DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.DNA methylation is essential for mammalian development (27, 37), playing an important role in maintaining transcriptional silencing of genes on the inactive X chromosome, imprinted genes, and parasitic elements (2, 5). In mammals, DNA methylation occurs predominantly on cytosines (m 5 C) in the context of the 5Ј-CpG-3Ј dinucleotide, and this epigenetic mark is propagated on both parent and nascent strands after DNA replication. Recent experiments suggest that DNA methylation may serve as a tag for the recruitment of methyl DNA binding domain proteins and the histone deacetylase complexes with which they interact (24, 35) to generate a chromatin structure that is repressive for transcription (33). However, why some genes or CpG sites are susceptible to methylation while others remain methylation free remains to be determined.Using in vitro-methylated constructs and transient-transfection assays, a number of studies have shown that enhancers and associated transcription factor complexes play an important role in overcoming the repressive effects of methylation (25, 51). Transcription of an ␣-globin reporter construct methylated at low density, for example, only occurs in the presence of an enhancer (6, 7), but even in the presence of this enhancer, dense methylation prevents transcription. Similarly, the degree of repression of an episomal Rous sarcoma virus promoter construct is correlated with increasing methylation density, with a high level of methylation extinguishing expression completely (20). While these experiments reveal the antagonistic relationship between DNA methylation density and transcriptional activity, the methods used preclude analysis of the dynamic relationship between trans...

A locus control region (LCR) is a cis-acting gene-regulatory element capable of transferring the expression characteristics of its gene locus of origin to a linked transgene. Furthermore, it can do this independently of the site of integration in the genome of transgenic mice. Although most LCRs contain subelements with classical transcriptional enhancer function, key aspects of LCR activity are supported by cis-acting sequences devoid of the ability to act as direct transcriptional enhancers. Very few of these "non-enhancer" LCR components have been characterized. Consequently, the sequence requirements and molecular bases for their functions, as well as their roles in LCR activity, are poorly understood. We have investigated these questions using the LCR from the mouse T cell receptor (TCR) ␣/Dad1 gene locus. Here we focus on DNase hypersensitive site (HS) 6 of the TCR␣ LCR. HS6 does not support classical enhancer activity, yet has gene regulatory activity in an in vivo chromatin context. We have identified three in vivo occupied factor-binding sites within HS6, two of which interact with Runx1 and Elf-1 factors. Deletion of these sites from the LCR impairs its activity in vivo. This mutation renders the transgene locus abnormally inaccessible in chromatin, preventing the normal function of other LCR subelements and reducing transgene mRNA levels. These data show these factor-binding sites are required for preventing heterochromatin formation and indicate that they function to maintain an active TCR␣ LCR assembly in vivo.