BAH Domain Interactions in the DIM-2 Methyltransferase Sabrina Z. Abdulla; Vincent T. Bicocca, PhD; Eric U. Selker, PhD University of Oregon, Institute of Molecular Biology Abstract DIM-2 may require specific interactions Analysis of BAH domain mutants In a prior experiment, targeting of the DIM-5, HP1, and DIM-2 proteins was conducted to gauge DNA methylation activity when each was recruited individually. Localization of DIM-5 and HP1 results in methylation, whereas direct localization of DIM-2 results in little to no methylation. A mutation within histone h3 (hH3) at arginine-2 to a leucine (R2L) results in normal DIM-5 activity, normal HP1 localization and subsequent DIM-2 recruitment, but lacks DIM-2 activity. This leads us to suspect that DIM-2 requires a specific interactions with chromatin for methylation activity, and the R2 residue may be the point of interaction Here, interactions within the DIM-2 methyltransferase protein are analyzed in the model organism Neurospora crassa, with a focus on interactions of the BAH (bromo-adjacent homology) domains and the R2 residue of histone H3 (hH3). In other organisms, BAH domains of proteins, including ORC1 and RSC, have been shown to directly interact with nucleosomes3, 4. As BAH domains have been identified within the DIM-2 protein, it raises the question of whether specific protein-nucleosome interactions are important for DIM-2 activity. Furthermore, R2 mutants of histone H3 have been identified to have normal DIM-2 recruitment patterns, yet lack DIM-2 activity, providing further evidence for a possible DIM-2/nucleosome interaction. We hypothesize that the DIM-2 BAH domains mediate these interactions between DIM-2 and R2 of histone H3 in N. crassa. To test this hypothesis, we performed site-directed mutagenesis experiments to introduce point mutations in the DIM-2 BAH domains, resulting in BAH 1, BAH 2, and BAH 1+2 mutants. Southern blot analysis of the normally methylated 8:A6 heterochromatic region show DNA methylation defects similar to that of ΔDIM-2. Western blot analysis of BAH domain mutants confirmed translation of mature protein. Further analysis of other known methylated regions, as well observing localization of a DIM-2-DAM (DNA adenosine methyltransferase) construct will provide a better idea as to the extent to which DIM-2 activity is affected by BAH domain mutations. Westerns: Southerns: ΔDIM-2 DIM-2 BAH 1 Mutants DIM-2 BAH 1 Mutants WT WT #2 #3 #9 #1 #2 #3 #4 #5 #9 #11 #14 DIM-2 Hypothesis DIM-2 requires a specific interaction for methylation activity The BAH domains of DIM-2 are required for this interaction DIM-2 interacts with the ‘R2’ residue of histone H3 Anti-3xflag Probe: 8:A6 ΔDIM-2 Background DIM-2 BAH 2 Mutants DIM-2 BAH 2 Mutants WT WT #10 #14 #10 #14 Structure of the BAH domains in DIM-2 ortholog, DNA methyltransferase 1 (DNMT-1) DNA in eukaryotes is wrapped around histones and packaged into chromosomes. DNA packed loosely into euchromatin is accessible for transcription, whereas DNA packaged tightly into heterochromatin is not. Heterochromatin formation is a feature important in gene regulation and gene silencing. DIM-2 Probe: 8:A6 Anti-3xflag DIM-2 BAH 1+2 Mutants ΔDIM-2 DIM-2 BAH 1+2 Mutants WT WT #18 #14 #14 #18 #19 #21 premedhq.com DIM-2 Recruitment of certain proteins to histones allows histone and DNA modifications at these sites, leading to heterochromatin formation. In N. crassa, heterochromatin DNA is A:T rich, which serves as a signal for the DIM-5 methyltransferase to trimethylate at lysine-9. HP1 (heterochromatin protein 1) localizes to the trimethylated site (H3K9me3), where it recruits the DIM-2 methyltransferase. DIM-2 is then able to methylate specific cytosines on the DNA (Figure 1). Anti-3xflag Probe: 8:A6 Southern samples were digested with AvaII, which is sensitive to cytosine methylation, then probed for the 8:A6 heterochromatic region. Figure 2. Crystal