139 Introduction DNA methylation is involved in DNA-protein interactions [13], protection of DNA against restriction endonucleases [33], and enhancement of mutation and recombination. Besides, it affects DNA structure [49], DNA replication [39], virus latency [4] and the regulation of gene expression in eukaryotic cells [5, 10, 13, 15, 16, 38, 43, 44]. How does DNA methylation modulate gene expression? Previous studies [13, 16] have established a correlation between undermethylation and unimpeded gene expression. They have also shown that the control of the expression of certain genes and the maintenance of a cellular differentiated stage are related to increased cytosine methylation levels [13, 15, 26, 38]. 5-Methylcytosine (MeCyt) appears to be the main modified base in eukaryotic DNA. For example, it occurs predominantly in the so called CpG islands; in animal DNA, an average 70% of all CpG pairs are methylated [3]. For some genes or groups of genes, frequency clusters of the dinucleotide CpG can be found either close to or in the promoter, as well as in the leader or in the 5´ region of these genes. Site-specific methylation studies involving both viral and eukaryotic gene promoters have shown [13] that methylation of only one or a few cytosines at CpG sites can alter transcriptional activity, and that, in most cases in which expression is affected, these methylated sites are part of binding sequences for transcription factors. Therefore, a positive correlation can be established between the following conditions: (i) existence of CpG islands in gene regulation sequences; (ii) cytosine methylation generally originated in these CpG islands; and (iii) methylation in these CpG islands blocks the corresponding coding gene expression by not allowing particular transcription factors to bind to the regulatory region of the gene (a diagram of this gene expression modulation mechanism is shown in Fig. 1). Recently, it has been suggested that DNA methylation suppresses rather than regulates gene expression, and that methylation causes permanent suppression of CpG island promoter activity. This hypothesis is supported by the genomic sequencing of inactive promoters and by observations that silenced genes rarely reactivate spontaneously, that methylation is regulated developmentally, and that in vitro methylation down-regulates the expression of reporter genes. In this article, we present for the first time an updated study of DNA methylation in ciliated protozoa that includes a general view of the ciliate nuclear system, the methylated bases present in it with regard to other eukaryotic microorganisms, the DNA methylation pattern changes during developmental or differentiation processes, and the role of DNA methylation in the regulation of ciliate gene expression. Ciliate nuclear system Nuclear dualism, a main feature of the nuclear system of ciliates, consists of the presence of two different types of nuclei in the same cytoplasm. These are called, according to their sizes, micronucleus (Mi) and macronucleus (Ma). In general, ciliates Juan C. Gutiérrez Sergio Callejas Sara Borniquel Ana Martín-González Department of Microbiology-III, Faculty of Biology, Complutense University of Madrid, Spain Received 20 April 2000 Accepted 3 July 2000 Correspondence to: Juan Carlos Gutiérrez. Departamento de Microbiología-III. Facultad de Biología. Universidad Complutense de Madrid. 28040 Madrid. Spain Tel.: +34-913944968 Fax: +34-913944964 E-mail: jgf00004@teleline.es REVIEW ARTICLE INTERNATL MICROBIOL (2000) 3:139–146 © Springer-Verlag Ibérica 2000 DNA methylation in ciliates: implications in differentiation processes Summary Much experimental evidence on the role of DNA methylation in gene expression has been reported. Here we review reports on DNA methylation in ciliated protozoa, emphasizing its implications in cell differentiation processes. Both types of methylated bases (adenine and cytosine) can be found in macronuclear DNA. The division cycle and conjugation have been studied with regard to adenine methylation, and several different functions have been assigned to the methylation changes detected in these processes. Cytosine methylation changes were analyzed during stomatogenesis of Paramecium and encystment of Colpoda inflata. A comparative analysis with other similar microbial eukaryotic differentiation processes is carried out. Key words DNA methylation · Conjugation · Stomatogenesis · Encystment · Ciliates brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Hemeroteca Cientifica Catalana