Functional and Structural Profiling of the Human Thrombopoietin Gene Promoter

Human thrombopoietin (TPO) is involved in cardiovascular disease as it regulates megakaryocyte development and enhances platelet adhesion/aggregation. The THPO promoter structure is still controversial. By reverse transcription-PCR, we confirm that THPO transcription is cell line-dependently initiated at two alternative promoters, which we newly designated P1a and P1. We subsequently electrophoretically scanned and resequenced these portions in 95 and 57 patients with cardiovascular disease, respectively, and identified seven variants (–1450/del58bp, C-920T [rs2855306], A-622G, C-413T [rs885838], C+5A, G+115A, and C+135T). After subcloning of 1032 bp of THPO P1 in pGL3-basic vector, five molecular haplotypes (MolHaps1–5) were observed: [A–622-C–413-C+5-G+115; wild type (wt)], [A–622-T–413-C+5-G+115], [G–622-T–413-C+5-G+115], [A–622-C–413-A+5-G+115], [A–622-C–413-C+5-A+115], and analyzed in reporter gene assays in HEK293T and HepG2 cells. MolHaps 2, 4, and 5 were significantly more active than wt (all p values ≤0.01) in HEK293T cells, MolHap3 exerted a substantial loss of promoter activity (p < 0.0001 in HEK293T and p < 0.01 in HepG2, compared with wt). Electrophoretic mobility shift assays revealed that A-622G and C-413T individually differed from MolHaps in their DNA-protein interaction patterns. Supershift and chromatin immunoprecipitation assays identified CCAAT/enhancer-binding protein δ as the binding protein exclusively for the –622A allelic portion. Human thrombopoietin (TPO) is involved in cardiovascular disease as it regulates megakaryocyte development and enhances platelet adhesion/aggregation. The THPO promoter structure is still controversial. By reverse transcription-PCR, we confirm that THPO transcription is cell line-dependently initiated at two alternative promoters, which we newly designated P1a and P1. We subsequently electrophoretically scanned and resequenced these portions in 95 and 57 patients with cardiovascular disease, respectively, and identified seven variants (–1450/del58bp, C-920T [rs2855306], A-622G, C-413T [rs885838], C+5A, G+115A, and C+135T). After subcloning of 1032 bp of THPO P1 in pGL3-basic vector, five molecular haplotypes (MolHaps1–5) were observed: [A–622-C–413-C+5-G+115; wild type (wt)], [A–622-T–413-C+5-G+115], [G–622-T–413-C+5-G+115], [A–622-C–413-A+5-G+115], [A–622-C–413-C+5-A+115], and analyzed in reporter gene assays in HEK293T and HepG2 cells. MolHaps 2, 4, and 5 were significantly more active than wt (all p values ≤0.01) in HEK293T cells, MolHap3 exerted a substantial loss of promoter activity (p < 0.0001 in HEK293T and p < 0.01 in HepG2, compared with wt). Electrophoretic mobility shift assays revealed that A-622G and C-413T individually differed from MolHaps in their DNA-protein interaction patterns. Supershift and chromatin immunoprecipitation assays identified CCAAT/enhancer-binding protein δ as the binding protein exclusively for the –622A allelic portion. The human glycoprotein hormone thrombopoietin (TPO), 3The abbreviations used are:TPOthrombopoietinTHPOthrombopoietin geneCVDcardiovascular diseasewtwild typeMImyocardial infarctionSNPsingle nucleotide polymorphismMolHapmolecular haplotypeECTIMEtude Cas-Témoins de l'Infarctus du MyocardeMolProMDMolecular Functional Profiling for Mechanism DetectionEMSAelectrophoretic mobility shift assayTFBStranscription factor binding siteChIPchromatin immunoprecipitationC/EBPCCAAT/enhancer-binding proteinHEKhuman embryonic kidneyPMAphorbol 12-myristate 13-acetateRTroom temperatureNF-1nuclear factor 1. 3The abbreviations used are:TPOthrombopoietinTHPOthrombopoietin geneCVDcardiovascular diseasewtwild typeMImyocardial infarctionSNPsingle nucleotide polymorphismMolHapmolecular haplotypeECTIMEtude Cas-Témoins de l'Infarctus du MyocardeMolProMDMolecular Functional Profiling for Mechanism DetectionEMSAelectrophoretic mobility shift assayTFBStranscription factor binding siteChIPchromatin immunoprecipitationC/EBPCCAAT/enhancer-binding proteinHEKhuman embryonic kidneyPMAphorbol 12-myristate 13-acetateRTroom temperatureNF-1nuclear factor 1. also known as myeloproliferative leukemia virus ligand (c-Mpl), is an important growth factor for the megakaryocyte lineage, controlling its proliferation and differentiation (1Kaushansky K. J. Clin. Investig. 2005; 115: 3339-3347Crossref PubMed Scopus (404) Google Scholar, 2Kaushansky K. N. Engl. J. Med. 1998; 339: 746-754Crossref PubMed Scopus (318) Google Scholar). TPO affects platelet function by stimulating platelet aggregation via phosphoinositide-3 kinase (3Kojima H. Shinagawa A. Shimizu S. Kanada H. Hibi M. Hirano T. Nagasawa T. Exp. Hematol. 