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Regulation of insulin gene transcription
seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 11, 2000: pp. 227–233 doi: 10.1006/10.1006/scdb.2000.0171, available online at http://www.idealibrary.com on
Regulation of insulin gene transcription Kinuko Ohnedaa , Hooi Eeb and Michael German
DNA technology, and other neuroendocrine cells can further process the proinsulin to mature insulin, package it, and secrete it in a regulated fashion.1 Therefore the mechanisms that control transcription of the insulin gene are essential determinants of both β-cell function and metabolic regulation. The insulin gene is a small gene, located on chromosome 11p15.5 in humans,2 and consisting of three exons and two introns (for reviews of the insulin gene see References 3 and 4). The sequences immediately upstream (50 flanking sequences) of the transcription start site are defined as the insulin promoter, and can by themselves restrict expression of a linked gene to the β-cell.5–7 Studies directed towards understanding how insulin expression is restricted to the β-cell have focused on a small fragment, approximately 400 base pairs, of the proximal insulin promoter (see Figure 1). As defined by mutagenesis studies, multiple sequences along the length of the promoter contribute to its overall activity, by functioning as binding sites for sequence-specific DNA-binding proteins found in the nucleus of the β-cell. Many of these transcription factors have been identified and are shown above their binding sites on the human insulin and rat insulin I promoters in Figure 1.
Recent studies of the insulin gene promoter and the transcription factors that regulate it have expanded our understanding of both how the production of insulin is restricted to the pancreatic β-cell, and how that production is regulated by physiologic signals such as glucose. A picture is emerging in which an elaborate set of transcription factors binds to specific sequences along the promoter and recruits additional transcriptional co-activators to build a functional transcriptional activation complex that is unique to β-cells. Surprisingly, however, genetic experiments in mice have demonstrated an unexpected degree of redundancy in the factors that control insulin gene expression, and have revealed the presence of a network of transcription factors that coordinate the expression of factors forming the insulin gene activation complex. Key words: insulin gene / transcription / glucose / neuroD1 / BETA2 c 2000 Academic Press
The insulin in the circulation of adult mammals is produced exclusively by the pancreatic β-cells, making the β-cells the central regulators of energy storage and use by the rest of the organism. This restriction of insulin production to the β-cell depends on constraints at the level of transcription of the insulin gene, the gene that encodes preproinsulin. In contrast, almost any eukaryotic cell can make proinsulin if it is engineered to transcribe an exogenously introduced insulin gene by recombinant
Formation of the transcription complex on the insulin promoter This linear view of the promoter as a series of binding sites and binding proteins, however, lacks the complexity and dynamic character of the functioning promoter in vivo. Missing is the additional dimension provided by the interactions among the transcription factors, DNA structural proteins, co-activators and repressors, intra-cellular signaling molecules and the RNA polymerase complex. This map also fails to show the upstream factors that regulate the expression and function of these proteins. The promoter should be viewed as a large complex formed by the interactions among many different proteins and the chromosome
From the Department of Medicine and Hormone Research Institute, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0534, USA. Email: [email protected] a Present address: Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Japan. b Present address: Gastroenterology Department, Queen Elizabeth II Medical Centre, Perth, Western Australia 6009. c
2000 Academic Press 1084–9521 / 00 / 040227+ 07 / $35.00/0 / 0
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Figure 1. The human insulin gene and rat insulin I gene promoters with known sequence elements and binding factors. The boxes represent characterized sequence elements. The new names are in boxes; the old names are shown below each promoter. The cloned binding proteins are circled above the promoter.
