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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

Hisashi Tamaru and Eric U. Selker*

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.

* Corresponding author. Mailing address: Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229. Phone: (541) 346-5193. Fax: (541) 346-5891. E-mail: selker{at}molbio.uoregon.edu.

Molecular and Cellular Biology, April 2003, p. 2379-2394, Vol. 23, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.7.2379-2394.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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