RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins
Summary Background Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt‐related transcription factor 1 (RUNX1) of FPD patients. Objectives To elucidate t...
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| Veröffentlicht in: | Journal of thrombosis and haemostasis 2013-09, Vol.11 (9), p.1742-1750 |
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| creator | Okada, Y. Watanabe, M. Nakai, T. Kamikawa, Y. Shimizu, M. Fukuhara, Y. Yonekura, M. Matsuura, E. Hoshika, Y. Nagai, R. Aird, W. C. Doi, T. |
| description | Summary
Background
Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt‐related transcription factor 1 (RUNX1) of FPD patients.
Objectives
To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1‐mediated induction of the FPD phenotype.
Methods
We assessed whether RUNX1 and its mutants, in combination with E26 transformation‐specific‐1 (ETS‐1), Core‐binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI‐1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real‐time RT‐PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos‐7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos‐7 cells.
Results
We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS‐1 and CBFβ, or FLI‐1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA‐binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS‐1, CBFβ, and FLI‐1.
Conclusions
RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities. |
| doi_str_mv | 10.1111/jth.12355 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_proquest_miscellaneous_1496889971</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>3069838221</sourcerecordid><originalsourceid>FETCH-LOGICAL-p3125-51742d248a97798b79d756f1f627d4301ce638f243e4138f0ee2e1f436f9ae923</originalsourceid><addsrcrecordid>eNqNkc1O3TAQhS1UVH7aBS9QWeqmCy7EHie2lxXipwgBKheJXeSbTMBXjhNip5B34KFr7oUuumI2c0bzaY5Gh5A9lh2wVIfL-HDAOOT5BtlmOaiZVFB8etcaYIvshLDMMqZznn0mWxyUUCKDbfLy-_byju3TxRip7yK1MdDGtNZZ42jvTESHkdY2dEONA23HaHwM-zRMHod7G6KtjHMTNVW0fxId6PWJoPfokeJzP2AItvPUelp17cJ6E1_HJxsf6PH8Zu000X7oIlofvpDNxriAX9_6Lrk9OZ4fnc0urk5_Hf28mPXAeD7LmRS85kIZLaVWC6lrmRcNawouawEZq7AA1XABKFgSGSJH1ggoGm1Qc9glP9Z3k_HjiCGWrQ0VOmc8dmMomdCFUlpL9gEUeMEkyCKh3_9Dl904-PTIigKejupEfXujxkWLddkPtjXDVL5HkoDDNfBkHU7_9iwrX7MuU9blKuvyfH62EvAX_06bOA</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1432329689</pqid></control><display><type>article</type><title>RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins</title><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Alma/SFX Local Collection</source><creator>Okada, Y. ; Watanabe, M. ; Nakai, T. ; Kamikawa, Y. ; Shimizu, M. ; Fukuhara, Y. ; Yonekura, M. ; Matsuura, E. ; Hoshika, Y. ; Nagai, R. ; Aird, W. C. ; Doi, T.</creator><creatorcontrib>Okada, Y. ; Watanabe, M. ; Nakai, T. ; Kamikawa, Y. ; Shimizu, M. ; Fukuhara, Y. ; Yonekura, M. ; Matsuura, E. ; Hoshika, Y. ; Nagai, R. ; Aird, W. C. ; Doi, T.</creatorcontrib><description>Summary
Background
Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt‐related transcription factor 1 (RUNX1) of FPD patients.
Objectives
To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1‐mediated induction of the FPD phenotype.
Methods
We assessed whether RUNX1 and its mutants, in combination with E26 transformation‐specific‐1 (ETS‐1), Core‐binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI‐1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real‐time RT‐PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos‐7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos‐7 cells.
Results
We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS‐1 and CBFβ, or FLI‐1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA‐binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS‐1, CBFβ, and FLI‐1.
