- Email: [email protected]
Gene Regulation: Insulating chromatin
ALAN
GENE REGULATION~~~~~~~~~~~~~~~~
RWOLFFE ALAN WOLFFE
GENE REGULATION
Insulating chromatin 'Insulator elements' have been identified that define the limits of transcriptionally active chromatin domains, protecting them against the repressive influence of neighboring heterochromatin. It is becoming increasingly recognized that eukaryotic chromosomes are organized into discrete functional domains. This is largely a result of experiments exploiting the phenomenon of position-effect variegation in Drosopbila I1], in which the expression state of a gene is affected by its chromosomal position - in particular its proximity to transcriptionally inactive, condensed heterochromatin. Thus, if a normally active gene is introduced into a chromosome at a position adjacent to a heterochromatin domain, its transcription will be significantly repressed. This is believed to occur through a spreading of heterochromatin structure into the normally active gene. This phenomenon provides a useful screen for genes that suppress or enhance positioneffect variegation, conveniently assayed using a suitable reporter gene, which is often chosen to be one influencing Drosophila eye coloration. Many of the genes influencing position-effect variegation have been characterized, and they have been found to encode either structural components of chromatin itself or enzymes capable of modifying chromatin organization [2]. Position-effect variegation is now recognized to be a universal phenomenon among eukaryotes. In yeast, genes integrated near the silent mating-type loci or chromosomal telomeres are repressed. This silencing effect can spread at least 5-10 kilobases (kb), but not as much as 20-30kb, in yeast. DNA is progressively compacted within the chromosome: each nucleosome contains - 166 bp of DNA, nucleosomal arrays compact
into the chromatin fibre with - 6 nucleosomes per turn and the chromatin fibre is arranged in loops of 5-100 kb. An effect that can spread over 5-10 kb would therefore involve many nucleosomes, a few turns of the chromatin fibre and usually less than one loop domain. Thus, modifications to the chromatin fibre or to a small chromatin loop domain could most economically account for the transmissibility of position effects. As in Drosophila, genes influencing position effects in yeast encode structural components of chromatin or enzymes associated with chromatin modification [2,3]. Position effects in mammalian chromosomes have been a recurrent problem for experiments involving transgenic animals, as reporter genes introduced randomly into the genome have highly variable transcription levels [4,5]. These effects of chromosomal position on transcription can be relieved by linking the transgene to sequences known as locus control regions (LCRs), which have a dominant, activating effect on transcription in a large chromatin domain (10-100kb). The mechanism by which LCRs exert this effect remains to be determined, though there is clear evidence that LCR activity is associated with the modification of chromatin structural components [2]. These observations on LCRs reinforce the view that chromosomes are compartmentalized into discrete functional units, which in their natural chromosomal context are prevented from influencing each other. The sequences that prevent the transmission of chromatin structural features associated
Fig. 1. Anatomy of an insulator. The complex of the Drosophila su(Hw) protein with a region of the gypsy transposable element has the properties of an insulator (see text). The model for the interaction between the su(Hw) protein and gypsy 10 bp repeats isbased on experimental data [1 7]. 85
86
Current Biology 1994, Vol 4 No 1 with repressed or active chromosomal domains have become known as 'insulators' [6]. The first evidence for chromosomal insulators came from genetic experiments with Drosophila. Each boundary of the Drosophila 87A7 heat-shock locus is defined by a pair of nuclease-hypersensitive sites bordering a 250-300bp segment of DNA. These specialized chromosome structures (scs) are located at the junctions between the decondensed chromatin of the actively transcribed 87A7 heat-shock locus and adjacent condensed chromatin. Three functional properties of the scs have been identified: first, they establish a domain of independent gene activity at many distinct chromosomal positions; second, scs elements are required at each boundary of the domain; and third, scs elements independently have neither inhibitory or stimulatory effects on transcriptional activity within the domain they