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.

Nuclear relocation of a transactivator subunit precedes target gene activation.

Murine erythroleukemia (MEL) cells are a model system to study reorganization of the eukaryotic nucleus during terminal differentiation. Upon chemical induction, MEL cells undergo erythroid differentiation, leading to activation of globin gene expression. Both processes strongly depend on the transcriptional activator NF-E2. Before induction of differentiation, both subunits of the NF-E2 heterodimer are present, but little DNA-binding activity is detectable. Using immunofluorescence microscopy, we show that the two NF-E2 subunits occupy distinct nuclear compartments in uninduced MEL cells; the smaller subunit NF-E2p18 is found primarily in the centromeric heterochromatin compartment, whereas the larger subunit NF-E2p45 occupies the euchromatin compartment. Concomitant with the commitment period of differentiation that precedes globin gene activation, NF-E2p18, along with other transcriptional repressors, relocates to the euchromatin compartment. Thus, relocation of NF-E2 p18 may be a rate-limiting step in formation of an active NF-E2 complex. To understand the mechanisms of NF-E2 localization, we show that centromeric targeting of NF-E2p18 requires dimerization, but not with an erythroid-specific partner, and that the transactivation domain of NF-E2p45 may be necessary and sufficient to prevent its localization in centromeric heterochromatin. Finally, using fluorescence in situ hybridization, we show that, upon differentiation, the beta-globin gene loci relocate away from heterochromatin compartments to euchromatin. This relocation correlates with both transcriptional activation of the globin locus and relocation of NF-E2p18 away from heterochromatin, suggesting that these processes are linked.

Targeted deletion of 5'HS1 and 5'HS4 of the beta-globin locus control region reveals additive activity of the DNaseI hypersensitive sites.

The mammalian beta-globin locus is a multigenic, developmentally regulated, tissue-specific locus from which gene expression is regulated by a distal regulatory region, the locus control region (LCR). The functional mechanism by which the beta-globin LCR stimulates transcription of the linked beta-like globin genes remains unknown. The LCR is composed of a series of 5 DNaseI hypersensitive sites (5'HSs) that form in the nucleus of erythroid precursors. These HSs are conserved among mammals, bind transcription factors that also bind to other parts of the locus, and compose the functional components of the LCR. To test the hypothesis that individual HSs have unique properties, homologous recombination was used to construct 5 lines of mice with individual deletions of each of the 5'HSs of the endogenous murine beta-globin LCR. Here it is reported that deletion of 5'HS1 reduces expression of the linked genes by up to 24%, while deletion of 5'HS4 leads to reductions of up to 27%. These deletions do not perturb the normal stage-specific expression of genes from this multigenic locus. In conjunction with previous studies of deletions of the other HSs and studies of deletion of the entire LCR, it is concluded that (1) none of the 5'HSs is essential for nearly normal expression; (2) none of the HSs is required for proper developmental expression; and (3) the HSs do not appear to synergize either structurally or functionally, but rather form independently and appear to contribute additively to the overall expression from the locus.

The murine beta-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes.

Locus control regions (LCRs) are defined by their ability to confer high-level tissue-specific expression to linked genes in transgenic assays. Previously, we reported that, at its native site, the murine beta-globin LCR is required for high-level beta-globin gene expression, but is not required to initiate an open chromatin conformation of the locus. To further investigate the mechanism of LCR-mediated transcriptional enhancement, we have analyzed allele-specific beta-globin expression and the pattern of histone acetylation in the presence and absence of the LCR. In single cells from mice heterozygous for a deletion of the LCR, beta-globin expression from the LCR-deleted allele is consistently low ( approximately 1-4% of wild type). Thus, the endogenous LCR enhances globin gene expression by increasing the rate of transcription from each linked allele rather than by increasing the probability of establishing transcription per se. Furthermore, in erythroid cells from mice homozygous for the highly expressing wild-type beta-globin locus, hyperacetylation of histones H3 and H4 is localized to the LCR and active genes. In mice homozygous for the LCR deletion reduced histone hyperacetylation is observed in LCR proximal sequences; however, deletion of the LCR has no effect on the localized hyperacetylation of the genes. Together, our results suggest that, in its native genomic context, the LCR follows the rate model of enhancer function, and that the developmentally specific hyperacetylation of the globin genes is independent of both the rate of transcription and the presence of the LCR.

Activation of beta-major globin gene transcription is associated with recruitment of NF-E2 to the beta-globin LCR and gene promoter.