Conclusions
RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.</description><identifier>ISSN: 1538-7933</identifier><identifier>EISSN: 1538-7836</identifier><identifier>DOI: 10.1111/jth.12355</identifier><identifier>PMID: 23848403</identifier><language>eng</language><publisher>England: Elsevier Limited</publisher><subject>Blood Platelet Disorders - genetics ; Cell Line ; Core Binding Factor Alpha 2 Subunit - genetics ; Core Binding Factor Alpha 2 Subunit - metabolism ; Differentiation ; Electrophoretic Mobility Shift Assay ; ETS1 protein, human ; FLI1 protein, human ; Gene expression ; Gene Expression Regulation - genetics ; Humans ; Medical research ; megakaryocytes ; Mutation ; platelet disorder, familial, with associated myeloid malignancy ; platelet factor 4 ; Platelet Factor 4 - genetics ; Proto-Oncogene Proteins c-ets - metabolism ; Real-Time Polymerase Chain Reaction ; RUNX1 protein, human ; Stem cells ; Subcellular Fractions - metabolism</subject><ispartof>Journal of thrombosis and haemostasis, 2013-09, Vol.11 (9), p.1742-1750</ispartof><rights>2013 International Society on Thrombosis and Haemostasis</rights><rights>2013 International Society on Thrombosis and Haemostasis.</rights><rights>Copyright © 2013 International Society on Thrombosis and Haemostasis</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23848403$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Okada, Y.</creatorcontrib><creatorcontrib>Watanabe, M.</creatorcontrib><creatorcontrib>Nakai, T.</creatorcontrib><creatorcontrib>Kamikawa, Y.</creatorcontrib><creatorcontrib>Shimizu, M.</creatorcontrib><creatorcontrib>Fukuhara, Y.</creatorcontrib><creatorcontrib>Yonekura, M.</creatorcontrib><creatorcontrib>Matsuura, E.</creatorcontrib><creatorcontrib>Hoshika, Y.</creatorcontrib><creatorcontrib>Nagai, R.</creatorcontrib><creatorcontrib>Aird, W. C.</creatorcontrib><creatorcontrib>Doi, T.</creatorcontrib><title>RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins</title><title>Journal of thrombosis and haemostasis</title><addtitle>J Thromb Haemost</addtitle><description>Summary
Background
Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt‐related transcription factor 1 (RUNX1) of FPD patients.
Objectives
To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1‐mediated induction of the FPD phenotype.
Methods
We assessed whether RUNX1 and its mutants, in combination with E26 transformation‐specific‐1 (ETS‐1), Core‐binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI‐1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real‐time RT‐PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos‐7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos‐7 cells.
Results
We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS‐1 and CBFβ, or FLI‐1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA‐binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS‐1, CBFβ, and FLI‐1.
Conclusions
RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.</description><subject>Blood Platelet Disorders - genetics</subject><subject>Cell Line</subject><subject>Core Binding Factor Alpha 2 Subunit - genetics</subject><subject>Core Binding Factor Alpha 2 Subunit - metabolism</subject><subject>Differentiation</subject><subject>Electrophoretic Mobility Shift Assay</subject><subject>ETS1 protein, human</subject><subject>FLI1 protein, human</subject><subject>Gene expression</subject><subject>Gene Expression Regulation - genetics</subject><subject>Humans</subject><subject>Medical research</subject><subject>megakaryocytes</subject><subject>Mutation</subject><subject>platelet disorder, familial, with associated myeloid malignancy</subject><subject>platelet factor 4</subject><subject>Platelet Factor 4 - genetics</subject><subject>Proto-Oncogene Proteins c-ets - metabolism</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>RUNX1 protein, human</subject><subject>Stem cells</subject><subject>Subcellular Fractions - metabolism</subject><issn>1538-7933</issn><issn>1538-7836</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1O3TAQhS1UVH7aBS9QWeqmCy7EHie2lxXipwgBKheJXeSbTMBXjhNip5B34KFr7oUuumI2c0bzaY5Gh5A9lh2wVIfL-HDAOOT5BtlmOaiZVFB8etcaYIvshLDMMqZznn0mWxyUUCKDbfLy-_byju3TxRip7yK1MdDGtNZZ42jvTESHkdY2dEONA23HaHwM-zRMHod7G6KtjHMTNVW0fxId6PWJoPfokeJzP2AItvPUelp17cJ6E1_HJxsf6PH8Zu000X7oIlofvpDNxriAX9_6Lrk9OZ4fnc0urk5_Hf28mPXAeD7LmRS85kIZLaVWC6lrmRcNawouawEZq7AA1XABKFgSGSJH1ggoGm1Qc9glP9Z3k_HjiCGWrQ0VOmc8dmMomdCFUlpL9gEUeMEkyCKh3_9Dl904-PTIigKejupEfXujxkWLddkPtjXDVL5HkoDDNfBkHU7_9iwrX7MuU9blKuvyfH62EvAX_06bOA</recordid><startdate>201309</startdate><enddate>201309</enddate><creator>Okada, Y.</creator><creator>Watanabe, M.</creator><creator>Nakai, T.</creator><creator>Kamikawa, Y.</creator><creator>Shimizu, M.</creator><creator>Fukuhara, Y.