Substitutions in the amino-terminal tail of neurospora histone H3 have varied effects on DNA methylation

Abstract

Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3(K9L) or hH3(K9R)) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3(WT)). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9.

Figure 1. Reactivation of a methylation-silenced transgene by amino acid substitutions in the N-terminal tail of histone H3.

(A) Sequence of N. crassa H3 amino-terminal tail and summary of results after transformation of strain N644 with hH3 alleles bearing indicated mutations. (See also Figure S1). (B) Scheme to test the possible role of H3 residues in DNA methylation. Strain N644 harbors a methylated, silenced hph transgene, which confers resistance to hygromycin when it is expressed. Transformation of this strain with mutant forms of hH3 normally result in a mixture of wild-type and mutant histone in the cell. If the tested amino acid residue is critical for DNA methylation this may cause a semi-dominant loss of DNA methylation and thus expression of hph. (C) Partial loss of DNA methylation in transformants with native hH3 plus ectopic mutant hH3 alleles. DNA from selected hygromycin-resistant transformants [hH3^R2L (N3079); hH3^K4L (N3081); hH3^R8A (N3082); hH3^K9L (N3084); hH3^S10A (N3085); hH3^T11A (N3087); hH3^G12P (N3088); hH3^+G13 (N3090); hH3^G13M (N3092); hH3^K14R (N3094); hH3^K14Q (N3096); hH3^K18R (N3098)] was digested with 5-methylcytosine-sensitive restriction enzymes (BamHI and EcoRI) and analyzed by Southern hybridization. The blots were probed for the 8:A6 region , which is normally methylated (m), giving rise to 4.0 and 3.1 kb fragments. Complete loss of methylation, as in the dim-2 mutant, gives rise to 2.8 and 0.3 kb fragments. The levels of DNA methylation in various strains, determined by the relative intensities of the 3.1 and 2.8 kb bands using ImageJ are indicated in the pie-charts (bottom). To convert the images into the approximate methylation levels conveyed in the pie graphs, we used densitometry to compare the intensity of bands representing methylated and unmethylated sites and normalized the results to that in wild-type (defined as 100%). Similar results were observed for other methylated regions including ψ₆₃, 2:B3 (see also Figure S2, Table S4), 5:B8, 8:G3, 1d21 and 8:F10 (data not shown).

Figure 2. DIM-5 activity on histone H3 peptides.

(A) Residues surrounding H3K9 are critical for DIM-5 activity. Histone methyltransferase assays were performed with GST-H3 (1–57) bound to glutathione-agarose and S-adenosyl [methyl-³H]-L-methionine as the methyl group donor and analyzed by gel electrophoresis and fluorography. The membrane was stained with Ponceau S to confirm amount of protein loaded (bottom panel). (B) DIM-5 is sensitive to modifications of the H3 tail. In vitro assays were performed with unmodified H3 peptide (residues 1–20) or with peptides bearing covalent modifications (K4me2, S10ph and K14ac) or a substitution (K4L). Incorporation of the methyl group was analyzed by fluorography (left panel) or by liquid scintillation counting (right panel, each bar represents an average of three reactions).

Figure 3. Substitutions in the H3 tail can cause recessive or semi-dominant loss of DNA methylation.

(A) Generalized genotypes of strains harboring two copies of hH3. Various substitution alleles (hH3^mutant) were integrated at an ectopic location (his-3) and combined with either the wild-type allele (hH3⁺; left) or the null allele (hH3^RIP1; right) at the native hH3 locus. A gene encoding HP1-GFP was integrated at the pan-2 locus to visualize heterochromatin by microscopy (Figure 4B). (B) Defective asexual development in stains containing mutant H3. The H3 mutants showed reduced growth relative to wild-type (N150) and reduced production of asexual spores. The strains tested carried the following mutations: hH3^R2L (N3520); hH3^A7M (N3537); hH3^R8A (N3542); hH3^S10A (N3474); hH3^A15M (N3553); hH3^P16A (N3556); hH3^R17L (N3560); hH3^K18R (N3565); hH3^K23R (N3568). All strains were grown on solidified minimal medium with 1.5% sucrose for 7 days at 32°C. (See also Figure S3). (C) Loss of DNA methylation caused by H3 substitutions. Southern blotting with DNA isolated from strains that contain either a mixture of wild-type and altered H3 [top panel: hH3^R2L (N3524); hH3^A7M (N3540); hH3^R8A (N3543); hH3^K9L (N3544); hH3^S10A (N3494); hH3^S10G (N3475); hH3^S10E (N3476); hH3^G12P (N3546); hH3^G13M (N3548); hH3^K14Q (N3550); hH3^A15M (N3554); hH3^P16A (N3558); hH3^R17L (N3562); hH3^K18R (N3566); hH3^K23R (N3570); hH3^K27L (N3572); hH3^S28A (N3573)] or just altered H3 [bottom panel: hH3^R2L (N3520); hH3^A7M (N3537); hH3^R8A (N3542); hH3^S10A (N3474); hH3^S10G (N3477); hH3^S10E (N3478); hH3^A15M (N3553); hH3^P16A (N3556); hH3^R17L (N3560); hH3^K18R (N3565); hH3^K23R (N3568)]. DNA was digested with the 5mC-sensitive restriction enzyme AvaII and the blots were probed for the methylated region 2:B3 . Levels of DNA methylation are summarized as in Figure 1 based on the relative intensitites of 2.3 and 0.9 kb bands. Similar results were observed for other methylated regions including 2:G12 and 8:A6 (see also Figures S4 and S5, Table S4).

Figure 4. Reduced H3K9 methylation and HP1 binding in selected hH3 substitution strains.

(A) Reduction in H3K9 methylation in the A7M and R8A substitution strains. Western blots of nuclear extracts from wild-type (N150), dim-5 (N2264), hH3^A7M (N3537) and hH3^R8A (N3542) strains. An antibody recognizing the C-terminus of H3 served as a loading control. (B) Apparently normal H3K9 methylation and HP1 binding in the hH3^R2L mutant. Western blots of nuclear extracts from wild-type (N150), dim-5 (N2264) and hH3^R2L (N3520) strains. An antibody recognizing the C-terminus of H3 served as a loading control. (C) Localization of HP1-GFP in H3 substitution strains. Duplicate sets of images generated by light (DIC) and fluorescence (HP1-GFP) microscopy on germinating conidia of wild type (N2534), dim-5 (N2542), hH3^A7M (N3537), hH3^R8A (N3542) and hH3^R2L (N3520) strains are shown.