Direct Interaction between DNA Methyltransferase DIM-2 and HP1 Is Required for DNA Methylation in Neurospora crassa

Abstract

DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Gregory Kothe for the construction of yeast two-hybrid vectors pGBDU-dim-2^1-366, pGBDU-dim-2^290-566, and pGBDU-dim-2^431-847 and Glen Cronan and Tamir Khlafallah for advice on fluorescence microscopy. We also thank Zachary Lewis, Keyur Adhvaryu, and T.K. for comments on the manuscript.

This work was supported by grant GM025690-22 to E.U.S. from the National Institutes of Health.