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Michael S. German - One of the best experts on this subject based on the ideXlab platform.
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lmx1b transactivation and expression in nail patella syndrome
Human Molecular Genetics, 2000Co-Authors: Sandra D. Dreyer, Kerby C. Oberg, Roy Morello, Andreas Winterpacht, Bernhard Zabel, Michael S. German, Gregory P. Lunstrum, William A. Horton, Brendan LeeAbstract:Lmx1b, a member of the LIM Homeodomain Protein family, is essential for the specification of dorsal LIMb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal LIMb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine LIMb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the LIMb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting Protein, LDB1, and the helix-loop-helix Protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B Proteins containing human mutations affecting each of the helices or the N-terminal arm of the Homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.
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the Homeodomain of pdx 1 mediates multiple Protein Protein interactions in the formation of a transcriptional activation complex on the insulin promoter
Molecular and Cellular Biology, 2000Co-Authors: Kinuko Ohneda, Juehu Wang, Jeffrey D. Johnson, Raghavendra G Mirmira, Michael S. GermanAbstract:Like expression of other cell-type-specific genes, expression of the insulin gene depends on the actions of a unique set of nuclear activators. These activators cooperate synergistically in building a transcriptional activation complex that binds to the regulatory domains of the gene and activates the basal RNA polymerase machinery (reviewed in reference 7). The complexity and specificity of the interactions among these activators LIMit the cell types capable of building a functional activation complex. Dissection of these interactions provides insight into the mechanism by which insulin expression is LIMited to the correct cell type. In adult mammals, activation of the insulin gene is tightly restricted to the β cells in the pancreatic islets of Langerhans, where it is expressed at high levels. This specificity is reflected in the restricted function of the insulin promoter, the proximal few hundred base pairs of which can replicate the specificity of the intact gene (19, 50). Because of the complexity of the intact promoter (9, 13, 24), a short portion of the rat insulin I promoter between bp −247 and −197 upstream from the transcription initiation site has been used as a model of the types of synergistic interactions that combine to give the characteristic activity of the full promoter (15). This 50-bp fragment contains at least three distinct DNA-binding sites named E2, A3, and A4 (13). The E and A elements synergize: neither has significant activity on its own, but in combination E and A elements produce β-cell specific transcriptional activation (15, 23). The E2 element functions as a recognition site for dimers of basic helix-loop-helix (bHLH) Proteins, including a heterodimer of the ubiquitous bHLH Protein E47/Pan1 and the neuroendocrine specific bHLH Protein BETA2/NeuroD1 (35). The A elements each contain the sequence TAAT and have been shown to bind to several Homeodomain Proteins found in β-cell nuclei (12, 16, 22, 32, 37). Two of these Homeodomain Proteins, PDX-1 (also known as IPF-1 [37], STF-1 [28], IDX-1 [32], IUF-1 [29], and GSF [30]) and Lmx1.1 can bind to the A3 and A4 (collectively referred to as A3/4) sites and activate the E2A3/4 minienhancer by synergizing with E47/Pan1 bound to the E2 site (16, 38, 40). The LIM Homeodomain Protein Lmx1.1 contains two LIM domains that form zinc-binding structures in the amino end of the molecule. The second of these two LIM domains (LIM2) directly binds to the bHLH domain of E47/Pan1 and mediates the synergy between Lmx1.1 and E47/Pan1. This interaction is specific, since analogous domains from other LIM Proteins and bHLH Proteins cannot substitute for the LIM2 domain of Lmx1.1 or the bHLH domain of E47/Pan1, respectively (20). PDX-1 plays an important role both in the development of the pancreas and in maintaining β-cell function. Mice with a targeted disruption of the pdx1 gene selectively lack a pancreas (2, 21, 36). Similar pancreatic agenesis has been found in a human patient with a single nucleotide deletion in the pdx1 gene (46). If the pancreas is allowed to develop with an intact pdx1 gene, and the pdx1 gene is disrupted only in mature β cells, diabetes ensues due to impaired β-cell function (3). This impairment presumably results from the loss of PDX-1 activation of β-cell genes, since PDX-1 has been shown to activate a variety of pancreatic islet-specific genes, including those encoding insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 (28, 32, 37, 44, 49, 51, 52). It should be noted, however, that the β cells lacking PDX-1 continue to express insulin (3). The molecular basis of the PDX-1–E47/Pan interaction is unknown. The DNA-binding domains of both Proteins, as well as the activation domains, are required. The transcription activation domain of PDX-1 is located in the amino