define [7]. Subsequently, scs elements were found to block enhancer activity when interposed between an enhancer and a promoter [8]. Thus, scs elements prevent the transmission of both repressive effects on transcription from heterochromatin and stimulatory effects on transcription from an enhancer. How this insulation is achieved has not yet been established and, moreover, the nature of the nucleoprotein complex that assembles on scs elements has yet to be determined. There is, however, a well-defined nucleoprotein complex in Drosophila with similar properties to scs elements. This is the complex that forms between the gypsy transposable element and the suppressor of Hairy wing (su(Hw)) protein [9]. Insertion of a gypsy element as far as 10-30kb from a promoter can cause a mutant phenotype [10]. The mutant phenotype requires the su(Hw) protein to interact with the inserted gypsy element at a 350bp region containing twelve copies of a 10bp sequence separated by AT-rich sequences (Fig. 1). The su(Hw) protein has a molecular weight of 100kD, and its sequence includes several motifs characteristic of eukaryotic transcription factors, including twelve zinc fingers, a leucine zipper and two acidic domains [11]. The complex of the su(Hw) protein with a gypsy element has many of the properties of an insulator element: the complex blocks enhancer activity when placed between an enhancer and a promoter [12,13], and when the complex is placed at the boundaries of a gene-containing fragment, the gene is protected from the repressive effects of heterochromatin on transcription [14]. Although in certain circumstances the su(Hw) protein does not independently stimulate or repress transcription of a reporter gene, the su(Hw) protein can occasionally function as a transcriptional activator [9]. This suggests that the function of an insulator may be conferred on sequences by DNA-binding proteins that might, under other circumstances, have more conventional roles in the transcription process. Although it is clear that the su(Hw) protein does not bind to scs elements [14], it seems likely that these elements will form large nucleoprotein complexes with a similar composition. Insulator elements have recently been defined at the boundary of the chicken -globin gene
Fig. 2. Models for the action of insulators. See text for details. chromosomal domain [6]; remarkably, these elements also function in Drosophila, suggesting that similar insulators exist at the boundaries of chromosomal domains in all organisms. How might insulators function? Any models must explain how characteristics of both repressive or active chromatin are restricted to particular chromosomal domains (Fig. 2). Several models have been suggested to explain the activities of LCRs and enhancers (Fig. 2). These elements might, for example, function as entry points for transcription factors, RNA polymerase or general activators such as the SWI1/2/3/snf5/6 complex, which might then track along the DNA until reaching the promoter. Alternatively, the LCR or enhancer complex might associate with the promoter complex by stable looping of the intervening DNA or chromatin, forming a complex that increases the efficiency with which RNA polymerase is recruited and used. Another possibility is that the LCR or enhancer
DISPATCH
complex might cause the gene to assemble into a chromatin structure capable of being transcribed by association with nuclear organelles that act as 'transcription factories' or associate with proteins, such as the SWI1/2/3/snf5/6 complex or histone acetyltransferases, that modify repressive chromatin structure by disrupting nucleosomes. A number of models have also been suggested to explain the repressive effects of heterochromatin (Fig. 2). Repressive chromatin proteins, such as the Drosopbila HP1 protein or histone deacetylases, may undergo local diffusion, enlarging theheterochromatin domain. Alternatively, heterochromatin may be sequestered in a transcriptionally incompetent region of the nucleus.
and accuracy with which transgenes are expressed in experimental animals. Insulation is here to stay. References 1. SCHAFFER CD, WALLRATH LL, ELGIN SCR: Regulating genes by packaging domains: bits of heterochromatin in euchromatin. Trends Genet 1993, 9:35-38. 2. WOLFFE AP, DIMITROV S: Histone modulated gene activity: developmental implications CRC. Crit Rev Euk Gene Exp 1993, 3:167-191. 3. LAURENSEN P, RINE J: Silencers, silencing and heritable transcriptional states. Microb Rev 1992, 56:543-592. 4.
Position-independent, high-level expression of the human -globin gene in transgenic mice. Cell 1987, 51:975-985. 5.