2001; 29: 616-622Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and it affects mature platelets by reducing the threshold levels of ADP, collagen, or thrombin necessary for aggregation (4Oda A. Miyakawa Y. Druker B.J. Ozaki K. Yabusaki K. Shirasawa Y. Handa M. Kato T. Miyazaki H. Shimosaka A. Ikeda Y. Blood. 1996; 87: 4664-4670Crossref PubMed Google Scholar, 5Pasquet J.M. Gross B.S. Gratacap M.P. Quek L. Pasquet S. Payrastre B. van Willigen G. Mountford J.C. Watson S.P. Blood. 2000; 95: 3429-3434Crossref PubMed Google Scholar) and stimulates platelet adhesion (6Van Os E. Wu Y.P. Pouwels J.G. Ijsseldijk M.J. Sixma J.J. Akkerman J.W. De Groot P.G. Van Willigen G. Br. J. Haematol. 2003; 121: 482-490Crossref PubMed Scopus (33) Google Scholar). In TPO-deficient mice platelet counts are reduced by ∼90% compared with wild type mice (wt) (7Murone M. Carpenter D.A. de Sauvage F.J. Stem Cells. 1998; 16: 1-6Crossref PubMed Scopus (83) Google Scholar). Circulating TPO levels are strongly influenced by inflammatory processes, including the presence of coronary atherosclerosis (8Cotton J.M. Hong Y. Hawe E. Mathur A. Humphries S.E. Brown A.S. Martin J.F. Erusalimsky J.D. Thromb. Haemostasis. 2003; 89: 538-543Crossref PubMed Scopus (8) Google Scholar). Although predominantly expressed in liver cells, TPO is also generated by kidney cells and bone marrow stroma as well as smooth muscle cells (9Sungaran R. Markovic B. Chong B.H. Blood. 1997; 89: 101-107Crossref PubMed Google Scholar). Recently, a genome-wide linkage analysis in a large Asian kindred proposed TPO as a candidate for platelet count variation (10Garner C. Best S. Menzel S. Rooks H. Spector T.D. Thein S.L. Eur. J. Hum. Genet. 2006; 14: 101-108Crossref PubMed Scopus (13) Google Scholar). The THPO locus has been mapped to chromosome 3q26.3-q27. Three groups independently reported that THPO comprises 6 exons and 5 introns spanning more than 6.2 kilobases (kb) (11Sohma Y. Akahori H. Seki N. Hori T. Ogami K. Kato T. Shimada Y. Kawamura K. Miyazaki H. FEBS Lett. 1994; 353: 57-61Crossref PubMed Scopus (240) Google Scholar, 12Foster D.C. Sprecher C.A. Grant F.J. Kramer J.M. Kuijper J.L. Holly R.D. Whitmore T.E. Heipel M.D. Bell L.A. Ching A.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13023-13027Crossref PubMed Scopus (134) Google Scholar, 13Kamura T. Handa H. Hamasaki N. Kitajima S. J. Biol. Chem. 1997; 272: 11361-11368Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), whereas Chang et al. (14Chang M.S. McNinch J. Basu R. Shutter J. Hsu R.Y. Perkins C. Mar V. Suggs S. Welcher A. Li L. J. Biol. Chem. 1995; 270: 511-514Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) proposed that it consists of 7 exons and 6 introns spanning 8 kb. Thus, the 5′-regulatory region consists of two alternative promoters which we designated P1a and the intronic promoter P1 with multiple transcription initiation sites, with 10% of all transcripts synthesized from P1a and 90% originating from P1 (15Ghilardi N. Wiestner A. Skoda R.C. Blood. 1998; 92: 4023-4030Crossref PubMed Google Scholar, 16Wiestner A. Schlemper R.J. van der Maas A.P. Skoda R.C. Nat. Genet. 1998; 18: 49-52Crossref PubMed Scopus (238) Google Scholar). Given the controversy with respect to the THPO structure, we analyzed the two alternative promoter regions and identified 7 exons instead of 6 and propose an alternative nomenclature for THPO architecture. Furthermore, we systematically scanned the promoter region P1 in 190 chromosomes of high risk myocardial infarction (MI) patients for genetic variation/single-nucleotide polymorphisms (SNPs) and resequenced this promoter portion in an additional set of 114 chromosomes of patients with cardiovascular disease (CVD) including MI and essential hypertension. We then performed molecular functional analyses of the identified promoter variations in single-allelic assays as well as for the molecular haplotypes (MolHaps), determined by individual subcloning procedures in the 57 CVD patients. Subsequently, in-depths reporter gene and band shift assays were performed in two different cell lines under three different stimulatory regimes. thrombopoietin thrombopoietin gene cardiovascular disease wild type myocardial infarction single nucleotide polymorphism molecular haplotype Etude Cas-Témoins de l'Infarctus du Myocarde Molecular Functional Profiling for Mechanism Detection electrophoretic mobility shift assay transcription factor binding site chromatin immunoprecipitation CCAAT/enhancer-binding protein human embryonic kidney phorbol 12-myristate 13-acetate room temperature nuclear factor 1. thrombopoietin thrombopoietin gene cardiovascular disease wild type myocardial infarction single nucleotide polymorphism molecular haplotype Etude Cas-Témoins de l'Infarctus du Myocarde Molecular Functional Profiling for Mechanism Detection electrophoretic mobility shift assay transcription factor binding site