interact through their DNA binding domains—the homeodomain and bHLH domains, respectively. This interaction leads to cooperative DNA binding, although with purified proteins in vitro the relative increase in binding affinity is small.26 But in vivo, there are many other proteins in the nucleus that can impact on the bHLH-PDX1 interaction. The structure of the chromosome is altered by the binding of histones and of small, abundant DNA-binding proteins, the high mobility group (HMG) proteins. The presence of these structural DNA-binding proteins changes the energetic cost of DNA binding by the transcription factors (for a review see Reference 27). In fact, we have found that in the presence of HMG I(Y), a small non-histone DNA-binding protein that can bind to the A3/4 region of the insulin promoter, co-binding of PDX1 and the bHLH dimer is greatly favored in vitro, and that HMG I(Y) increases PDX–bHLH transcriptional synergy in vivo.26 While cooperative DNA binding by the PDX1/ bHLH/HMG I(Y) complex may increase the occupancy of the E–A sites on the insulin promoter, transcriptional activation requires interaction with the basal transcription machinery. The grouping of transcription factors on DNA creates clusters of protein interaction sites that cooperatively recruit or stabilize binding of the RNA polymerase II transcription initiation complex (reviewed in Reference 28). Non-DNA-binding co-activators promote this process by linking the DNA-bound transcription factors with the basal transcription machinery. The increased stability provided by co-activators probably plays an
in dynamic equilibrium with forces acting from within and outside the cell. As an example of the interactions that generate this complex, we have studied the interactions among proteins that bind to the juxtaposed E and A elements in the insulin gene promoter. Neither the E elements nor the A elements by themselves have any significant transcriptional activity in β-cells; but together, they can dramatically boost the activity of a linked promoter in a β-cell-specific fashion.8, 9 The synergy between these elements results from interactions among the proteins that bind to them and non-DNA-binding transcriptional co-activators. The E elements are binding sites for protein dimers formed by heterodimerization between two members of the family of basic helix-loop-helix (bHLH) proteins: an ubiquitous class A bHLH protein (products of the E2A gene, E12, E47, and E2/5, or HEB), and a cell-type restricted bHLH protein (neuroD1/BETA2).10–16 The A elements are binding sites for several proteins of the homeodomain family, the most abundant in β-cells being PDX1 (also called IPF1, STF1, IDX1, IUF1 or GSF).17–23 A bHLH dimer on the E element can synergize with PDX1 bound to the A element and activate transcription of an E–A minienhancer in a non-β-cell engineered to express these proteins.24, 25 This interaction requires both DNA binding sites, the DNA binding domains of both PDX1 and the bHLH dimer, and the transcriptional activation domains of both PDX1 and the bHLH dimer24, 25 (K. Ohneda and M. German, unpublished data). PDX1 and the bHLH proteins E47 and neuroD1/BETA2 physically 228
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a
b
Figure 3. Glucose regulation of insulin gene expression. Proposed pathways for glucose regulation of insulin gene expression are shown. E represents E2A proteins, and β2 represents the selectively expressed heterodimer partner BETA2/neuroD1. Question marks indicate unknown steps in the pathway or the probable intersection with other pathways.
Figure 2. PDX1 function in β-cells. In (a) an outline is shown of the functional domains of the PDX1 protein. The regions labeled A–E represent conserved domains within the activation domain.25 In (b) interactions are shown between PDX1, the bHLH proteins, and HMGI(Y) that result in transcriptional activation of the E–A minienhancer.
and increases the activation potential of the PDX1 activation domain.41 In addition, glucose increases the binding of the bHLH dimer to the E elements,34 which in turn cooperates with the activated PDX1 bound at the A site to increase insulin gene transcription. But sequences outside the E–A elements are also important for the response to glucose, and the binding of as yet unidentified proteins at the C1 and Za sites is also stimulated by glucose. The overall increase in insulin gene transcription in response to glucose results from the combination of all of these effects (see Figure 3).
essential role in the formation and the function of the insulin promoter transcription activation complex (see Figure 2). The identities of these co-activators in the insulin gene transcription complex are unknown, although both the E2A proteins and neuroD1 can interact with the ubiquitous p300 co-activator.29–31
Glucose regulation of the transcription complex How rigid is the constitution of the transcription complex?