end, upstream of the Homeodomain (38), and is composed of five subdomains which are conserved between PDX-1 and the related Xenopus homeoProtein XIHbox8 (40). Interestingly, when the unrelated activation domain of herpesvirus VP16 is substituted for the PDX-1 activation domain, the chimeric VP16–PDX-1 Protein can still synergize with E47/Pan1, even though the only portion of PDX-1 that is retained in the chimeric Protein is the DNA-binding domain, the Homeodomain (38). This result suggests that the synergy between PDX-1 and E47/Pan1 could result from cooperative DNA binding and the formation of a stable DNA-binding complex with both Proteins. Previous studies, however, have been unable to demonstrate cooperative DNA binding in vitro (38). In this study, we directly compare the transcriptional activation properties of PDX-1 and Lmx1.1. Even though both homeoProteins interact with a common factor, E47/Pan1, and activate insulin gene transcription through the same promoter element, the two Proteins interact with E47/Pan1 by distinct mechanisms. We also demonstrate that the β-cell-specific bHLH factor BETA2/NeuroD1 and the nuclear high-mobility-group Protein I(Y) [HMG I(Y)] contribute to PDX-1–E47/Pan1 synergy through direct interactions with the Homeodomain of PDX-1. These results demonstrate that PDX-1 acts multifunctionally as a center of Protein-Protein interactions in an intricate complex controlling β-cell-specific gene expression in vivo.
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Transcriptional synergy between LIM-Homeodomain Proteins and basic helix-loop-helix Proteins: the LIM2 domain determines specificity.
Molecular and cellular biology, 1997Co-Authors: Jeffrey D. Johnson, Wei Zhang, A Rudnick, William J. Rutter, Michael S. GermanAbstract:LIM-Homeodomain Proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-Homeodomain Protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) Protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two Proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 Proteins and to characterize the physical interactions between Lmx1 Proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 Proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-Homeodomain Protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-Homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues.
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synergistic activation of the insulin gene by a LIM homeo domain Protein and a basic helix loop helix Protein building a functional insulin minienhancer complex
Genes & Development, 1992Co-Authors: Michael S. German, Juehu Wang, Robert B Chadwick, William J. RutterAbstract:The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a beta-cell-specific transcriptional enhancer. We have isolated several cDNAs encoding Proteins that bind to the FLAT element. Two of these cDNAs, cdx-3 and lmx-1, represent homeo box containing mRNAs with restricted patterns of expression. The Protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich "LIM" domains. Both cdx-3 and lmx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected beta-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix Protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-Homeodomain Protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. We conclude that beta-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These Proteins form the minimal components for a functional minienhancer complex.
Chunyan Zhou - One of the best experts on this subject based on the ideXlab platform.
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The LIM-Homeodomain Protein ISL1 activates insulin gene promoter directly through synergy with BETA2.
Journal of Molecular Biology, 2009Co-Authors: Hui Zhang, Weiping Wang, Ting Guo, Jichun Yang, Ping Chen, Youfei Guan, Chunyan ZhouAbstract:Abstract The LIM-Homeodomain transcription factor ISL1 (islet factor 1) is essential for pancreatic islet cell and dorsal mesenchyme development. Mutations in ISL1 are associated with maturity-onset diabetes of the young and type 2 diabetes. Whether ISL1 plays a role in the insulin gene expression has not been fully elucidated. In the present study, we show that ISL1 can synergistically activate insulin gene transcription with BETA2 in pancreatic β cells. The Protein–Protein interactions of ISL1 and BETA2 are directly mediated by the LIM domains of ISL1 and the basic helix–loop–helix domain of BETA2. Deletion of the two LIM domains of ISL1 enhances the transcriptional activation of the insulin gene, indicating a key role for the Homeodomain in activating the insulin promoter. Furthermore, ISL1 can bind with the A3/4 box in the rat insulin gene І promoter through its Homeodomain. ISL1 expression is up-regulated at the mRNA level in type 2 diabetes (db/db mouse model) but down-regulated by dexamethasone in rat insulinoma cells. These results suggest that ISL1 is a transcriptional activator for insulin gene expression, and the interactions of ISL1 with BETA2 are required for the transcriptional activity of the insulin gene. Reduction in Isl1 gene expression appears to be involved in the impairment of insulin expression mediated by dexamethasone.