chromatin immunoprecipitation CCAAT/enhancer-binding protein human embryonic kidney phorbol 12-myristate 13-acetate room temperature nuclear factor 1. Study Populations—The current investigation was based on the Etude Cas-Témoins de l'Infarctus du Myocarde (ECTIM) and Münster Molecular Functional Profiling for Mechanism Detection (MolProMD) studies. ECTIM is a study of patients with MI (n = 1000) from several regions covered by World Health Organization MONICA (MONItoring trends and determinants in CArdiovascular disease) registers and controls (n = 1000) representative of each geographic area. The ECTIM Study design was originally described in 1992 (17Cambien F. Poirier O. Lecerf L. Evans A. Cambou J.P. Arveiler D. Luc G. Bard J.M. Bara L. Ricard S. Tiret L. Amouyel P. AlhencGelas F. Soubrier F. Nature. 1992; 359: 641-644Crossref PubMed Scopus (1905) Google Scholar). Genetic informed consent was obtained from all study subjects. Ninety-five high risk MI patients (positive family history for CVD, MI ≤ 55 years of age) from the ECTIM study were selected for a preliminary scan of the THPO promoter region. An additional sample of 57 CVD patients recruited in Münster hospitals (ongoing recruitment) as well as 35 healthy controls was used for molecular haplotyping and further functional profiling studies. The Münster MolProMD study is a running study of patients with CVD (MI, essential hypertension) and families aimed at studying molecular genetic mechanism of CVD. The study was approved by the ethics committee of the Medical Faculty, Westfälische Wilhelms-University of Münster, and written informed consent was obtained from all study subjects. Scanning of the THPO Promoter P1 for Genetic Variants—Genomic DNA was prepared from white blood cells using either phenol-chloroform extraction or a salting out method (18Miller S.A. Dykes D.D. Polesky H.F. Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17628) Google Scholar). From the published sequences of the THPO gene (accession number D32046), 15 overlapping fragments <300 bp in length from 95 MI patients (190 alleles) of the ECTIM study were amplified to cover 2084 bp of the upstream region. Specific amplification protocols can be obtained at GeneCanvas INSERM website. Single-strand Conformation Polymorphism analysis was performed as previously described (19Herrmann S.M. Funke-Kaiser H. Schmidt-Petersen K. Nicaud V. Gautier-Bertrand M. Evans A. Kee F. Arveiler D. Morrison C. Orzechowski H.D. A. P. F. M. Thromb. Biol. 2001; PubMed Scopus Google Scholar). DNA from patients different on the were then DNA with and using an of the THPO Promoter P1 and of Molecular THPO promoter 1032 bp of the promoter region P1 of DNA from 57 CVD patients from the Münster study were amplified and and as described embryonic kidney cell was in with 10% and Human cells were in the cells were with or for of and of from cells was with the the the 5 were used for the of performed with the were for and for of THPO gene of the was by for protein of were generated using human DNA from patients either the or and the –622A or from the nucleotide of number as to the nucleotide of the was at of and the was were the reporter gene pGL3-basic the reporter the and –622A the and the of C-413T and the of The presence and of the sequences were by as described cells were with the reporter gene in which transcription is by a promoter as a and the pGL3-basic as a and using HepG2 cells were with the as using and were in cell using the assay using a and for electrophoretic mobility shift assay were synthesized at a of and by high DNA was using the DNA with or the for each of and were in at 95 for 5 and then to to of were by a of the published by et al. E. P. Nucleic Acids Res. PubMed Scopus Google Scholar). of nuclear protein was by and were performed using the each 5 of nuclear protein were with of as a of as and with binding 5 for 5 at DNA in with was with the of After the of of the THPO were for at or on of the was and for an additional 15 at were as a in or and using the Nucleic Detection and was performed as described previously J. J. S.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, S. S. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were by to a of and by for at cells were with The was and and were and the chromatin an length of After the was with of binding protein and at for The of were and further at for After the was from the subsequently were and were with at DNA was by with in with the to the DNA and used for and for the promoter A-622G were and was performed using the with and a were was by analyses using In of and of bp bp either of the were to analyses using the N. In Biol. Google Scholar). the of bp of THPO promoter region P1a were analyzed using the of THPO at and the promoter of THPO in HEK293T and HepG2, we and and exons and for reverse We THPO in cell lines and under all stimulatory and with and THPO transcription initiated from promoter P1a was more in the embryonic cell HEK293T than in the liver cell HepG2 for cells. of the THPO Promoter two of P1a and and subsequently DNA of 57 MI patients to MolHaps of promoter variants of promoter P1 The P1a region was amplified from to the nucleotide of number this was we subsequently performed a are as regions of DNA with a and a of to of M. M. J. Biol. PubMed Scopus Google Scholar). We analyzed bp of THPO promoter region P1a from to (accession number and a of with an of strongly the presence of of in the THPO Promoter of A-622G, C+5A, and with the several of which were by the presence of different of Genetic in the THPO Promoter and of Molecular the using polymorphism analysis in 95 MI patients from the ECTIM study and of the THPO promoter P1 in an additional set of 57 CVD patients from the MolProMD we identified 6 and a Three variants were in the [rs2855306], A-622G, and C-413T and three in the region G+115A, and C+135T). The identified in CVD patients is and number bp upstream of The the A-622G site to of the and which to the protein transcription factor is also in a binding site for the of this binding site is for bp by the the the to a of the which is involved in the of different as or type D. D. B. J. Biol. Google Scholar). binding site for a of the family of transcription was identified by and is for The C-413T for the of a of the nuclear factor family and the a binding site for The for this transcription factor to of and G+115A, which are in 1. The is in a binding site for the hormone of for of the protein more which has a structure T. H. C. T. T. J. M. S. J. PubMed Scopus Google Scholar). the we also a binding site for of the transcription factor family and protein a transcription The of the family to a of the transcription factor family Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). of the THPO Promoter the identified genetic variants A-622G, C+5A, and for their on promoter DNA fragments to the five MolHaps were pGL3-basic were performed in HEK293T and HepG2 cells, these cell lines expressed THPO In the activity of was significantly under (p < and under stimulatory (p = and (p = which was in HepG2 cells under stimulatory activity was compared with wt (p = also a significantly activity compared with (wt) in HEK293T cells under all stimulatory p < p < p < which was in HepG2 cells under with and in a activity p = p = in the MolProMD and in the ECTIM sample and in MolProMD and were further In HEK293T cells, under and stimulatory was significantly more active than wt p = p = p = In HepG2 cells was in activity of and MolHap3 a in promoter activity in cell in HEK293T p < p < p = in HepG2 p < p = p = compared with C-920T allelic in promoter activity in assays using HEK293T and HepG2 cells and further for A-622G and C-413T with from HEK293T and of A-622G the THPO promoter P1 by in an DNA-protein binding with nuclear from HEK293T and HepG2 with nuclear from HEK293T cells in a interaction that was more for the under and and band single the interaction with HepG2 nuclear an band different of transcription in the two different cell Supershift assays with a in a shift of the with nuclear from HEK293T be by which identified as of the binding at from HepG2 cells to that in this cell or is to a the in of an site for the assays using as well as and to with nuclear from HEK293T or HepG2 for C-413T in interaction with nuclear from HEK293T and HepG2 for and stimulatory and whereas the binding of the to be with nuclear from with for transcription and in a of and cell lines with the and The and the DNA-protein that a of the is involved in THPO promoter binding in this region. Supershift assays with a as well as with the binding instead of nuclear in that is either involved in DNA binding or at high levels to Supershift assays with were also for with from HEK293T and with we the interaction of a that in with nuclear from HEK293T and HepG2 by that we amplified fragments by that three haplotypes either with the or with individual with either or The of nuclear from HEK293T in the presence of two band single was by the or by the the or to with the the –622A or band in the –622A was in the with the band was significantly more with The band was by the with either that the regions are with nuclear and that this DNA-protein interaction on the presence of variants in a The with nuclear from HepG2 a was also a band that was with the with either single and it in the presence of the In the of genome-wide R. A. N. A. R. D.A. L.A. A. E. J.C. PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar), which the of of with genetic disease, and molecular functional profiling of genetic variation in gene region is In the study we analyzed the of the human THPO and propose a nomenclature for its molecular and We confirm that THPO consists of seven exons and introns and that transcription is initiated at two alternative The groups reported that THPO of 6 exons and 5 introns obtained their by and amplification of were performed with liver cell lines and revealed a single promoter region with multiple transcription initiation D.C. Sprecher C.A. Grant F.J. Kramer J.M. Kuijper J.L. Holly R.D. Whitmore T.E. Heipel M.D. Bell L.A. Ching A.