Transcriptional enhancers--on/off gene regulation as an adaptation to silencing in higher eukaryotic nuclei.

Transcriptional enhancers are DNA elements that act at a distance from a gene to regulate its expression. Principally, they seem to antagonize gene silencing, but they can also influence transcription rate. The relative importance and functional relationship of these two processes remains unclear. Cell differentiation requires a stable program of gene expression, with some genes active and others silent. Enhancers could function in this process by preventing incorporation of genes into heterochromatin, thus allowing the expression of genes characteristic of the cell lineage. This hypothesis takes into account the ability of enhancers to antagonize silencing, the association of silenced genes with heterochromatin in differentiated cells, and disruption of that association by enhancers. Comparison with yeast suggests that in higher eukaryotes, stronger and more numerous activating elements have been made necessary by the formation of a powerfully repressive nuclear compartment during cell differentiation.

Retrotransposons as epigenetic mediators of phenotypic variation in mammals.

Phenotypic variation in mammals is frequently attributed to the action of quantitative trait loci (QTL) or the environment, but may also be epigenetic in origin. Here we consider a mechanism for phenotypic variation based on interference of transcription by somatically active retrotransposons. Transcriptionally competent retrotransposons may number in the tens of thousands in mammalian genomes. We propose that silencing of retrotransposons occurs by cosuppression during early embryogenesis, but that this process is imperfect and produces a mosaic pattern of retrotransposon expression in somatic cells. Transcriptional interference by active retrotransposons perturbs expression of neighboring genes in somatic cells, in a mosaic pattern corresponding to activity of each retrotransposon. The epigenotype of retrotransposon activity is reset in each generation, but incomplete resetting can lead to heritable epigenetic effects. The stochastic nature of retrotransposon activity, and the very large number of genes that may be affected, produce subtle phenotypic variations even between genetically identical individuals, which may affect disease risk and be heritable in a non-mendelian fashion.

Reactivation of heritably silenced gene expression in mice.

Epigenetic modifications that suppress gene activity in mammals are generally considered to be cleared in the germline, restoring totipotency of the genome. Here we report the germline inheritance of transcriptional silencing in mice, and reversion to activity after as many as three generations in the silent state. In a series of lines made with a LacZ transgene, one line exhibits variable expressivity: genotypically identical littermates have proportions of beta-Gal-positive erythrocytes that vary over at least four orders of magnitude, and in some offspring expression is completely silenced. The silent state of the transgene is inherited for multiple generations in the founder strain irrespective of the sex of the parent, implying maintenance of the epigenetic state through meiosis. Crosses of silenced mice with C57BL/6 mice result in reactivation of the transgene in approximately a third of F(1) littermates. The silencing involves a stochastic, all-or-none mechanism. Furthermore, silencing is transcriptional and correlates with methylation of the transgene as well as an inaccessible chromatin structure; these changes are reversed when expression is reactivated. This work supports the notion that silent genetic information in mammals can be inherited and later reactivated, and implies a mode of phenotypic inheritance that is less stable than Mendelian inheritance.

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.

To be or not to be active: the stochastic nature of enhancer action.

Transcriptional enhancers are traditionally considered to regulate the rate at which a linked promoter transcribes mRNA, but recent experiments suggest a reevaluation of this model is necessary. Single-cell assays of transgenes reveal that enhancers increase the probability that a reporter gene will be active, but have little or no effect on the transcription rate once a gene has been activated. These results raise the question of how enhancers affect gene expression in their native contexts. A simple interpretation is that enhancers act in a stochastic fashion to increase the probability that a regulated gene will be transcribed; such a model is compatible with programs of cell differentiation in which multiple similar cells subject to similar environmental stimuli do not respond uniformly.

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?

Epigenetic inheritance at the agouti locus in the mouse.

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

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.

The chicken beta-globin 5'HS4 boundary element blocks enhancer-mediated suppression of silencing.

A constitutive DNase I-hypersensitive site 5' of the chicken beta-globin locus, termed 5'HS4 or cHS4, has been shown to insulate a promoter from the effect of an upstream enhancer and to reduce position effects on mini-white expression in Drosophila cells; on the basis of these findings, it has been designated a chromatin insulator. We have examined the effect of the cHS4 insulator in a system that assays both the level of gene expression and the rate of transcriptional silencing. Because transgenes flanked by insulator elements are shielded from position effects in Drosophila cells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector with the cHS4 insulator in a colony assay did not increase the number of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the effect of cHS4 on the silencing of an integrated beta-geo reporter at three genomic sites in K562 erythroleukemia cells. In this assay, enhancers act to suppress silencing but do not increase expression levels. While cHS4 blocked enhancement at each integration site, the strength of the effect varied from site to site. Furthermore, at some sites, cHS4 inhibited the enhancer effect either when placed between the enhancer and the promoter or when placed upstream of the enhancer. These results suggest that the activity of cHS4 is not dominant in all contexts and is unlikely to prevent silencing at all genomic integration sites.

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.

The beta-globin LCR is not necessary for an open chromatin structure or developmentally regulated transcription of the native mouse beta-globin locus.

