{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/16867"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/16867","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"A functional analysis of the epigenetic regulator CTCF","abstract":"The highly conserved protein CTCF (CCCTC-binding factor) participates in many epigenetic regulatory functions, including insulation, imprinting, X chromosome inactivation, and both transcriptional activation and repression. Recent data has also suggested a prominent role for CTCF in mediating interactions between chromosomes and in the global organization of the nucleus. In this work, we initiated a functional analysis of CTCF to determine the regions of the protein necessary for regulating transcription and altering chromatin structure. As a reporter for changes in chromatin, we employed a cell line bearing a lac operator array that can dramatically unfold its normally condensed chromatin when targeted by transcriptional proteins fused to a GFP-tagged lac repressor DNA binding domain. With this system, we demonstrated that CTCF-lac repressor fusions induced substantial decondensation of the array and we have mapped the opening activity to portions of the N- and C-terminal regions of the protein. In addition, we mapped a transcriptional activation domain in CTCF‟s NT-region (NTAD) that recruits HATs and chromatin remodeling proteins similar to acidic activators. The C-terminal also has two opening domains, but they do not activate transcription. Moreover, arrays opened by the CT region adopt structures morphologically distinct from the NTAD and other activator proteins. The unique CT-induced structures require an AT-hook motif that appears to bring disparate portions of the array together. Thus, the conserved AT-hook may contribute to the tethering activity of CTCF during DNA loop formation at endogenous loci. In addition to assessing chromatin opening, we have demonstrated the utility of the array system as an in vivo method for cofactor recruitment by confirming co-localization of known CTCF protein partners to a targeted array. Therefore, this system also provided an assay to identify the recruitment domains for each partner. We revealed that the cohesin complex is recruited by CTCF through the NTAD, which provides the first evidence that an activation domain can recruit the cohesin complex. We also clarified CTCF‟s interaction to the remodeling protein CHD8 by showing its functional recruitment to the array through one of the CT-opening domains. Additionally, we differentiated the three domains in CTCF that are sufficient to initiate decondensation of chromatin structure by ascertaining the co-factors recruited to the array by each domain. Finally, with this system we found and analyzed SUMO-motif sites in the NT- and CT-regions. Our data suggests that sumoylation of the NTAD reduces its transactivation and chromatin opening activities, while sumoylation of the CT region demonstrated enhancer blocking activity. This is the first study to elucidate an enhancer blocking domain for CTCF and suggests a mechanism for directing this activity to specific loci via a post-translational SUMO-modification. We have demonstrated a direct role in altering chromatin structure, elucidated several of the diverse transcriptional regulatory domains in CTCF and further characterized the co-factors potentially involved with CTCF function.","abstract_html":"The highly conserved protein CTCF (CCCTC-binding factor) participates in many epigenetic regulatory functions, including insulation, imprinting, X chromosome inactivation, and both transcriptional activation and repression. Recent data has also suggested a prominent role for CTCF in mediating interactions between chromosomes and in the global organization of the nucleus. In this work, we initiated a functional analysis of CTCF to determine the regions of the protein necessary for regulating transcription and altering chromatin structure. As a reporter for changes in chromatin, we employed a cell line bearing a lac operator array that can dramatically unfold its normally condensed chromatin when targeted by transcriptional proteins fused to a GFP-tagged lac repressor DNA binding domain. With this system, we demonstrated that CTCF-lac repressor fusions induced substantial decondensation of the array and we have mapped the opening activity to portions of the N- and C-terminal regions of the protein. In addition, we mapped a transcriptional activation domain in CTCF‟s NT-region (NTAD) that recruits HATs and chromatin remodeling proteins similar to acidic activators. The C-terminal also has two opening domains, but they do not activate transcription. Moreover, arrays opened by the CT region adopt structures morphologically distinct from the NTAD and other activator proteins. The unique CT-induced structures require an AT-hook motif that appears to bring disparate portions of the array together. Thus, the conserved AT-hook may contribute to the tethering activity of CTCF during DNA loop formation at endogenous loci. In addition to assessing chromatin opening, we have demonstrated the utility of the array system as an in vivo method for cofactor recruitment by confirming co-localization of known CTCF protein partners to a targeted array. Therefore, this system also provided an assay to identify the recruitment domains for each partner. We revealed that the cohesin complex is recruited by CTCF through the NTAD, which provides the first evidence that an activation domain can recruit the cohesin complex. We also clarified CTCF‟s interaction to the remodeling protein CHD8 by showing its functional recruitment to the array through one of the CT-opening domains. Additionally, we differentiated the three domains in CTCF that are sufficient to initiate decondensation of chromatin structure by ascertaining the co-factors recruited to the array by each domain. Finally, with this system we found and analyzed SUMO-motif sites in the NT- and CT-regions. Our data suggests that sumoylation of the NTAD reduces its transactivation and chromatin opening activities, while sumoylation of the CT region demonstrated enhancer blocking activity. This is the first study to elucidate an enhancer blocking domain for CTCF and suggests a mechanism for directing this activity to specific loci via a post-translational SUMO-modification. We have demonstrated a direct role in altering chromatin structure, elucidated several of the diverse transcriptional regulatory domains in CTCF and further characterized the co-factors potentially involved with CTCF function.","abstract_has_math":false,"creators":["Kitchen, Neal S."],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Cell and Developmental Biology","degree_department":null,"school":null,"contributors":["Schoenherr, Christopher J.","Chen, Jie","Belmont, Andrew S.","Kemper, Byron W.","Freeman, Brian C."