The preceding discussion presents a model by which insulin gene transcription in the β-cell results from the formation of a transcription complex from an interacting set of nuclear proteins. Since formation of an effective complex depends on all of the interacting proteins, the net activity of the promoter can be regulated by altering the concentration or function of any one protein in the complex. Glucose, however, appears to regulate insulin gene transcription through multiple effects on several of the proteins in the transcription activation complex.32–37 Glucose acutely stimulates an increase in PDX1 binding to the A elements,22, 23, 33 due in part to phosphorylation of PDX1 through a pathway that involves PI3 kinase and the stress-activated protein kinase 2 (SAPK2).22, 38 Glucose also causes a shift in the cellular distribution of PDX1 to the nucleus39, 40
If insulin gene transcription proceeds from the formation on the promoter of a transcription complex from an interacting set of nuclear protein factors, then the β-cell specificity of insulin gene transcription results from the specificity of this complex. No single protein member of the complex needs to be restricted in expression to the β-cell if the set of protein factors is unique to the β-cell, and the formation of the complex depends on the presence of all the members of the set. This model for β-cell-specific insulin gene transcription makes two predictions: first, the protein interactions that form the complex should be highly specific, so that different proteins present in other cell nuclei cannot be substituted in the interaction. Second, in the absence of any one of 229
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The ability of the Lmx1 proteins to synergize with the bHLH dimer suggests that other proteins in the β-cell nucleus may be able to substitute for PDX1 in the transcription activation complex on the insulin promoter. Further evidence that PDX1 may not be essential for insulin gene transcription comes from genetic studies in mice. Mice that are homozygous for a targeted disruption of the PDX1 gene fail to develop a pancreas beyond the initial bud stage;44–46 but rare insulin-producing cells do appear in the bud.46 If the PDX1 gene is inactivated after the pancreas forms and the β-cells have differentiated, the β-cell mass decreases and β-cell function is impaired, but the remaining β-cells continue to produce insulin.47 Furthermore, levels of PDX1 protein can be dramatically reduced in β-cell lines using antisense oligonucleotides without causing any reduction in insulin gene transcription.48 If the assumption that PDX1 is a member of the insulin gene transcription complex is correct, then other related proteins such as the Lmx1 proteins or the related HB9 homeodomain protein49 must be able to substitute for PDX1 in its absence. Interestingly, in addition to PDX1, neither the E2A and HEB genes nor the neuroD1/BETA2 gene are required for insulin gene transcription. Mice carrying homozygous null mutations in either the E2A or HEB gene transcribe the insulin gene at normal rates in the pancreas,50 suggesting that either protein can function equally well in the insulin gene transcription complex. Studies of neuroD1/BETA2 mutant mice show that this class B bHLH protein is important for maintenance of the β-cells, but is not required for insulin gene transcription.51 This result suggests that other class B bHLH proteins expressed in the pancreas can substitute for neuroD1/BETA2. In support of this hypothesis, we have recently detected two closely related members of the neuroD family in β-cells.52 But if none of these transcription factors is required for insulin gene transcription, and several other factors can replace their function, how is insulin gene transcription restricted to the β-cell, since many other cells probably have some combination of these redundant factors? Several explanations are possible: first, non-β-cells could contain proteins in their nuclei that interfere with formation of the insulin gene transcription complex. For example, the homeodomain protein pbx can associate with PDX1 and alter its DNA binding specificity, thereby preventing it from binding to the insulin promoter A sites53 (K. Ohneda and M. German, unpublished data).
b
Figure 4. Lmx1.1 function in β-cells. In (a) an outline is shown of the functional domains of the lmx1.1 protein. In (b) interactions are shown between lmx1.1 and the bHLH proteins that result in transcriptional activation of the E–A minienhancer.
the protein factors the complex should not be formed and insulin gene transcription should be silenced. Surprisingly, neither of these predictions is entirely true. First, PDX1 is not the only protein that can bind to the A elements and cooperate with the bHLH dimer to activate insulin gene transcription. Many different homeodomain transcription factors can bind to the A elements; and while most of these cannot synergize with the bHLH dimer, two can. These are the LIM-homeodomain proteins Lmx1.1 and Lmx1.2; and their interactions with the bHLH dimer actually give higher levels of transcriptional activation than that produced by the PDX1–bHLH combination. Interestingly, the mechanism by which the Lmx1 proteins synergize with the bHLH dimer is quite distinct from that employed by PDX1. The second LIM domain of the Lmx1 proteins contacts the HLH domain of the bHLH dimer. This interaction releases an allosteric inhibition of the Lmx1 activation domain42, 43 and also stimulates the second activation domain (AD2) of the E2A proteins.26 These added interactions, in addition to any cooperative DNA binding or clustering of activation domains, produce the extra transcriptional activation capacity of the Lmx1–bHLH combination relative to the PDX1–bHLH combination. 230
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of the insulin gene transcription complex (H. Ee and M. German, unpublished data). Therefore, Nkx2.2 probably drives the expression of as yet unidentified transcription factors, such as the proteins that bind to the C1 and Za elements on the insulin promoter. Given the degree of redundancy of most of these factors, it seems likely that Nkx2.2 may lie upstream of more than one insulin gene transcription factor. The Nkx2.2 experiments point out the role of upstream factors that are not part of the insulin gene transcription complex, but are required for the expression of other proteins that are part of the complex. As a result, insulin gene expression is regulated by an entire network of factors directly and indirectly involved in the formation of the insulin gene transcription complex.
