The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation
Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that cre...
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| Veröffentlicht in: | Nucleic acids research 2002-02, Vol.30 (4), p.958-965 |
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| creator | Birke, Marco Schreiner, Silke García-Cuéllar, María-Paz Mahr, Kerstin Titgemeyer, Fritz Slany, Robert K |
| description | Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct. |
| doi_str_mv | 10.1093/nar/30.4.958 |
| format | Article |
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The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.</description><identifier>ISSN: 1362-4962</identifier><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/30.4.958</identifier><identifier>PMID: 11842107</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford Publishing Limited (England)</publisher><subject>Amino Acid Sequence ; Base Sequence ; Binding Sites ; Deoxyribonuclease I - chemistry ; DNA Footprinting ; DNA Methylation ; DNA Modification Methylases - chemistry ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - genetics ; DNA-Binding Proteins - metabolism ; Electrophoretic Mobility Shift Assay ; Glutathione Transferase - genetics ; Histone-Lysine N-Methyltransferase ; Humans ; MLL protein ; Molecular Sequence Data ; Myeloid-Lymphoid Leukemia Protein ; Oligodeoxyribonucleotides - metabolism ; Protein Structure, Tertiary ; Proto-Oncogene Proteins - chemistry ; Proto-Oncogene Proteins - genetics ; Proto-Oncogene Proteins - metabolism ; Proto-Oncogenes ; Recombinant Fusion Proteins - metabolism ; Substrate Specificity ; Surface Plasmon Resonance ; Transcription Factors ; Transcriptional Activation ; VP16 protein</subject><ispartof>Nucleic acids research, 2002-02, Vol.30 (4), p.958-965</ispartof><rights>Copyright Oxford University Press(England) Feb 15, 2002</rights><rights>Copyright © 2002 Oxford University Press 2002</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c503t-678b988d399c4169d86d36d64f41bae20cbf223d7eb75291bb95cb4458e6d34f3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC100340/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC100340/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11842107$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Birke, Marco</creatorcontrib><creatorcontrib>Schreiner, Silke</creatorcontrib><creatorcontrib>García-Cuéllar, María-Paz</creatorcontrib><creatorcontrib>Mahr, Kerstin</creatorcontrib><creatorcontrib>Titgemeyer, Fritz</creatorcontrib><creatorcontrib>Slany, Robert K</creatorcontrib><title>The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.</description><subject>Amino Acid Sequence</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Deoxyribonuclease I - chemistry</subject><subject>DNA Footprinting</subject><subject>DNA Methylation</subject><subject>DNA Modification Methylases - chemistry</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Electrophoretic Mobility Shift Assay</subject><subject>Glutathione Transferase - genetics</subject><subject>Histone-Lysine N-Methyltransferase</subject><subject>Humans</subject><subject>MLL protein</subject><subject>Molecular Sequence Data</subject><subject>Myeloid-Lymphoid Leukemia Protein</subject><subject>Oligodeoxyribonucleotides - metabolism</subject><subject>Protein Structure, Tertiary</subject><subject>Proto-Oncogene Proteins - chemistry</subject><subject>Proto-Oncogene Proteins - genetics</subject><subject>Proto-Oncogene Proteins - metabolism</subject><subject>Proto-Oncogenes</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Substrate Specificity</subject><subject>Surface Plasmon Resonance</subject><subject>Transcription Factors</subject><subject>Transcriptional Activation</subject><subject>VP16 