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ISL1 physically interacts with BETA2 to promote insulin gene transcriptional synergy in non-β cells
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2005Co-Authors: Siyang Peng, Hui Zhang, Weiping Wang, Ping Chen, Jun Meng, Chunyan ZhouAbstract:Abstract ISL1 is a LIM Homeodomain Protein that plays an important role in insulin gene transcriptional activation and islet cell formation. BETA2 is a transcription factor in the basic helix–loop–helix (bHLH) family, which activates expression of tissue-specific genes in several developmental systems. In this study, we investigated the functional and physical interactions of ISL1 and BETA2 in promoting insulin gene transcription in non-β cells. Using the luciferase assay, we demonstrated that ISL1 and BETA2 could activate insulin gene transcription synergistically. Co-immunoprecipitation also supported that ISL1 and BETA2 appear in one complex and this physical interaction mediates the synergy between these two Proteins.
Ernie M.h.f. Bongers - One of the best experts on this subject based on the ideXlab platform.
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Identification of entire LMX1B gene deletions in nail patella syndrome: evidence for haploinsufficiency as the main pathogenic mechanism underlying dominant inheritance in man.
European journal of human genetics : EJHG, 2008Co-Authors: Ernie M.h.f. Bongers, Ilse J. De Wijs, Carlo Marcelis, Lies H. Hoefsloot, Nine V A M KnoersAbstract:Heterozygous mutations in the LMX1B gene cause nail patella syndrome (NPS) that is associated with nail and skeletal malformations, nephropathy, and glaucoma. Previous phenotype studies of Lmx1b null mice revealed dorsal LIMb and renal anomalies similar to human NPS, which contributed to the identification of heterozygous mutations in this LIM-Homeodomain Protein LMX1B as the genetic defect responsible for NPS. Despite advanced insight into the role of the Lmx1b transcription factor in a broad range of animal developmental programs, the pathogenic mechanism underlying dominant inheritance of NPS in man remained unclear. Here, we describe for the first time the detection of two entire LMX1B gene deletions and one smaller exonic LMX1B deletion by multiplex ligation-dependent probe amplification (MLPA) in a series of eight unrelated families with classical features of NPS in whom no pathogenic LMX1B mutation was found by sequence analysis. The identification of entire LMX1B deletions strongly confirms that haploinsufficiency is the principal pathogenetic mechanism of NPS and suggests a difference in dosage sensitivity for this gene between mice and man.
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in vivo expression of putative lmx1b targets in nail patella syndrome kidneys
American Journal of Pathology, 2003Co-Authors: Laurence Heidet, Ernie M.h.f. Bongers, Mireille Sich, Shaoyu Zhang, Chantal Loirat, Alain Meyrier, M Broyer, Gerard Landthaler, Bernadette Faller, Yoshikazu SadoAbstract:The nail-patella syndrome (NPS) is characterized by nail and bone abnormalities, associated with glomerular involvement in ∼40% of patients. Typical glomerular changes consist of fibrillar material in the irregularly thickened glomerular basement membrane. NPS is inherited as an autosomal dominant trait and caused by heterozygous loss of function mutations in LMX1B, a member of the LIM Homeodomain Protein family. Mice with homozygous inactivation of the gene exhibit nail and skeletal defects, similar to those observed in patients, associated with glomerular abnormalities. Strong reduction in the glomerular expression of the α3 and α4 chains of type IV collagen, and of podocin and CD2AP, two podocyte Proteins critical for glomerular function, has been observed in Lmx1b null mice. The expression of these Proteins appeared to be regulated by Lmx1b. To determine whether these changes in podocyte gene expression are involved in the development of NPS nephropathy, using immunohistological techniques, we analyzed the podocyte phenotype and the renal distribution of type IV collagen chains in the kidneys of seven NPS patients with severe glomerular disease. We also examined the nature of the fibrillar material present within the glomerular extracellular matrix. The glomerular basement membrane fibrillar material was specifically labeled with anti-type III collagen antibodies, suggesting a possible regulation of type III collagen expression by LMX1B. The expression of the α3 and α4 chains of type IV collagen, and of podocin and CD2AP, was found to be normal in the seven patients. These findings indicate that heterozygous mutations of LMX1B do not appear to dramatically affect the expression of type IV collagen chains, podocin, or CD2AP in NPS patients.
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In vivo expression of putative LMX1B targets in nail-patella syndrome kidneys.