Second, the relative functional levels of the proteins that make up the complex may be very important. Given the degree of synergy observed with insulin gene transcription factors, it can be assumed that transcription rates in vivo do not vary linearly with the functional level of specific factors. As a result, small differences in functional factor concentration may make exponential differences in the rate of insulin gene transcription. If this effect is combined with a minimal threshold for transcription, the observed specificity could be explained by functional factor concentration alone. An additional explanation may be that insulin gene transcription depends on the physical state of the chromosome around the insulin gene as well as on the presence of the correct set of transcription factors. Modifications such as methylation or structural changes induced by histone binding could control the availability of the gene for binding to the transcription complex.
Conclusions As we learn more about the molecular mechanisms underlying insulin gene transcription, it is becoming obvious that expression of insulin at such a high level in a single cell type requires the integrated contributions of many factors to build a functional transcription complex. The very complexity of these interactions ensures that they cannot be completely replicated in other cell types. At the same time, the constitution of the complex is not rigid, as there is some flexibility and redundancy built into the design of the complex. Given these variables, we should not assume that we have as yet a complete picture of how insulin gene transcription is achieved in vivo.
Upstream factors While the targeted disruption of genes encoding several putative members of the insulin gene transcription complex has demonstrated that none of these factors are essential for insulin gene transcription, one other factor, the homeodomain protein Nkx2.2, is essential for insulin production at all stages of pancreatic development. Targeted disruption of the Nkx2.2 gene results in the formation of endocrine cells in the pancreas that make several β-cell products such as islet amyloid polypeptide (IAPP) and PDX1, but fail to produce any insulin.54 Insulin gene transcription in the pancreases of these animals is reduced by at least two orders of magnitude (J. Wang, L. Sussel, and M. German, unpublished data). There are no consensus Nkx2.2 binding sites within the insulin promoter, and we have been unable to detect binding to the insulin promoter by in vitro produced Nkx2.2 or by native Nkx2.2 in β-cell nuclear extracts (H. Ee and M. German, unpublished data). Why then does the absence of Nkx2.2 cause the suppression of insulin gene transcription? We presume that Nkx2.2 is required for the expression of other βcell nuclear proteins that form essential components of the insulin gene transcription complex. However, the only known β-cell transcription factor that we cannot detect in the Nkx2.2 null animals is the related homeodomain transcription factor Nkx6.1,54 a protein that we do not believe forms an important part
Acknowledgements We would like to thank the many members of the Rutter and German laboratories that contributed to the work discussed here. The work from our laboratory was supported by NIH grants DK-21344 and DK-48281 and grants from the Juvenile Diabetes Foundation.
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286:82–84 3. German M (1994) Insulin gene structure and regulation, in Molecular Biology of Diabetes (Draznin B, Leroith D, eds) pp. 91–117. Humana Press, Totowa, NJ 4. Steiner DF, Chan SJ, Welsh JM, Kwok SC (1985) Structure and evolution of the insulin gene. Annu Rev Genet 19:463–484 5. Walker MD, Edlund T, Boulet AM, Rutter WJ (1983) Cellspecific expression controlled by the 50 flanking regions of the insulin and chymotrypsin genes. Nature 306:557–581 6. Edlund T, Walker MD, Barr PJ, Rutter WJ (1985) Cell-specific expression of the rat insulin gene: evidence for role of two distinct 50 flanking elements. Science 230:912–916 7. Hanahan D (1985) Heritable formation of pancreatic βcell tumors in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315:115–122 8. German MS, Moss LG, Wang J, Rutter WJ (1992) The insulin and islet amyloid polypeptide genes contain similar cellspecific promoter elements that bind identical β-cell nuclear complexes. Mol Cell Biol 12:1777–1788 9. Karlsson O, Walker MD, Rutter WJ, Edlund T (1989) Individual protein-binding domains of the insulin gene enhancer positively activate β-cell-specific transcription. Mol Cell Biol 9:823–827 10. Aronheim A, Ohlsson H, Park CW, Edlund T, Walker MD (1991) Distribution and characterization of helix-loophelix enhancer-binding proteins from pancreatic β-cells and lymphocytes. Nucleic Acids Res 19:3893–3899 11. Sheih S, Tsai M (1991) Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene. J Biol Chem 266:16708–16714 12. German MS, Blanar MA, Nelson C, Moss LG, Rutter WJ (1991) Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind to the insulin enhancer. Mol Endocrinol 5:292–299 13. Cordle SR, Henderson E, Masuoka H, Weil PA, Stein R (1991) Pancreatic β-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNAbinding proteins. Mol Cell Biol 11:1734–1738 14. Peyton M, Moss LG, Tsai MJ (1994) Two distinct class A helix-loop-helix transcription factors, E2A and BETA1, form separate DNA binding complexes on the insulin gene E box. J Biol Chem 269:25936–25941 15. Naya FJ, Stellrecht CM, Tsai MJ (1995) Tissue-specific regulation of the insulin gene by a basic helix-loop-helix transcription factor. Genes Dev 9:1009–1019 16. Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N, Weintraub H (1995) Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268:836–844 17. Ohlsson H, Karlsson K, Edlund T (1993) IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 12:4251–4259 18. Leonard J, Peers B, Johnson T, Ferreri K, Lee S, Montminy MR (1993) Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol 7:1275–1283 19. Petersen HV, Serup P, Leonard J, Michelsen BK, Madsen OD (1994) Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes. Proc Natl Acad Sci USA 91:10465– 10469 20. Miller CP, McGehee RE Jr, Habener JF (1994) IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin
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upstream factor 1 and insulin gene transcription by high glucose in pancreatic beta-cells. J Biol Chem 272:20936–20944 Macfarlane WM, McKinnon CM, Felton-Edkins ZA, Cragg H, James RF, Docherty K (1999) Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells. J Biol Chem 274:1011–1016 Rafiq I, Kennedy HJ, Rutter GA (1998) Glucose-dependent translocation of insulin promoter factor-1 (IPF-1) between the nuclear periphery and the nucleoplasm of single MIN6 betacells. J Biol Chem 273:23241–23247 Petersen HV, Peshavaria M, Pedersen AA et al. (1998) Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor. FEBS Lett 431:362–366 German MS, Wang J, Chadwick RB, Rutter WJ (1992) Synergistic activation of the insulin gene by a LIM-homeodomain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex. Genes Dev 6:2165– 2176 Johnson JD, Zhang W, Rudnick A, Rutter WJ, German MS (1997) Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity. Mol Cell Biol 17:3488–3496 Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulinpromoter-factor 1 is required for pancreas development in mice. Nature 371:606–609 Ahlgren U, Jonsson J, Edlund H (1996) The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 122:1409–1416 Offield MF, Jetton TL, Labosky PA et al. (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983–995
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Regulation of insulin gene transcription Kinuko Ohnedaa , Hooi Eeb and Michael German
DNA technology, and other neuroendocrine cells can further process the proinsulin to mature insulin, package it, and secrete it in a regulated fashion.1 Therefore the mechanisms that control transcription of the insulin gene are essential determinants of both β-cell function and metabolic regulation. The insulin gene is a small gene, located on chromosome 11p15.5 in humans,2 and consisting of three exons and two introns (for reviews of the insulin gene see References 3 and 4). The sequences immediately upstream (50 flanking sequences) of the transcription start site are defined as the insulin promoter, and can by themselves restrict expression of a linked gene to the β-cell.5–7 Studies directed towards understanding how insulin expression is restricted to the β-cell have focused on a small fragment, approximately 400 base pairs, of the proximal insulin promoter (see Figure 1). As defined by mutagenesis studies, multiple sequences along the length of the promoter contribute to its overall activity, by functioning as binding sites for sequence-specific DNA-binding proteins found in the nucleus of the β-cell. Many of these transcription factors have been identified and are shown above their binding sites on the human insulin and rat insulin I promoters in Figure 1.
Recent studies of the insulin gene promoter and the transcription factors that regulate it have expanded our understanding of both how the production of insulin is restricted to the pancreatic β-cell, and how that production is regulated by physiologic signals such as glucose. A picture is emerging in which an elaborate set of transcription factors binds to specific sequences along the promoter and recruits additional transcriptional co-activators to build a functional transcriptional activation complex that is unique to β-cells. Surprisingly, however, genetic experiments in mice have demonstrated an unexpected degree of redundancy in the factors that control insulin gene expression, and have revealed the presence of a network of transcription factors that coordinate the expression of factors forming the insulin gene activation complex. Key words: insulin gene / transcription / glucose / neuroD1 / BETA2 c 2000 Academic Press
The insulin in the circulation of adult mammals is produced exclusively by the pancreatic β-cells, making the β-cells the central regulators of energy storage and use by the rest of the organism. This restriction of insulin production to the β-cell depends on constraints at the level of transcription of the insulin gene, the gene that encodes preproinsulin. In contrast, almost any eukaryotic cell can make proinsulin if it is engineered to transcribe an exogenously introduced insulin gene by recombinant
Formation of the transcription complex on the insulin promoter This linear view of the promoter as a series of binding sites and binding proteins, however, lacks the complexity and dynamic character of the functioning promoter in vivo. Missing is the additional dimension provided by the interactions among the transcription factors, DNA structural proteins, co-activators and repressors, intra-cellular signaling molecules and the RNA polymerase complex. This map also fails to show the upstream factors that regulate the expression and function of these proteins. The promoter should be viewed as a large complex formed by the interactions among many different proteins and the chromosome
From the Department of Medicine and Hormone Research Institute, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0534, USA. Email: [email protected] a Present address: Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Japan. b Present address: Gastroenterology Department, Queen Elizabeth II Medical Centre, Perth, Western Australia 6009. c
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Figure 1. The human insulin gene and rat insulin I gene promoters with known sequence elements and binding factors. The boxes represent characterized sequence elements. The new names are in boxes; the old names are shown below each promoter. The cloned binding proteins are circled above the promoter.
