Targeting silence: the use of site-specific recombination to introduce in vitro methylated DNA into the genome.

DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by the lack of experimental systems that permit control of the methylation state of genes in a chromosomal context. Here, we describe a novel technique that allows for efficient introduction of methylated and unmethylated DNA into defined sites in the mammalian genome. This protocol utilizes bacterial CpG methyltransferases to methylate the DNA of interest in vitro, followed by site-specific targeting using Cre recombinase. Long-term maintenance of the methylation state in vivo allows analysis of the biological consequences of methylation by direct comparison of the methylated and unmethylated state in the same genomic position.

Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation.

The majority of 5-methylcytosine in mammalian DNA resides in endogenous transposable elements and is associated with the transcriptional silencing of these parasitic elements. Methylation also plays an important role in the silencing of exogenous retroviruses. One of the difficulties inherent in the study of proviral silencing is that the sites in which proviruses randomly integrate influence the probability of de novo methylation and expression. In order to compare methylated and unmethylated proviruses at the same genomic site, we used a recombinase-based targeting approach to introduce an in vitro methylated or unmethylated Moloney murine leukemia-based provirus in MEL cells. The methylated and unmethylated states are maintained in vivo, with the exception of the initially methylated proviral enhancer, which becomes demethylated in vivo. Although the enhancer is unmethylated and remodeled, the methylated provirus is transcriptionally silent. To further analyze the repressed state, histone acetylation status was determined by chromatin immunoprecipitation (ChIP) analyses, which revealed that localized histone H3 but not histone H4 hyperacetylation is inversely correlated with proviral methylation density. Since members of the methyl-CpG binding domain (MBD) family of proteins recruit histone deacetylase activity, these proteins may play a role in proviral repression. Interestingly, only MBD3 and MeCP2 are expressed in MEL cells. ChIPs with antibodies specific for these proteins revealed that only MeCP2 associates with the provirus in a methylation-dependent manner. Taken together, our results suggest that MeCP2 recruitment to a methylated provirus is sufficient for transcriptional silencing, despite the presence of a remodeled enhancer.

Position effects are influenced by the orientation of a transgene with respect to flanking chromatin.

We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression.

Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.

Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.

Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.