In considering these models, it is important to recognize that the eukaryotic nucleus is a highly organized structure in which DNA is compacted by its association with histone proteins into nucleosomes and the chromatin fibre. Although it is possible that insulators prevent protein tracking or diffusion between active and repressive domains (Fig. 2), it is difficult to envisage how this might occur in the nucleus - where DNA segments separated linearly by many kilobases can be juxtaposed by folding of the DNA helical axis in three dimensions - without invoking some specific attachment of inactive chromatin domains, insulators and active chromatin domains to a nuclear framework. Similar attachments might be required to prevent the juxtaposition, as a result of DNA looping, of LCRs, enhancers and promoters. Perhaps the most economical suggestion is that insulators are nucleoprotein complexes that associate neither with regions or structures in the nucleus where 'transcription factories' load onto DNA [15], nor with regions or structures from which the transcriptional machinery is excluded [16] - instead, the insulators associate with other 'neutral' nuclear structures. The 'neutral' nuclear structures would tether promoter elements where the transmissible activating effects of enhancers or silencing effects of heterochromatin could not occur. This absence of transmissible effects could be accounted for by the exclusion from the 'neutral' nuclear structures of particular transcriptional coactivators normally associated with communication between promoters and enhancers, or of chromatin modification proteins or enzymes normally associated with heterochromatin. The resolution of these issues will require purification of the proteins that associate with insulators, and further analysis of the organization of insulators within functional eukaryotic nuclei. The definition of an insulator as a discrete entity in eukaryotic chromosomes not only provides a useful tool for the further dissection of nuclear architecture and function, but also has important practical implications for the efficiency
GROSVELD F, VAN ASSENDELFT GB, GREAVES D, KOLLIAS G:
STEIF A, WINTER DM, STRATLING WH, SIPPEL AE: A nuclear DNA
attachment element mediates elevated and position-independent gene activity. Nature 1989, 341:343-345. 6.
CHUNG JH, WHITELEY M, FELSENFELD G: A 5' element of the
chicken -globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophtla. Cell 1993, 74:505-514. 7. KELLUM R, SCHEDL P: A position-effect assay for boundaries of higher-order chromosomal domains. Cell 1991, 64:941-950. 8. KELLUM R, SCHEDL P: A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 1992, 12:2424-2431. 9. CORCES VG, GEYER PK: Interactions of retrotransposons with the host genome: the case of the gypsy element of Drosophila. Trends Genet 1991, 7:69-73. 10. JACK J, DORSETT D, DELOTTO Y, LU S: Expression of the Cut locus in the Drosophla wing margin is required for cell type specification and is regulated by a distal enhancer. Development 1991, 113:735-748. 11.
PARKHURST SM, HARRISON DA, REMINGTON MP, SPANA C, KELLEY
RL, COYNE RS, CORCES VG: The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA binding protein. Genes Dev 1988, 2:1205-1215. 12. GEYER PK, CORCES VG: DNA position-effect repression of transcription by a Drosophlla zinc-finger protein. Genes Dev 1992, 6:1865-1873. 13.
14.
15.
HOLDRIDGE C, DORSETT D: Repression of hsp70 heat shock
gene transcription by the suppressor of Hairy-wing protein of Drosophila melanogaster. Mol Cell Biol 1991, 11:1894-1900. ROSEMAN RR, PIRROTrrA V, GEYER PK: The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J 1993, 12:435-442. JACKSON DA, HASSAN AB, ERRINGTON RJ, COOK PR: Visualization
of focal sites of transcription within human nuclei. EMBOJ 1993, 12:1059-1065. 16.
PALLADINO F, LAROCHE T, GILSON E, AXELROD A, PILLUSL, GASSER
SM: SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres. Cell 1993, 75:543-555. 17.
SPANA C, HARRISON DA, CORCES VG: The DrosophIla
melanogaster suppressor of Hairy wing protein binds to specific sequences of the gypsy retrotransposon. Genes Dev 1988, 2:1414-1423.
Alan P. Wolffe, Laboratory of Molecular Embryology, NICHHD, NIH, Bethesda, Maryland 20892, USA.
