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Molecular and Cellular Biology, April 2003, p. 2379-2394, Vol. 23, No. 7
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.7.2379-2394.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Synthesis of Signals for De Novo DNA Methylation in Neurospora crassa

Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229,

Received 30 September 2002/ Returned for modification 26 November 2002/ Accepted 2 January 2003

Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA)_n or (TTAA)_n were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5' of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals.

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