DNA Methylation and Cellular Differentiation

In 1977 I wrote a grant proposal in which I applied to study developmental patterns in enzymatic methylation of DNA in eukaryotes. One part of the proposal was to assay cells at different embryonic developmental stages for maintenance and de novo type methylase activity. With one exception the referees, probably developmental biologists, recommended that the work not be supported because there was no evidence that methylation plays any role in eukaryotic gene regulation. Aside from proving that innovative ideas can seldom be used to successfully compete for grant funds, the skepticism of biologists toward methylation as a regulatory mechanism was, and still is, widespread even among some of those who investigate the problem. That is a healthy situation for all points of view should be brought to bear on a problem of such importance. However, to deny funds to investigate a problem because one has already formed an opinion without evidence is hardly commendable. The great skepticism about the significance of DNA methylation is based in part on the evidence that it is absent or very little used in Drosophila, a favorite organism for genetic and developmental studies. There now remains little doubt that methylation of cytosine in certain CpG sites can strikingly affect the transcription of sequences 3' to the methylated doublet. How this inhibition operates and to what extent it is utilized in cells is still debatable.

Inhalt
I. DNA Methylation and Cell Differentiation: An Overview.- A. Introduction.- B. What New Properties Does Methylation Confer on DNA?.- C. The Origin and Maintenance of Methyl Cytosine in DNA.- D. Differentiation: The Problem Posed.- E. Genome Modifications Which Can Be Associated with Differentiation.- a) Examples of Mobile Genetic Elements in Com.- b) The Discovery of a Mutable Gene.- II. DNA Methylation and Transposable Genetic Elements.- A. Discovery of Enzymatic Methylation of DNA.- B. The First Demonstrated Role of DNA Methylation: Restriction-Modification Systems in Bacteria.- C. Hypotheses for Roles of Methylation in Eukaryotes.- D. Transposable Genetic Elements.- III. Differentiating Systems and Their Methylation Patterns.- A. Differentiation of the Hemopoietic System and Organization of Hemoglobin Genes..- B. Methylation Patterns of Hemoglobin Genes.- C. Differentiation of Lymphocytes and the Role of Methylation.- D. Suppression of Integrated Viral Genomes in Cells by DNA Methylation.- E. The Vitellogenin Genes in Xenopus Are Methylated, But Can Be Expressed Without a Detectable Change in the Pattern.- F. Integrated Retroviruses Are Methylated Early in Development.- G. Suppression of Metallothionein Genes by Methylation.- H. Other Genes That Are Suppressed by Methylation.- I. Correlation Between DNase I Sensitivity of Chromatin änd Cytosine Methylation.- J. The Hpall Sites in an Ovalbumin Gene.- K. The 5' End of the Rat Albumin Gene Is Undermethylated in Cells in Which It Is Expressed.- L. Methylation of Human Growth Hormone and Somatotropin Genes.- M. Methylation of Ribosomal Genes.- IV. DNA Methylation and the Inactive X Chromosome of Mammals.- A. A Brief History of X Chromosome Inactivation Studies.- B. A New Methylation Model.- V. Mechanisms ofSuppression by DNA Methylation.- A. Expression of the Late Viral Protein of SV-40 (VP-1) Is Reduced by Methylation at One Hpall Site.- B. Transcription of a Cloned Adenovirus Gene Is Inhibited by in vitro Methylation.- C. Transcription of a Cloned Human Gamma Globin Gene Is Inhibited by Methylation at the 5' Region Flanking the Structural Gene.- D. The Mechanism by Which Methylated CpG Sites Inhibit Transcription.- E. How Are Methylation Patterns Established and Maintained?.- F. The Specificity of Methylases and the Maintenance of Methylation Patterns.- G. Properties of Eukaryotic Methylases.- H. The Maintenance of Methylation Patterns Imposed in vitro.- I. Deletion of Methylation Patterns Düring Differentiation.- VI. Evolution, Stability and Regulation of Methylation Patterns.- A. The Evolutionary Aspects of DNA Methylation.- B. DNA Methylation and Repair.- C. How Stable Is a Pattern of Methylation?.- D. Overview on the Role of DNA Methylation.- E. An Hypothesis for the Control of Methylation Patterns.- F. A Pyramid of Controls in Vertebrate Cells.- References.