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010-08-20T18:00:20Z","date_published":"2010-08-20T18:00:20Z","updated_at":"2026-07-22T22:25:09Z","subjects":["CCCTC-binding factor (zinc finger protein) (CTCF)","large-scale chromatin","Small Ubiquitin-like MOdifier (SUMO)","insulator","chromatin boundary","imprinting"],"languages":["en"],"rights":["Copyright 2010 Neal S. Kitchen"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/16867","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Schoenherr, Christopher J.","Chen, Jie","Belmont, Andrew S.","Kemper, Byron W.","Freeman, Brian C."]},{"key":"dc:creator","label":"Author","values":["Kitchen, Neal S."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2010-08-20T18:00:20Z","2010-08"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Cell and Developmental Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["CCCTC-binding factor (zinc finger protein) (CTCF)","large-scale chromatin","Small Ubiquitin-like MOdifier (SUMO)","insulator","chromatin boundary","imprinting"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2010 Neal S. 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As a reporter for changes in chromatin, we employed a cell line bearing a lac operator array that can dramatically unfold its normally condensed chromatin when targeted by transcriptional proteins fused to a GFP-tagged lac repressor DNA binding domain. With this system, we demonstrated that CTCF-lac repressor fusions induced substantial decondensation of the array and we have mapped the opening activity to portions of the N- and C-terminal regions of the protein. In addition, we mapped a transcriptional activation domain in CTCF‟s NT-region (NTAD) that recruits HATs and chromatin remodeling proteins similar to acidic activators. The C-terminal also has two opening domains, but they do not activate transcription. Moreover, arrays opened by the CT region adopt structures morphologically distinct from the NTAD and other activator proteins. The unique CT-induced structures require an AT-hook motif that appears to bring disparate portions of the array together. Thus, the conserved AT-hook may contribute to the tethering activity of CTCF during DNA loop formation at endogenous loci. In addition to assessing chromatin opening, we have demonstrated the utility of the array system as an in vivo method for cofactor recruitment by confirming co-localization of known CTCF protein partners to a targeted array. Therefore, this system also provided an assay to identify the recruitment domains for each partner. We revealed that the cohesin complex is recruited by CTCF through the NTAD, which provides the first evidence that an activation domain can recruit the cohesin complex. We also clarified CTCF‟s interaction to the remodeling protein CHD8 by showing its functional recruitment to the array through one of the CT-opening domains. Additionally, we differentiated the three domains in CTCF that are sufficient to initiate decondensation of chromatin structure by ascertaining the co-factors recruited to the array by each domain. Finally, with this system we found and analyzed SUMO-motif sites in the NT- and CT-regions. Our data suggests that sumoylation of the NTAD reduces its transactivation and chromatin opening activities, while sumoylation of the CT region demonstrated enhancer blocking activity. This is the first study to elucidate an enhancer blocking domain for CTCF and suggests a mechanism for directing this activity to specific loci via a post-translational SUMO-modification. 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In this work, we initiated a functional analysis of CTCF to determine the regions of the protein necessary for regulating transcription and altering chromatin structure. As a reporter for changes in chromatin, we employed a cell line bearing a lac operator array that can dramatically unfold its normally condensed chromatin when targeted by transcriptional proteins fused to a GFP-tagged lac repressor DNA binding domain. With this system, we demonstrated that CTCF-lac repressor fusions induced substantial decondensation of the array and we have mapped the opening activity to portions of the N- and C-terminal regions of the protein. In addition, we mapped a transcriptional activation domain in CTCF‟s NT-region (NTAD) that recruits HATs and chromatin remodeling proteins similar to acidic activators. The C-terminal also has two opening domains, but they do not activate transcription. Moreover, arrays opened by the CT region adopt structures morphologically distinct from the NTAD and other activator proteins. The unique CT-induced structures require an AT-hook motif that appears to bring disparate portions of the array together. Thus, the conserved AT-hook may contribute to the tethering activity of CTCF during DNA loop formation at endogenous loci. In addition to assessing chromatin opening, we have demonstrated the utility of the array system as an in vivo method for cofactor recruitment by confirming co-localization of known CTCF protein partners to a targeted array. Therefore, this system also provided an assay to identify the recruitment domains for each partner. We revealed that the cohesin complex is recruited by CTCF through the NTAD, which provides the first evidence that an activation domain can recruit the cohesin complex. We also clarified CTCF‟s interaction to the remodeling protein CHD8 by showing its functional recruitment to the array through one of the CT-opening domains. Additionally, we differentiated the three domains in CTCF that are sufficient to initiate decondensation of chromatin structure by ascertaining the co-factors recruited to the array by each domain. Finally, with this system we found and analyzed SUMO-motif sites in the NT- and CT-regions. Our data suggests that sumoylation of the NTAD reduces its transactivation and chromatin opening activities, while sumoylation of the CT region demonstrated enhancer blocking activity. This is the first study to elucidate an enhancer blocking domain for CTCF and suggests a mechanism for directing this activity to specific loci via a post-translational SUMO-modification. 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