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13023-13027Crossref PubMed Scopus (134) Google Scholar, 13Kamura T. Handa H. Hamasaki N. Kitajima S. J. Biol. Chem. 1997; 272: 11361-11368Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In the HepG2 cells used in transcription was exclusively at the to transcripts upstream the which to the nomenclature of exons (accession number the Chang et al. (14Chang M.S. McNinch J. Basu R. Shutter J. Hsu R.Y. Perkins C. Mar V. Suggs S. Welcher A. Li L. J. Biol. Chem. 1995; 270: 511-514Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) reported that THPO seven exons and In embryonic HEK293T cells we that transcription is also strongly by an upstream promoter region of an to the nomenclature of the human gene B. R. R. 1994; PubMed Scopus Google Scholar, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we this as and the nomenclature of for the The promoter which we in the are promoter upstream of and promoter upstream of 1. the molecular for this promoter we and bp of promoter P1a and of 57 patients with CVD. The THPO promoter P1a is and several it a for with or it is that transcription from P1a be by or to as in P1 is the the promoter in HepG2 cells and for THPO in the is in with from et al. (15Ghilardi N. Wiestner A. Skoda R.C. Blood. 1998; 92: 4023-4030Crossref PubMed Google Scholar) that in human liver 90% of the transcripts from P1. The intronic promoter is exons and the region for its is By subcloning and we identified the [A–622-C–413-C+5-G+115; [A–622-T–413-C+5-G+115], and [G–622-T–413-C+5-G+115], and were or in 57 respectively, we analyses on which were by allelic the promoter A-622G and of these in the of the P1 revealed a activity in HEK293T and HepG2, variants in functional promoter Furthermore, we were to that single genetic and molecular variation in the THPO promoter P1 and functional for transcription as well as DNA-protein as identified by promoter analyses and MolHap3 to a loss in activity in cell lines under and including inflammatory and cell differentiation compared with the wt In to HepG2, was significantly more active compared with the wt in HEK293T cells. these functional sequences with transcription we performed in which revealed that A-622G is a binding site for a family of with either or S.L. PubMed Scopus Google Scholar). The activity of transcription a on the of these their interaction with and their After in that DNA-protein interaction revealed that to the –622A in HEK293T cells of the stimulatory We were to confirm a binding at this site by the in of an site for the assays using as well as and to with nuclear from HEK293T or By the C-413T with of and we that in cell The a binding site for and a significantly promoter activity compared with the wt in HEK293T cells, whereas in the MolHap3 the promoter activity in cell lines was that a as a with the of we were to that nuclear with the region the A-622G and C-413T and that this interaction was more in the presence of the in HEK293T cells and in the presence of in cell a loss of protein which the activity of MolHap3 in We and cells in protein In the we were to that protein binding differed promoter single variation and in the embryonic cell HEK293T and for all three MolHaps we identified and binding which were with the as well as C-413T to the that DNA-protein were exclusively in the presence of in the promoter In the liver cell HepG2 we this DNA-protein which a different protein binding in these two cell differed from single as the was to the DNA-protein binding of In analyses to protein binding for the of on the of to a a to and all involved in promoter binding and the of their the THPO locus on chromosome has been with Nature. PubMed Scopus Google Scholar), and a THPO has been with type and the disease in genome-wide these used in genome-wide are in linkage with functional it be for these to functional MolHaps are with these disease In in the analyses we and the alternative promoter P1a and propose to the as we identified three functional which influenced its We that A-622G and C-413T are functional as in an active region of THPO promoter P1. of MolHaps with TPO levels in cells from as well as gene and to the interaction of different transcription the THPO respect to protein protein to candidate that in to THPO with