protein</subject><issn>1362-4962</issn><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkTtvFDEUhS0EIg_oqJFFQZXZ-DljFxTRkgSkDTRLbfk1u45m7MX2Rsq_x1FWENKk8pXPd67uvQeADxgtMJL0POp8TtGCLSQXr8Axpj3pmOzJ6yf1ETgp5RYhzDBnb8ERxoIRjIZjMK23Ht6soUuzDhGmEdb2scuppi5Fmx4q34Sb1QqaEF2BNcHl7rqzKdbmCHEDv_64gDo66EKxOcwh6uoL1JsmlwpnX7f3k64hxXfgzain4t8f3lPw6-pyvfzWrX5ef19erDrLEa1dPwgjhXBUSstwL53oHe1dz0aGjfYEWTMSQt3gzcCJxMZIbg1jXPgGspGegi-PfXd7M3tnfaxZT2rXhtP5XiUd1P9KDFu1SXcKI0QZav7PB39Ov_e-VDW31fw06ejTvqgBM95z_DKIBRFUItrAT8_A27TPsR1BEYT4IDkjDTp7hGxOpWQ__p0YI_WQtWpZK4oUUy3rhn98uuU_-BAu_QOUMaXF</recordid><startdate>20020215</startdate><enddate>20020215</enddate><creator>Birke, Marco</creator><creator>Schreiner, Silke</creator><creator>García-Cuéllar, María-Paz</creator><creator>Mahr, Kerstin</creator><creator>Titgemeyer, Fritz</creator><creator>Slany, Robert K</creator><general>Oxford Publishing Limited (England)</general><general>Oxford University Press</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20020215</creationdate><title>The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation</title><author>Birke, Marco ; Schreiner, Silke ; García-Cuéllar, María-Paz ; Mahr, Kerstin ; Titgemeyer, Fritz ; Slany, Robert K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c503t-678b988d399c4169d86d36d64f41bae20cbf223d7eb75291bb95cb4458e6d34f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Amino Acid Sequence</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Deoxyribonuclease I - chemistry</topic><topic>DNA Footprinting</topic><topic>DNA Methylation</topic><topic>DNA Modification Methylases - chemistry</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Electrophoretic Mobility Shift Assay</topic><topic>Glutathione Transferase - genetics</topic><topic>Histone-Lysine N-Methyltransferase</topic><topic>Humans</topic><topic>MLL protein</topic><topic>Molecular Sequence Data</topic><topic>Myeloid-Lymphoid Leukemia Protein</topic><topic>Oligodeoxyribonucleotides - metabolism</topic><topic>Protein Structure, Tertiary</topic><topic>Proto-Oncogene Proteins - chemistry</topic><topic>Proto-Oncogene Proteins - genetics</topic><topic>Proto-Oncogene Proteins - metabolism</topic><topic>Proto-Oncogenes</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Substrate Specificity</topic><topic>Surface Plasmon Resonance</topic><topic>Transcription Factors</topic><topic>Transcriptional Activation</topic><topic>VP16 protein</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Birke, Marco</creatorcontrib><creatorcontrib>Schreiner, Silke</creatorcontrib><creatorcontrib>García-Cuéllar, María-Paz</creatorcontrib><creatorcontrib>Mahr, Kerstin</creatorcontrib><creatorcontrib>Titgemeyer, Fritz</creatorcontrib><creatorcontrib>Slany, Robert K</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Birke, Marco</au><au>Schreiner, Silke</au><au>García-Cuéllar, María-Paz</au><au>Mahr, Kerstin</au><au>Titgemeyer, Fritz</au><au>Slany, Robert K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2002-02-15</date><risdate>2002</risdate><volume>30</volume><issue>4</issue><spage>958</spage><epage>965</epage><pages>958-965</pages><issn>1362-4962</issn><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.</abstract><cop>England</cop><pub>Oxford Publishing Limited (England)</pub><pmid>11842107</pmid><doi>10.1093/nar/30.4.958</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| source | Oxford Journals Open Access Collection; MEDLINE; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence Base Sequence Binding Sites Deoxyribonuclease I - chemistry DNA Footprinting DNA Methylation DNA Modification Methylases - chemistry DNA-Binding Proteins - chemistry DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism Electrophoretic Mobility Shift Assay Glutathione Transferase - genetics Histone-Lysine N-Methyltransferase Humans MLL protein Molecular Sequence Data Myeloid-Lymphoid Leukemia Protein Oligodeoxyribonucleotides - metabolism Protein Structure, Tertiary Proto-Oncogene Proteins - chemistry Proto-Oncogene Proteins - genetics Proto-Oncogene Proteins - metabolism Proto-Oncogenes Recombinant Fusion Proteins - metabolism Substrate Specificity Surface Plasmon Resonance Transcription Factors Transcriptional Activation VP16 protein |
| title | The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation |
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