The mouse beta-globin gene locus control region (LCR), located upstream of the beta-globin gene cluster, is essential for the activated transcription of genes in the cluster. The LCR contains multiple binding sites for transactivators, including Maf-recognition elements (MAREs). However, little is known about the specific proteins that bind to these sites or the time at which they bind during erythroid differentiation. We have performed chromatin immunoprecipitation experiments to determine the recruitment of the erythroid-specific transactivator p45 NF-E2/MafK (p18 NF-E2) heterodimer and small Maf proteins to various regions in the globin gene locus before and after the induction of murine erythroleukemia (MEL) cell differentiation. We report that, before induction, the LCR is occupied by small Maf proteins, and, on erythroid maturation, the NF-E2 complex is recruited to the LCR and the active globin promoters, even though the promoters do not contain MAREs. This differentiation-coupled recruitment of NF-E2 complex correlates with a greater than 100-fold increase in beta-major globin transcription, but is not associated with a significant change in locus-wide histone H3 acetylation. These findings suggest that the beta-globin gene locus exists in a constitutively open chromatin conformation before terminal differentiation, and we speculate that recruitment of NF-E2 complex to the LCR and active promoters may be a rate-limiting step in the activation of beta-globin gene expression.

Vitamin D(3) enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts.

I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D(3), specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D(3) upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D(3) increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D(3) enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D(3).

Inhibition of Tcf3 binding by I-mfa domain proteins.

We have determined that I-mfa, an inhibitor of several basic helix-loop-helix (bHLH) proteins, and XIC, a Xenopus ortholog of human I-mf domain-containing protein that shares a highly conserved cysteine-rich C-terminal domain with I-mfa, inhibit the activity and DNA binding of the HMG box transcription factor XTcf3. Ectopic expression of I-mfa or XIC in early Xenopus embryos inhibited dorsal axis specification, the expression of the Tcf3/beta-catenin-regulated genes siamois and Xnr3, and the ability of beta-catenin to activate reporter constructs driven by Lef/Tcf binding sites. I-mfa domain proteins can regulate both the Wnt signaling pathway and a subset of bHLH proteins, possibly coordinating the activities of these two critical developmental pathways.

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse beta-globin gene clusters.

By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.

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.

Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region.

We have generated mice with a targeted deletion of the beta-globin locus control region (LCR). Mice homozygous for the deletion die early in embryogenesis but can be rescued with a YAC containing the human beta-globin locus. After germline passage, deletion of the LCR leads to a severe reduction in expression of all mouse beta-like globin genes, but no alteration in the developmental specificity of expression. Furthermore, a DNase I-sensitive "open" chromatin conformation of the locus is established and maintained. Thus, the dominant role of the LCR in the native locus is to confer high-level transcription, and elements elsewhere in the locus are sufficient to establish and maintain an open conformation and to confer developmentally regulated globin gene expression.

Long-distance control of origin choice and replication timing in the human beta-globin locus are independent of the locus control region.

DNA replication in the human beta-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the beta-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5'HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5'HS2-5 in the normal chromosomal context of the human beta-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5'HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5'HS2-5 in the classically defined LCR do not control replication in the human beta-globin locus. Recent studies also show that targeted deletion of 5'HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

Independent formation of DnaseI hypersensitive sites in the murine beta-globin locus control region.

Mammalian beta-globin loci are composed of multiple orthologous genes whose expression is erythroid specific and developmentally regulated. The expression of these genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR). The LCR encompasses 5 major HSs, each of which is highly homologous among humans, mice, and other mammals. To analyze the function of individual HSs in the endogenous murine beta-globin LCR, we have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In this report, we demonstrate that deletion of the conserved region of 5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion site but has no influence on the formation of the remaining HSs in the LCR. Therefore, in the endogenous murine locus, there is no dominant or initiating site whose formation must precede the formation of the other HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of beta-globin regulation.

Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human beta-globin locus.

We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous beta-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed beta-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the beta-globin genes.

Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression.

The mouse beta-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the beta(min) and beta(maj) genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.

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.

Nuclear compartmentalization and gene activity.

The regulated expression of genes during development and differentiation is influenced by the availability of regulatory proteins and accessibility of the DNA to the transcriptional apparatus. There is growing evidence that the transcriptional activity of genes is influenced by nuclear organization, which itself changes during differentiation. How do these changes in nuclear organization help to establish specific patterns of gene expression?

A functional enhancer suppresses silencing of a transgene and prevents its localization close to centrometric heterochromatin.

To explore the mechanism by which enhancers maintain gene expression, we have assessed the ability of an enhancer and derivative mutants to influence silencing and nuclear location of a transgene. Using site-specific recombination to place different constructs at the same integration sites, we find that disruption of core enhancer motifs impairs the enhancer's ability to suppress silencing. FISH analysis reveals that active transgenes linked to a functional enhancer localize away from centromeres. However, enhancer mutations that result in increased rates of transgene silencing fail to localize the transgene away from centromeric heterochromatin, even when the transgene is in an active state. These mutations thus dissociate transcriptional activity and subnuclear location. Together, our results suggest that the functional enhancer antagonizes gene silencing by preventing localization of a gene near centromeric heterochromatin.