87
GENE REGULATION~~~~~~~~~~~~~~~~
RWOLFFE ALAN WOLFFE
GENE REGULATION
Insulating chromatin 'Insulator elements' have been identified that define the limits of transcriptionally active chromatin domains, protecting them against the repressive influence of neighboring heterochromatin. It is becoming increasingly recognized that eukaryotic chromosomes are organized into discrete functional domains. This is largely a result of experiments exploiting the phenomenon of position-effect variegation in Drosopbila I1], in which the expression state of a gene is affected by its chromosomal position - in particular its proximity to transcriptionally inactive, condensed heterochromatin. Thus, if a normally active gene is introduced into a chromosome at a position adjacent to a heterochromatin domain, its transcription will be significantly repressed. This is believed to occur through a spreading of heterochromatin structure into the normally active gene. This phenomenon provides a useful screen for genes that suppress or enhance positioneffect variegation, conveniently assayed using a suitable reporter gene, which is often chosen to be one influencing Drosophila eye coloration. Many of the genes influencing position-effect variegation have been characterized, and they have been found to encode either structural components of chromatin itself or enzymes capable of modifying chromatin organization [2]. Position-effect variegation is now recognized to be a universal phenomenon among eukaryotes. In yeast, genes integrated near the silent mating-type loci or chromosomal telomeres are repressed. This silencing effect can spread at least 5-10 kilobases (kb), but not as much as 20-30kb, in yeast. DNA is progressively compacted within the chromosome: each nucleosome contains - 166 bp of DNA, nucleosomal arrays compact
into the chromatin fibre with - 6 nucleosomes per turn and the chromatin fibre is arranged in loops of 5-100 kb. An effect that can spread over 5-10 kb would therefore involve many nucleosomes, a few turns of the chromatin fibre and usually less than one loop domain. Thus, modifications to the chromatin fibre or to a small chromatin loop domain could most economically account for the transmissibility of position effects. As in Drosophila, genes influencing position effects in yeast encode structural components of chromatin or enzymes associated with chromatin modification [2,3]. Position effects in mammalian chromosomes have been a recurrent problem for experiments involving transgenic animals, as reporter genes introduced randomly into the genome have highly variable transcription levels [4,5]. These effects of chromosomal position on transcription can be relieved by linking the transgene to sequences known as locus control regions (LCRs), which have a dominant, activating effect on transcription in a large chromatin domain (10-100kb). The mechanism by which LCRs exert this effect remains to be determined, though there is clear evidence that LCR activity is associated with the modification of chromatin structural components [2]. These observations on LCRs reinforce the view that chromosomes are compartmentalized into discrete functional units, which in their natural chromosomal context are prevented from influencing each other. The sequences that prevent the transmission of chromatin structural features associated
Fig. 1. Anatomy of an insulator. The complex of the Drosophila su(Hw) protein with a region of the gypsy transposable element has the properties of an insulator (see text). The model for the interaction between the su(Hw) protein and gypsy 10 bp repeats isbased on experimental data [1 7]. 85
86
Current Biology 1994, Vol 4 No 1 with repressed or active chromosomal domains have become known as 'insulators' [6]. The first evidence for chromosomal insulators came from genetic experiments with Drosophila. Each boundary of the Drosophila 87A7 heat-shock locus is defined by a pair of nuclease-hypersensitive sites bordering a 250-300bp segment of DNA. These specialized chromosome structures (scs) are located at the junctions between the decondensed chromatin of the actively transcribed 87A7 heat-shock locus and adjacent condensed chromatin. Three functional properties of the scs have been identified: first, they establish a domain of independent gene activity at many distinct chromosomal positions; second, scs elements are required at each boundary of the domain; and third, scs elements independently have neither inhibitory or stimulatory effects on transcriptional activity within the domain they define [7]. Subsequently, scs elements