interact through their DNA binding domains—the homeodomain and bHLH domains, respectively. This interaction leads to cooperative DNA binding, although with purified proteins in vitro the relative increase in binding affinity is small.26 But in vivo, there are many other proteins in the nucleus that can impact on the bHLH-PDX1 interaction. The structure of the chromosome is altered by the binding of histones and of small, abundant DNA-binding proteins, the high mobility group (HMG) proteins. The presence of these structural DNA-binding proteins changes the energetic cost of DNA binding by the transcription factors (for a review see Reference 27). In fact, we have found that in the presence of HMG I(Y), a small non-histone DNA-binding protein that can bind to the A3/4 region of the insulin promoter, co-binding of PDX1 and the bHLH dimer is greatly favored in vitro, and that HMG I(Y) increases PDX–bHLH transcriptional synergy in vivo.26 While cooperative DNA binding by the PDX1/ bHLH/HMG I(Y) complex may increase the occupancy of the E–A sites on the insulin promoter, transcriptional activation requires interaction with the basal transcription machinery. The grouping of transcription factors on DNA creates clusters of protein interaction sites that cooperatively recruit or stabilize binding of the RNA polymerase II transcription initiation complex (reviewed in Reference 28). Non-DNA-binding co-activators promote this process by linking the DNA-bound transcription factors with the basal transcription machinery. The increased stability provided by co-activators probably plays an
in dynamic equilibrium with forces acting from within and outside the cell. As an example of the interactions that generate this complex, we have studied the interactions among proteins that bind to the juxtaposed E and A elements in the insulin gene promoter. Neither the E elements nor the A elements by themselves have any significant transcriptional activity in β-cells; but together, they can dramatically boost the activity of a linked promoter in a β-cell-specific fashion.8, 9 The synergy between these elements results from interactions among the proteins that bind to them and non-DNA-binding transcriptional co-activators. The E elements are binding sites for protein dimers formed by heterodimerization between two members of the family of basic helix-loop-helix (bHLH) proteins: an ubiquitous class A bHLH protein (products of the E2A gene, E12, E47, and E2/5, or HEB), and a cell-type restricted bHLH protein (neuroD1/BETA2).10–16 The A elements are binding sites for several proteins of the homeodomain family, the most abundant in β-cells being PDX1 (also called IPF1, STF1, IDX1, IUF1 or GSF).17–23 A bHLH dimer on the E element can synergize with PDX1 bound to the A element and activate transcription of an E–A minienhancer in a non-β-cell engineered to express these proteins.24, 25 This interaction requires both DNA binding sites, the DNA binding domains of both PDX1 and the bHLH dimer, and the transcriptional activation domains of both PDX1 and the bHLH dimer24, 25 (K. Ohneda and M. German, unpublished data). PDX1 and the bHLH proteins E47 and neuroD1/BETA2 physically 228
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a
b
Figure 3. Glucose regulation of insulin gene expression. Proposed pathways for glucose regulation of insulin gene expression are shown. E represents E2A proteins, and β2 represents the selectively expressed heterodimer partner BETA2/neuroD1. Question marks indicate unknown steps in the pathway or the probable intersection with other pathways.
