HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6(Gag) and EIAV p9(Gag) proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.

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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.In vertebrates, methylation of DNA occurs predominantly at the cytosine of CpG dinucleotides. This reversible modification is required for mouse development (33), plays an active role in X-chromosome inactivation and imprinting (25), and may be involved in tissue-specific gene repression (4) and in the silencing of parasitic sequences (52). Dynamic changes in methylation have been implicated in malignant transformation (26), and thus far two genetic disorders have been correlated to defects in genes involved in maintenance of methylation and methylation-induced repression (18).The predominant consequence of methylation is transcriptional repression, which can be mediated either directly, by blocking the binding of transcription factors to CpG containing binding sites (23), or indirectly by proteins that specifically bind to methylated DNA via a methyl-CpG-binding domain (MDB) (37). Recently, several MBD-containing proteins have been described (19), of which four have been implicated in transcriptional repression. These proteins are thought to modify chromatin structure by recruiting histone deacetylase (HDAC) activity to methylated DNA, resulting in a repressive nucleosomal structure (reviewed in references 1 and 43).The repressive effect of methylation on a given gene depends on the nature of its control elements (such as enhancer and promoter) (2), the density of methylated CpGs (21), the protein environment of a given cell type, and the chromosomal context of the gene, which can support or rep...

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.