain (N3120) carrying the telVR hph allele were plated in the presence or absence of 0.5 mg/ml Hygromycin to assay the effect of the drugs 5-azacytidine (5AC), Trichostatin A (TSA) and NAM on telomeric silencing. C) Same as B except strain N3441 carrying the tel VIIL bar allele was plated in the presence and absence of 4 mg/ml Basta. Control plates lacking Basta show a lawn of growth.

Figure 5

Histone H4 acetylation is increased in nst mutants. A) Western blots of nuclear extracts from strains N3120 (nst⁺) and N3132 (nst-1^RIP1nst-3^RIP1nst-5^RIP1) probed for the indicated epitopes. Different exposure times were used (not indicated) to avoid saturation of signal. B)In vitro NAD⁺-dependent deacetylase assays with the indicated H3 or H4 peptide substrates (NT, unmodified N-terminal peptide). Grey bars show activity for purified NST-1 protein; white bars represent GST (only) control.

Figure 6

Chromatin immunoprecipitation (ChIP) to compare histone modifications near telomeres in WT and nst triple mutant strains. Telomeric regions investigated are shown in the schematics at left. Vertical striped bars represent telomeric (TTAGGG)_n repeats and horizontal striped bars in A represent Pogo LTRs. Black bars represent open reading frames and are labeled according to the Broad Institute database. Strains N3120 (nst-1⁺) and N3132 (nst-1^RIP1nst-3^RIP1nst-5^RIP1) show increased acetylation of H3 and H4 and decreased histone methylation at the derepressed hph gene at telomere VR. A-D) Immunoprecipitated chromatin was amplified with primers for the indicated regions, shown as gray bars. Values in the figure indicate enrichment of specific modifications at telomeric regions relative to euchromatic (hH4) regions (A-C). To determine these values bands were quantified using ImageQuant software and ratios were calculated relative to input DNA. D) We also tested a non-telomeric heterochromatic region with methylated DNA (8:F10) [27].

Figure 7

Analyses of DNA methylation near telomeres. DNA was digested with isoschizomers DpnII (D) and Sau3AI (S), separated on agarose gels, blotted, and probed with telomere proximal regions to assay DNA methylation in these regions. Strains for the TelVR hph panel: N2292, the primary transformant with hph targeted to tel VR (T); N3120, a nst⁺ strain (WT); N3004, an hpo strain (hpo); N3132, the triple nst-1, -3, -5 mutant (nst). The hph probe was the first 600 bp of the coding region. Strains for the tel VIIL bar panel: N3440, the primary transformant with bar targeted to tel VIIL (T); N3441 (nst⁺); N3443 (hpo). The bar probe was the entire coding region. For the other panels, DNA samples of a wildtype strain, N150 (WT), an hpo mutant, N3004 (hpo), and N3132, a triple nst-1 nst-3 nst-5 mutant (nst) were processed as for the other panels and probed for: telomere IL (Tel IL; 226–1249 bp from end); telomere IVL (TelIVL; 2500–3657 bp from end); telomere VR (Tel VR; 1130–2887 bp from end); telomere VIIL (Tel VIIL; 324–1306 bp from end); a methylated repeat called 8:F10 (see text).

Figure 8

Expression of genes near telomeres is unaffected by nst mutations. Total RNA was extracted [47] from N3120 (nst⁺) and N3132 (nst-1^RIP1nst-3^RIP1nst-5^RIP1) and 20 μg was separated by agarose gel, blotted, and probed as described [48]. The 28S rRNA panel shows equivalent loading in both lanes by methylene blue staining of a blot. Other panels are autoradiograms after probing with the coding region of the gene closest to telomere IL (NCU010129.3), VIL (NCU07143.3), or VIIL (NCU09306.3).