HP1 Is Essential for DNA Methylation in Neurospora Michael Freitag, Patrick C. Hickey, Tamir K. Khlafallah, Nick D. Read, Eric U. Selker  Molecular Cell  Volume 13, Issue 3, Pages 427-434 (February 2004) DOI: 10.1016/S1097-2765(04)00024-3 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Structure of the hpo Gene (A) The hpo gene on linkage group VI consists of four exons (black boxes), interrupted by three introns. The positions of the chromodomain (CD) and chromoshadow domain (CSD) are indicated. The red asterisk, circle, and squares indicate the positions of nonsense mutations in the hpoRIP1, hpoRIP2, and hpoRIP3 alleles, respectively (see Supplemental Figure S2 on Molecular Cell's website). Primers are indicated by arrows. (B) Comparison of the CD and CSD of HP1 proteins from Neurospora crassa (Nc), Magnaporthe grisea (Mg), Schizosaccharomyces pombe (Sp), Drosophila melanogaster (Dm), Homo sapiens (Hs), and Arabidopsis thaliana (At). Residues identical in all HP1 proteins are indicated by blue asterisks. Colons and periods indicate strong and weak conservation, respectively. (C) All HP1 proteins share the same domain structure, an amino-terminal chromodomain (CD, dark blue) and carboxy-terminal chromoshadow domain (CSD, light blue) interrupted by a hinge region of varying length. The amino terminus of all HP1 proteins contains a run of acidic residues. The CD and CSD are the most conserved regions within HP1 proteins. Molecular Cell 2004 13, 427-434DOI: (10.1016/S1097-2765(04)00024-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 HP1 Is Essential for DNA Methylation Samples of genomic DNA (0.5 μg/lane) of a wild-type strain (wt), a hpo pre-RIP duplication strain (dup), an hpoRIP3 strain (hpo), a dim-2 strain, and a dim-5 strain were digested with 5mC-sensitive Sau3AI (S) or a 5mC-insensitive isoschizomer, DpnII (D), fractionated by agarose gel electrophoresis, stained with ethidium bromide (EtBr), blotted to nylon membrane, and probed sequentially for known methylated regions (8:F10, 9:E1, 11:E5, 9a20, 1d21, 8:G3, 5:B8, rDNA) (see Selker et al., 2003). High-molecular weight fragments indicative of DNA methylation were only detected in Sau3AI digests of genomic DNA from wild-type and pre-RIP strains. Complete digestion was verified by probing with unmethylated regions (e.g., Bml). All samples were loaded on a single gel; replicate center lanes were removed for clarity. In some hpo and dim-5 strain backgrounds, RFLPs can be detected with certain probes when compared to the wild-type strain (e.g., 11:E5, 1d21, and 9a20 for dim-5, and 1d21 and rDNA for hpo). We also observed no DNA methylation in hpoRIP strains in control digests with additional enzymes that can be affected by DNA methylation in Neurospora (data not shown). Positions of size standards (kb) are shown on left. Molecular Cell 2004 13, 427-434DOI: (10.1016/S1097-2765(04)00024-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 HP1-GFP Is Localized to Heterochromatin (A) Fluorescence microscopy on hpo+ strains N2534 (top), N2540 (bottom left), and N2559 (bottom right) reveals that HP1-GFP (GFP) localizes to foci that coincide with regions of dense staining with the DNA dye Hoechst 33258 (DNA and merge). (B) Fluorescence microscopy on dim-5 strains N2541 (left) and N2542 (right) shows that HP1-GFP is largely mislocalized in dim-5 strains. Confocal microscopy (C–G) reveals strong foci of HP1-GFP in hpo+ strain N2540 (C), loss of HP1-GFP localization in dim-5 strain N2541 (D), but retention of the localization in dim-2 strain N2543 (E), and reestablishment of localization in the complemented hpoRIP2 mutant strain N2559 (F). (G) Histone H1-GFP localization is punctate but different from that of HP1-GFP. Scale bar shown in (C) applies also to (D)–(G); scale bar, 1 μm. Molecular Cell 2004 13, 427-434DOI: (10.1016/S1097-2765(04)00024-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 HP1 Is Essential for Normal Growth (A) Radial growth at 32°C of wild-type (blue; N = 6), dim-5 (green; N = 8), and hpo (n = 16) strains. Growth of hpo strains was more variable than that of the other strains, as indicated by the standard deviations (bars). (B) Formation of asexual spores and aerial hyphae observed in a wild-type strain (N150) and hpo+ siblings from hpo × wild-type crosses (N2547, N2549) is greatly retarded in hpo mutants (N2537, N2538, N2552) and somewhat delayed in dim-5 strains (N2264, N2229, N2231). Both hpo and dim-5 strains show irregular radial growth. Cultures were grown 4 days at 32°C. Molecular Cell 2004 13, 427-434DOI: (10.1016/S1097-2765(04)00024-3) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 DNA Methylation in the hpoRIP2 Mutant N2556 Is Restored to Wild-Type Levels by Complementation with the hpo-sgfp Fusion Gene The hpo-sgfp gene under control of the ccg-1 promoter was targeted to the his-3 locus. Genomic DNA (0.5 or 1 μg/lane) of wild-type strain N150 (wt), N1877 (dim-2), N2556 (hpo), and the complemented hpo strain N2559 (hpo+-sgfp+) was digested with 5mC-sensitive Sau3AI (S) or the 5mC-insensitive isoschizomer DpnII (D), fractionated by agarose gel electrophoresis, stained with ethidium bromide (EtBr), blotted to nylon membranes, and probed for known methylated regions (8:F10 and 9:E1; compare to Figure 2 and see Selker et al., 2003). We used an unmethylated control region (Bml) to ascertain complete digestion (data not shown). The complemented transformant also exhibits normal growth (data not shown) and HP1-GFP at heterochromatic foci (see Figures 3A and 3G). Position of size standards (kb) is shown on left. Molecular Cell 2004 13, 427-434DOI: (10.1016/S1097-2765(04)00024-3) Copyright © 2004 Cell Press Terms and Conditions