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.

Notch1 and Notch2 inhibit myeloid differentiation in response to different cytokines.

We have compared the ability of two mammalian Notch homologs, mouse Notchl and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.

Repeat-induced gene silencing in mammals.

In both plants and Drosophila melanogaster, expression from a transgenic locus may be silenced when repeated transgene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the lox/Cre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.

A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate.

c-Jun inhibits NF-E2 transcriptional activity in association with p18/maf in Friend erythroleukemia cells.

We have reported previously that antisense c-jun overcomes a block of Friend erythroleukemia cells to differentiation suggesting that the factor c-Jun may be an important negative regulator of erythroid differentiation. The recently described erythroid transcription factor NF-E2 plays an important role in the regulation of the transcription of globin genes and recognizes a sequence containing an AP-1 site. NF-E2 is a complex of two bZip proteins, p45 and p18/Maf. In order to determine whether c-Jun can interact with NF-E2/AP-1 sites to regulate transcriptional activation from them, we have compared the activity of AP-1 and NF-E2 in transient transcriptional assays, in erythroid and nonerythroid cells in the presence of c-jun sense and antisense expression vectors. In non-erythroid cells, c-Jun activates and NF-E2p18 inhibits both AP-1 and NF-E2 activities, suggesting that NF-E2/AP-1 sites function as AP-1 binding sites in these cells. In contrast, NF-E2p18 is a positive regulator of NF-E2 activity in erythroid cells. c-Jun alone is also a positive regulator of NF-E2 activity in erythroid cells but in association with NF-E2p18 inhibits this activity. Moreover antisense c-jun increases endogenous NF-E2 activity in erythroid cells. These results suggest that c-Jun could act as a repressor of NF-E2 transcriptional activity by forming inactive c-Jun/NF-E2p18 heterocomplexes which interfer with the transcription of globin genes in Friend erythroleukemia cells.

The vagaries of variegating transgenes.

Expression of transgenes in mice, when examined with assays that can distinguish individual cells, is often found to be heterocellular, or variegated. Line-to-line variations in expression of a transgene may be due largely to differences in the proportion of cells in which it is expressed. Variegated silencing by centromeric heterochromatin is well described, but other factors may also affect transgene silencing in mice. Tandem arrays of transgenes themselves form heterochromatin, and some cell lineages may tend to silence transgenes because of extensive facultative heterochromatin in their nuclei. The cis-acting transcriptional control elements within a transgene inhibit silencing, and strain-specific differences in chromatin proteins may strongly influence the extent of variegation. The accessibility of multiple differentiated cell lineages in mice suggests that they may provide a tool for dissecting the role of chromatin-mediated silencing in cell differentiation and tissue-specific gene expression.

Inhibition of granulocytic differentiation by mNotch1.

Effective hematopoiesis requires the commitment of pluripotent and multipotent stem cells to distinct differentiation pathways, proliferation and maturation of cells in the various lineages, and preservation of pluripotent progenitors to provide continuous renewal of mature blood cells. While the importance of positive and negative cytokines in regulating proliferation and maturation of hematopoietic cells has been well documented, the factors and molecular processes involved in lineage commitment and self-renewal of multipotent progenitors have not yet been defined. In other developmental systems, cellular interactions mediated by members of the Notch gene family have been shown to influence cell fate determination by multipotent progenitors. We previously described the expression of the human Notch1 homolog, TAN-1, in immature hematopoietic precursors. We now demonstrate that constitutive expression of the activated intracellular domain of mouse Notch1 in 32D myeloid progenitors inhibits granulocytic differentiation and permits expansion of undifferentiated cells, findings consistent with the known function of Notch in other systems.

An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression.

The mouse metallothionein-I (mMT-I) promoter is activated by the metal response element-binding transcription factor (MTF), which binds metal response elements (MREs) when stimulated with heavy metals. We analyzed eight K562 erythroleukemia cell clones, each carrying a single integrated copy of an mMT-I/beta-geo construct, using a system that can independently assess the level of beta-geo expression and the rate at which it is silenced. In these clones, basal expression and rate of silencing vary widely and independently with integration site. This implies that the rates of transcription and of silencing are separate properties determined by interaction of the regulatory elements of the transgene with the site of integration. Induction of the mMT-I promoter with zinc both increases expression level and strongly retards silencing of beta-geo expression. At a given integration site, expression level and silencing are affected coordinately by induction. Taken together with earlier studies of distant metal-responsive elements, these results suggest that distance from the promoter may determine whether a factor can increase transcription rate. Stimulation of an MRE can both increase transcription and overcome repressive effects of chromatin; we suggest that these functions are linked.

Regulation of beta-globin gene expression: straightening out the locus.

A casual examination of the globin literature would leave most readers with the impression that all aspects of beta-globin gene regulation are controlled by the upstream locus control region (LCR). There is no clear evidence, however, that the LCR affects transcription in the beta-globin locus other than by altering its topology to maintain it in a state permissive for expression of the globin genes. Developmental switching of the globin genes may be independent of the LCR, relying only on elements close to the genes and the arrangement of the genes with respect to each other.