Structure of BAH domains in DNMT-1. Two BAH domains are linked by an alpha helix, which is hypothesized to allow the domains to work together to contact histones. (Song et al. 2011) Conclusions Are one or both BAH domains required for DIM-2 methylation activity in N. crassa? Figure 1. Model of heterochromatin formation in N. crassa. Important factors involve DIM-5, HP1, and DIM-2 (Lewis et al. 2010) Multiple sequence alignment of BAH domains in various organisms and proteins Mutations within either of the BAH domains result in a loss of methylation phenotype, indicating that both BAH domains are required for DIM-2 activity. Future Directions Southern blot analysis of other known methylated regions to see if the methylation defect is consistent throughout the genome. If only specific regions are affected, whole genome bisulfite sequencing will be performed to determine where these regions are within the genome of N. crassa. BAH-DAM construct: Tagging DIM-2 with DAM allows DAM to methylate adenosines in areas of DIM-2 localization. We will be able to use the DIM-2/BAH-DAM construct to identify whether BAH mutations affect its ability to localize to these regions. DIM-2/nucleosome interactions: nucleosome pulldown and yeast two hybrid assays will be conducted to observe interactions between DIM-2 and the nucleosome. Methylation of the 5 position carbon on specific cytosines (5mC) is performed by DNA methyltransferases (DNMTs). 5 methyl group Figure 3. Three amino acid residues are conserved between species. Proline, phenylalanine, and glutamate residues are conserved, suggesting they play a role in BAH domain functionality (Oliver et al. 2005) While many higher order eukaryotes utilize multiple DNMTs, the model organism Neurospora crassa relies on the DIM-2 (defective in methylation) methyltransferase for cytosine methylation. Past studies6 have found that mutation of the conserved glutamate residue to a lysine disrupts the BAH domains. The same idea was applied here, resulting in the creation of BAH 1, BAH 2, and BAH 1+2 mutants. 5)  Lewis ZA, Adhvaryu KK, Honda S, Shiver AL, Knip M, Sack R, et al. (2010) DNA Methylation and Normal Chromosome Behavior in Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC. PLoS Genet 6(11) 6) Noguchi, K., Vassilev, A., Ghosh, S., Yates, J. L., & DePamphilis, M. L. (2006). The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo. The EMBO Journal, 25(22), 5372–5382. 7) Adhvaryu, K. K., Berge, E., Tamaru, H., Freitag, M., & Selker, E. U. (2011). Substitutions in the Amino-Terminal Tail of Neurospora Histone H3 Have Varied Effects on DNA Methylation. PLoS Genetics, 7(12). Oliver AW, Jones SA, Roe SM, Matthews S, Goodwin GH, Pearl LH (2005) Crystal structure of the proximal BAH domain of the polybromo protein. Biochem J 389: 657–664 5) Kuo, A. J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J. K., … Gozani, O. (2012). The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome. Nature, 484(7392), 115–119. Acknowledgements References Thank you to the Selker Lab and the SPUR program at the University of Oregon for advice and opportunity, as well as to NICDH for providing the necessary funding. R25 Program and OURS: NICHD Summer Research Program at the University of Oregon (NIH-1R25HD070817) 1) Kouzminova, E., & Selker, E. U. (2001). dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora. The EMBO Journal, 20(15), 4309–4323. 2) Moss, T. J., & Wallrath, L. L. (2007). Connections between Epigenetic Gene Silencing and Human Disease. Mutation Research, 618(1-2), 163–174. 3) Kuo, A. J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J. K., … Gozani, O. (2012). The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome. Nature, 484(7392), 115–119. 4) Chambers, A. L., Pearl, L. H., Oliver, A. W., & Downs, J. A. (2013). The BAH domain of Rsc2 is a histone H3 binding domain. Nucleic Acids Research, 41(19), 9168–9182.