The American journal of pathology, 2003Co-Authors: Laurence Heidet, Ernie M.h.f. Bongers, Mireille Sich, Shaoyu Zhang, Chantal Loirat, Alain Meyrier, M Broyer, Gerard Landthaler, Bernadette Faller, Yoshikazu SadoAbstract:The nail-patella syndrome (NPS) is characterized by nail and bone abnormalities, associated with glomerular involvement in approximately 40% of patients. Typical glomerular changes consist of fibrillar material in the irregularly thickened glomerular basement membrane. NPS is inherited as an autosomal dominant trait and caused by heterozygous loss of function mutations in LMX1B, a member of the LIM Homeodomain Protein family. Mice with homozygous inactivation of the gene exhibit nail and skeletal defects, similar to those observed in patients, associated with glomerular abnormalities. Strong reduction in the glomerular expression of the alpha3 and alpha4 chains of type IV collagen, and of podocin and CD2AP, two podocyte Proteins critical for glomerular function, has been observed in Lmx1b null mice. The expression of these Proteins appeared to be regulated by Lmx1b. To determine whether these changes in podocyte gene expression are involved in the development of NPS nephropathy, using immunohistological techniques, we analyzed the podocyte phenotype and the renal distribution of type IV collagen chains in the kidneys of seven NPS patients with severe glomerular disease. We also examined the nature of the fibrillar material present within the glomerular extracellular matrix. The glomerular basement membrane fibrillar material was specifically labeled with anti-type III collagen antibodies, suggesting a possible regulation of type III collagen expression by LMX1B. The expression of the alpha3 and alpha4 chains of type IV collagen, and of podocin and CD2AP, was found to be normal in the seven patients. These findings indicate that heterozygous mutations of LMX1B do not appear to dramatically affect the expression of type IV collagen chains, podocin, or CD2AP in NPS patients.
Nine V A M Knoers - One of the best experts on this subject based on the ideXlab platform.
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Identification of entire LMX1B gene deletions in nail patella syndrome: evidence for haploinsufficiency as the main pathogenic mechanism underlying dominant inheritance in man.
European journal of human genetics : EJHG, 2008Co-Authors: Ernie M.h.f. Bongers, Ilse J. De Wijs, Carlo Marcelis, Lies H. Hoefsloot, Nine V A M KnoersAbstract:Heterozygous mutations in the LMX1B gene cause nail patella syndrome (NPS) that is associated with nail and skeletal malformations, nephropathy, and glaucoma. Previous phenotype studies of Lmx1b null mice revealed dorsal LIMb and renal anomalies similar to human NPS, which contributed to the identification of heterozygous mutations in this LIM-Homeodomain Protein LMX1B as the genetic defect responsible for NPS. Despite advanced insight into the role of the Lmx1b transcription factor in a broad range of animal developmental programs, the pathogenic mechanism underlying dominant inheritance of NPS in man remained unclear. Here, we describe for the first time the detection of two entire LMX1B gene deletions and one smaller exonic LMX1B deletion by multiplex ligation-dependent probe amplification (MLPA) in a series of eight unrelated families with classical features of NPS in whom no pathogenic LMX1B mutation was found by sequence analysis. The identification of entire LMX1B deletions strongly confirms that haploinsufficiency is the principal pathogenetic mechanism of NPS and suggests a difference in dosage sensitivity for this gene between mice and man.
Hideki Sezutsu - One of the best experts on this subject based on the ideXlab platform.
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LIM Homeodomain transcription factor awh is a key component activating all three fibroin genes fibh fibl and fhx in the silk gland of the silkworm bombyx mori
Insect Biochemistry and Molecular Biology, 2015Co-Authors: Mai Kimoto, Takuya Tsubota, Hideki Sezutsu, Keiro Uchino, Shigeharu TakiyaAbstract:In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue Proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-Homeodomain Protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.
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Silk Gland Factor-2, Involved in Fibroin Gene Transcription, Consists of LIM Homeodomain, LIM-interacting, and Single-stranded DNA-binding Proteins
Journal of Biological Chemistry, 2013Co-Authors: Kaoru Ohno, Takuya Tsubota, Jun-ichi Sawada, Shigeharu Takiya, Keiro Uchino, Mai Kimoto, Akiko Matsumoto, Hideki SezutsuAbstract:SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM Homeodomain Protein Awh, LIM domain-binding Protein (Ldb), a sequence-specific single-stranded DNA-binding Protein (Lcaf), and the silk Protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf Proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf Protein complex serves as a scaffold to facilitate communication between transcriptional control elements.