were found to block enhancer activity when interposed between an enhancer and a promoter [8]. Thus, scs elements prevent the transmission of both repressive effects on transcription from heterochromatin and stimulatory effects on transcription from an enhancer. How this insulation is achieved has not yet been established and, moreover, the nature of the nucleoprotein complex that assembles on scs elements has yet to be determined. There is, however, a well-defined nucleoprotein complex in Drosophila with similar properties to scs elements. This is the complex that forms between the gypsy transposable element and the suppressor of Hairy wing (su(Hw)) protein [9]. Insertion of a gypsy element as far as 10-30kb from a promoter can cause a mutant phenotype [10]. The mutant phenotype requires the su(Hw) protein to interact with the inserted gypsy element at a 350bp region containing twelve copies of a 10bp sequence separated by AT-rich sequences (Fig. 1). The su(Hw) protein has a molecular weight of 100kD, and its sequence includes several motifs characteristic of eukaryotic transcription factors, including twelve zinc fingers, a leucine zipper and two acidic domains [11]. The complex of the su(Hw) protein with a gypsy element has many of the properties of an insulator element: the complex blocks enhancer activity when placed between an enhancer and a promoter [12,13], and when the complex is placed at the boundaries of a gene-containing fragment, the gene is protected from the repressive effects of heterochromatin on transcription [14]. Although in certain circumstances the su(Hw) protein does not independently stimulate or repress transcription of a reporter gene, the su(Hw) protein can occasionally function as a transcriptional activator [9]. This suggests that the function of an insulator may be conferred on sequences by DNA-binding proteins that might, under other circumstances, have more conventional roles in the transcription process. Although it is clear that the su(Hw) protein does not bind to scs elements [14], it seems likely that these elements will form large nucleoprotein complexes with a similar composition. Insulator elements have recently been defined at the boundary of the chicken -globin gene
Fig. 2. Models for the action of insulators. See text for details. chromosomal domain [6]; remarkably, these elements also function in Drosophila, suggesting that similar insulators exist at the boundaries of chromosomal domains in all organisms. How might insulators function? Any models must explain how characteristics of both repressive or active chromatin are restricted to particular chromosomal domains (Fig. 2). Several models have been suggested to explain the activities of LCRs and enhancers (Fig. 2). These elements might, for example, function as entry points for transcription factors, RNA polymerase or general activators such as the SWI1/2/3/snf5/6 complex, which might then track along the DNA until reaching the promoter. Alternatively, the LCR or enhancer complex might associate with the promoter complex by stable looping of the intervening DNA or chromatin, forming a complex that increases the efficiency with which RNA polymerase is recruited and used. Another possibility is that the LCR or enhancer
DISPATCH
complex might cause the gene to assemble into a chromatin structure capable of being transcribed by association with nuclear organelles that act as 'transcription factories' or associate with proteins, such as the SWI1/2/3/snf5/6 complex or histone acetyltransferases, that modify repressive chromatin structure by disrupting nucleosomes. A number of models have also been suggested to explain the repressive effects of heterochromatin (Fig. 2). Repressive chromatin proteins, such as the Drosopbila HP1 protein or histone deacetylases, may undergo local diffusion, enlarging theheterochromatin domain. Alternatively, heterochromatin may be sequestered in a transcriptionally incompetent region of the nucleus.
and accuracy with which transgenes are expressed in experimental animals. Insulation is here to stay. References 1. SCHAFFER CD, WALLRATH LL, ELGIN SCR: Regulating genes by packaging domains: bits of heterochromatin in euchromatin. Trends Genet 1993, 9:35-38. 2. WOLFFE AP, DIMITROV S: Histone modulated gene activity: developmental implications CRC. Crit Rev Euk Gene Exp 1993, 3:167-191. 3. LAURENSEN P, RINE J: Silencers, silencing and heritable transcriptional states. Microb Rev 1992, 56:543-592. 4.
Position-independent, high-level expression of the human -globin gene in transgenic mice. Cell 1987, 51:975-985. 5.