Figure 2. PDX1 function in β-cells. In (a) an outline is shown of the functional domains of the PDX1 protein. The regions labeled A–E represent conserved domains within the activation domain.25 In (b) interactions are shown between PDX1, the bHLH proteins, and HMGI(Y) that result in transcriptional activation of the E–A minienhancer.
and increases the activation potential of the PDX1 activation domain.41 In addition, glucose increases the binding of the bHLH dimer to the E elements,34 which in turn cooperates with the activated PDX1 bound at the A site to increase insulin gene transcription. But sequences outside the E–A elements are also important for the response to glucose, and the binding of as yet unidentified proteins at the C1 and Za sites is also stimulated by glucose. The overall increase in insulin gene transcription in response to glucose results from the combination of all of these effects (see Figure 3).
essential role in the formation and the function of the insulin promoter transcription activation complex (see Figure 2). The identities of these co-activators in the insulin gene transcription complex are unknown, although both the E2A proteins and neuroD1 can interact with the ubiquitous p300 co-activator.29–31
Glucose regulation of the transcription complex How rigid is the constitution of the transcription complex?
The preceding discussion presents a model by which insulin gene transcription in the β-cell results from the formation of a transcription complex from an interacting set of nuclear proteins. Since formation of an effective complex depends on all of the interacting proteins, the net activity of the promoter can be regulated by altering the concentration or function of any one protein in the complex. Glucose, however, appears to regulate insulin gene transcription through multiple effects on several of the proteins in the transcription activation complex.32–37 Glucose acutely stimulates an increase in PDX1 binding to the A elements,22, 23, 33 due in part to phosphorylation of PDX1 through a pathway that involves PI3 kinase and the stress-activated protein kinase 2 (SAPK2).22, 38 Glucose also causes a shift in the cellular distribution of PDX1 to the nucleus39, 40
If insulin gene transcription proceeds from the formation on the promoter of a transcription complex from an interacting set of nuclear protein factors, then the β-cell specificity of insulin gene transcription results from the specificity of this complex. No single protein member of the complex needs to be restricted in expression to the β-cell if the set of protein factors is unique to the β-cell, and the formation of the complex depends on the presence of all the members of the set. This model for β-cell-specific insulin gene transcription makes two predictions: first, the protein interactions that form the complex should be highly specific, so that different proteins present in other cell nuclei cannot be substituted in the interaction. Second, in the absence of any one of 229
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The ability of the Lmx1 proteins to synergize with the bHLH dimer suggests that other proteins in the β-cell nucleus may be able to substitute for PDX1 in the transcription activation complex on the insulin promoter. Further evidence that PDX1 may not be essential for insulin gene transcription comes from genetic studies in mice. Mice that are homozygous for a targeted disruption of the PDX1 gene fail to develop a pancreas beyond the initial bud stage;44–46 but rare insulin-producing cells do appear in the bud.46 If the PDX1 gene is inactivated after the pancreas forms and the β-cells have differentiated, the β-cell mass decreases and β-cell function is impaired, but the remaining β-cells continue to produce insulin.47 Furthermore, levels of PDX1 protein can be dramatically reduced in β-cell lines using antisense oligonucleotides without causing any reduction in insulin gene transcription.48 If the assumption that PDX1 is a member of the insulin gene transcription complex is correct, then other related proteins such as the Lmx1 proteins or the related HB9 homeodomain protein49 must be able to substitute for PDX1 in its absence. Interestingly, in addition to PDX1, neither the E2A and HEB genes nor the neuroD1/BETA2 gene are required for insulin gene transcription. Mice carrying homozygous null mutations in either the E2A or HEB gene transcribe the insulin gene at normal rates in the pancreas,50 suggesting that either protein can function equally well in the insulin gene transcription complex. Studies of neuroD1/BETA2 mutant mice show that this class B bHLH protein is important for maintenance of the β-cells, but is not required for insulin gene transcription.51 This result suggests that other class B bHLH proteins expressed in the pancreas can substitute for neuroD1/BETA2. In support of this hypothesis, we have recently detected two closely related members of the neuroD family in β-cells.52 But if none of these transcription factors is required for insulin gene transcription, and several other factors can replace their function, how is insulin gene transcription restricted to the β-cell, since many other cells probably have some combination of these redundant factors? Several explanations are possible: first, non-β-cells could contain proteins in their nuclei that interfere with formation of the insulin gene transcription complex. For example, the homeodomain protein pbx can associate with PDX1 and alter its DNA binding specificity, thereby preventing it from binding to the insulin promoter A sites53 (K. Ohneda and M. German, unpublished data).
b
Figure 4. Lmx1.1 function in β-cells. In (a) an outline is shown of the functional domains of the lmx1.1 protein. In (b) interactions are shown between lmx1.1 and the bHLH proteins that result in transcriptional activation of the E–A minienhancer.