</creator><creator>Yonekura, M.</creator><creator>Matsuura, E.</creator><creator>Hoshika, Y.</creator><creator>Nagai, R.</creator><creator>Aird, W. C.</creator><creator>Doi, T.</creator><general>Elsevier Limited</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7T5</scope><scope>H94</scope><scope>K9.</scope><scope>7X8</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>201309</creationdate><title>RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins</title><author>Okada, Y. ; Watanabe, M. ; Nakai, T. ; Kamikawa, Y. ; Shimizu, M. ; Fukuhara, Y. ; Yonekura, M. ; Matsuura, E. ; Hoshika, Y. ; Nagai, R. ; Aird, W. C. ; Doi, T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p3125-51742d248a97798b79d756f1f627d4301ce638f243e4138f0ee2e1f436f9ae923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Blood Platelet Disorders - genetics</topic><topic>Cell Line</topic><topic>Core Binding Factor Alpha 2 Subunit - genetics</topic><topic>Core Binding Factor Alpha 2 Subunit - metabolism</topic><topic>Differentiation</topic><topic>Electrophoretic Mobility Shift Assay</topic><topic>ETS1 protein, human</topic><topic>FLI1 protein, human</topic><topic>Gene expression</topic><topic>Gene Expression Regulation - genetics</topic><topic>Humans</topic><topic>Medical research</topic><topic>megakaryocytes</topic><topic>Mutation</topic><topic>platelet disorder, familial, with associated myeloid malignancy</topic><topic>platelet factor 4</topic><topic>Platelet Factor 4 - genetics</topic><topic>Proto-Oncogene Proteins c-ets - metabolism</topic><topic>Real-Time Polymerase Chain Reaction</topic><topic>RUNX1 protein, human</topic><topic>Stem cells</topic><topic>Subcellular Fractions - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Okada, Y.</creatorcontrib><creatorcontrib>Watanabe, M.</creatorcontrib><creatorcontrib>Nakai, T.</creatorcontrib><creatorcontrib>Kamikawa, Y.</creatorcontrib><creatorcontrib>Shimizu, M.</creatorcontrib><creatorcontrib>Fukuhara, Y.</creatorcontrib><creatorcontrib>Yonekura, M.</creatorcontrib><creatorcontrib>Matsuura, E.</creatorcontrib><creatorcontrib>Hoshika, Y.</creatorcontrib><creatorcontrib>Nagai, R.</creatorcontrib><creatorcontrib>Aird, W. C.</creatorcontrib><creatorcontrib>Doi, T.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>MEDLINE - Academic</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Journal of thrombosis and haemostasis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Okada, Y.</au><au>Watanabe, M.</au><au>Nakai, T.</au><au>Kamikawa, Y.</au><au>Shimizu, M.</au><au>Fukuhara, Y.</au><au>Yonekura, M.</au><au>Matsuura, E.</au><au>Hoshika, Y.</au><au>Nagai, R.</au><au>Aird, W. C.</au><au>Doi, T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins</atitle><jtitle>Journal of thrombosis and haemostasis</jtitle><addtitle>J Thromb Haemost</addtitle><date>2013-09</date><risdate>2013</risdate><volume>11</volume><issue>9</issue><spage>1742</spage><epage>1750</epage><pages>1742-1750</pages><issn>1538-7933</issn><eissn>1538-7836</eissn><abstract>Summary
Background
Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt‐related transcription factor 1 (RUNX1) of FPD patients.
Objectives
To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1‐mediated induction of the FPD phenotype.
Methods
We assessed whether RUNX1 and its mutants, in combination with E26 transformation‐specific‐1 (ETS‐1), Core‐binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI‐1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real‐time RT‐PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos‐7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos‐7 cells.
Results
We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS‐1 and CBFβ, or FLI‐1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA‐binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS‐1, CBFβ, and FLI‐1.
Conclusions
RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.</abstract><cop>England</cop><pub>Elsevier Limited</pub><pmid>23848403</pmid><doi>10.1111/jth.12355</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Blood Platelet Disorders - genetics Cell Line Core Binding Factor Alpha 2 Subunit - genetics Core Binding Factor Alpha 2 Subunit - metabolism Differentiation Electrophoretic Mobility Shift Assay ETS1 protein, human FLI1 protein, human Gene expression Gene Expression Regulation - genetics Humans Medical research megakaryocytes Mutation platelet disorder, familial, with associated myeloid malignancy platelet factor 4 Platelet Factor 4 - genetics Proto-Oncogene Proteins c-ets - metabolism Real-Time Polymerase Chain Reaction RUNX1 protein, human Stem cells Subcellular Fractions - metabolism |
| title | RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins |
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