In considering these models, it is important to recognize that the eukaryotic nucleus is a highly organized structure in which DNA is compacted by its association with histone proteins into nucleosomes and the chromatin fibre. Although it is possible that insulators prevent protein tracking or diffusion between active and repressive domains (Fig. 2), it is difficult to envisage how this might occur in the nucleus - where DNA segments separated linearly by many kilobases can be juxtaposed by folding of the DNA helical axis in three dimensions - without invoking some specific attachment of inactive chromatin domains, insulators and active chromatin domains to a nuclear framework. Similar attachments might be required to prevent the juxtaposition, as a result of DNA looping, of LCRs, enhancers and promoters. Perhaps the most economical suggestion is that insulators are nucleoprotein complexes that associate neither with regions or structures in the nucleus where 'transcription factories' load onto DNA [15], nor with regions or structures from which the transcriptional machinery is excluded [16] - instead, the insulators associate with other 'neutral' nuclear structures. The 'neutral' nuclear structures would tether promoter elements where the transmissible activating effects of enhancers or silencing effects of heterochromatin could not occur. This absence of transmissible effects could be accounted for by the exclusion from the 'neutral' nuclear structures of particular transcriptional coactivators normally associated with communication between promoters and enhancers, or of chromatin modification proteins or enzymes normally associated with heterochromatin. The resolution of these issues will require purification of the proteins that associate with insulators, and further analysis of the organization of insulators within functional eukaryotic nuclei. The definition of an insulator as a discrete entity in eukaryotic chromosomes not only provides a useful tool for the further dissection of nuclear architecture and function, but also has important practical implications for the efficiency
GROSVELD F, VAN ASSENDELFT GB, GREAVES D, KOLLIAS G:
STEIF A, WINTER DM, STRATLING WH, SIPPEL AE: A nuclear DNA
attachment element mediates elevated and position-independent gene activity. Nature 1989, 341:343-345. 6.
CHUNG JH, WHITELEY M, FELSENFELD G: A 5' element of the
chicken -globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophtla. Cell 1993, 74:505-514. 7. KELLUM R, SCHEDL P: A position-effect assay for boundaries of higher-order chromosomal domains. Cell 1991, 64:941-950. 8. KELLUM R, SCHEDL P: A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 1992, 12:2424-2431. 9. CORCES VG, GEYER PK: Interactions of retrotransposons with the host genome: the case of the gypsy element of Drosophila. Trends Genet 1991, 7:69-73. 10. JACK J, DORSETT D, DELOTTO Y, LU S: Expression of the Cut locus in the Drosophla wing margin is required for cell type specification and is regulated by a distal enhancer. Development 1991, 113:735-748. 11.
PARKHURST SM, HARRISON DA, REMINGTON MP, SPANA C, KELLEY
RL, COYNE RS, CORCES VG: The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA binding protein. Genes Dev 1988, 2:1205-1215. 12. GEYER PK, CORCES VG: DNA position-effect repression of transcription by a Drosophlla zinc-finger protein. Genes Dev 1992, 6:1865-1873. 13.
14.
15.
HOLDRIDGE C, DORSETT D: Repression of hsp70 heat shock
gene transcription by the suppressor of Hairy-wing protein of Drosophila melanogaster. Mol Cell Biol 1991, 11:1894-1900. ROSEMAN RR, PIRROTrrA V, GEYER PK: The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J 1993, 12:435-442. JACKSON DA, HASSAN AB, ERRINGTON RJ, COOK PR: Visualization
of focal sites of transcription within human nuclei. EMBOJ 1993, 12:1059-1065. 16.
PALLADINO F, LAROCHE T, GILSON E, AXELROD A, PILLUSL, GASSER
SM: SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres. Cell 1993, 75:543-555. 17.
SPANA C, HARRISON DA, CORCES VG: The DrosophIla
melanogaster suppressor of Hairy wing protein binds to specific sequences of the gypsy retrotransposon. Genes Dev 1988, 2:1414-1423.
Alan P. Wolffe, Laboratory of Molecular Embryology, NICHHD, NIH, Bethesda, Maryland 20892, USA.
87