the protein factors the complex should not be formed and insulin gene transcription should be silenced. Surprisingly, neither of these predictions is entirely true. First, PDX1 is not the only protein that can bind to the A elements and cooperate with the bHLH dimer to activate insulin gene transcription. Many different homeodomain transcription factors can bind to the A elements; and while most of these cannot synergize with the bHLH dimer, two can. These are the LIM-homeodomain proteins Lmx1.1 and Lmx1.2; and their interactions with the bHLH dimer actually give higher levels of transcriptional activation than that produced by the PDX1–bHLH combination. Interestingly, the mechanism by which the Lmx1 proteins synergize with the bHLH dimer is quite distinct from that employed by PDX1. The second LIM domain of the Lmx1 proteins contacts the HLH domain of the bHLH dimer. This interaction releases an allosteric inhibition of the Lmx1 activation domain42, 43 and also stimulates the second activation domain (AD2) of the E2A proteins.26 These added interactions, in addition to any cooperative DNA binding or clustering of activation domains, produce the extra transcriptional activation capacity of the Lmx1–bHLH combination relative to the PDX1–bHLH combination. 230
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of the insulin gene transcription complex (H. Ee and M. German, unpublished data). Therefore, Nkx2.2 probably drives the expression of as yet unidentified transcription factors, such as the proteins that bind to the C1 and Za elements on the insulin promoter. Given the degree of redundancy of most of these factors, it seems likely that Nkx2.2 may lie upstream of more than one insulin gene transcription factor. The Nkx2.2 experiments point out the role of upstream factors that are not part of the insulin gene transcription complex, but are required for the expression of other proteins that are part of the complex. As a result, insulin gene expression is regulated by an entire network of factors directly and indirectly involved in the formation of the insulin gene transcription complex.
Second, the relative functional levels of the proteins that make up the complex may be very important. Given the degree of synergy observed with insulin gene transcription factors, it can be assumed that transcription rates in vivo do not vary linearly with the functional level of specific factors. As a result, small differences in functional factor concentration may make exponential differences in the rate of insulin gene transcription. If this effect is combined with a minimal threshold for transcription, the observed specificity could be explained by functional factor concentration alone. An additional explanation may be that insulin gene transcription depends on the physical state of the chromosome around the insulin gene as well as on the presence of the correct set of transcription factors. Modifications such as methylation or structural changes induced by histone binding could control the availability of the gene for binding to the transcription complex.
Conclusions As we learn more about the molecular mechanisms underlying insulin gene transcription, it is becoming obvious that expression of insulin at such a high level in a single cell type requires the integrated contributions of many factors to build a functional transcription complex. The very complexity of these interactions ensures that they cannot be completely replicated in other cell types. At the same time, the constitution of the complex is not rigid, as there is some flexibility and redundancy built into the design of the complex. Given these variables, we should not assume that we have as yet a complete picture of how insulin gene transcription is achieved in vivo.
Upstream factors While the targeted disruption of genes encoding several putative members of the insulin gene transcription complex has demonstrated that none of these factors are essential for insulin gene transcription, one other factor, the homeodomain protein Nkx2.2, is essential for insulin production at all stages of pancreatic development. Targeted disruption of the Nkx2.2 gene results in the formation of endocrine cells in the pancreas that make several β-cell products such as islet amyloid polypeptide (IAPP) and PDX1, but fail to produce any insulin.54 Insulin gene transcription in the pancreases of these animals is reduced by at least two orders of magnitude (J. Wang, L. Sussel, and M. German, unpublished data). There are no consensus Nkx2.2 binding sites within the insulin promoter, and we have been unable to detect binding to the insulin promoter by in vitro produced Nkx2.2 or by native Nkx2.2 in β-cell nuclear extracts (H. Ee and M. German, unpublished data). Why then does the absence of Nkx2.2 cause the suppression of insulin gene transcription? We presume that Nkx2.2 is required for the expression of other βcell nuclear proteins that form essential components of the insulin gene transcription complex. However, the only known β-cell transcription factor that we cannot detect in the Nkx2.2 null animals is the related homeodomain transcription factor Nkx6.1,54 a protein that we do not believe forms an important part
Acknowledgements We would like to thank the many members of the Rutter and German laboratories that contributed to the work discussed here. The work from our laboratory was supported by NIH grants DK-21344 and DK-48281 and grants from the Juvenile Diabetes Foundation.
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