{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/72980"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/72980","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Mechanisms and functional significance of nuclear compartmentalization","abstract":"The mammalian cell nucleus is a complex yet highly organized structure. There is significant amount of knowledge about the organization of nucleus; however, the influence of such structural complexity of the nucleus in gene regulation is poorly understood. For better understanding of the effects of nuclear repositioning of a gene locus on gene expression, I investigated the physiological significance and mechanism of association of a gene locus with a nuclear sub-compartment. In chapter 1, I showed an actin dependent association of HSP70 transgenes with nuclear speckles after heat shock. I visualized HSP70 transgenes moving curvilinearly towards nuclear speckles over ~0.5-6 μm distances at velocities of 1-2 μm min-1. Observation of chromatin stretching in the direction of movement demonstrated a force generating mechanism. Transcription in nearly all cases increased noticeably only after initial contact with a nuclear speckle. The time lag between initial speckle contact and increased transcription was inversely proportional to the speckle size at first contact with the HSP70 transgene and after initial contact with small speckles an increase in speckle size typically preceded increased transcription. Our results demonstrate the existence of a still to be revealed machinery for moving chromatin in a direct path over long distances towards nuclear speckles in response to transcriptional activation. In Chapter 2, we used autonomous targeting of BAC transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs), including the 6 kb PTR1, target the ~100 kb beta-globin gene cluster and LCR region to the nuclear periphery and confer increased H3K9me3 modification, as assayed both by immunofluorescence and chromatin immunoprecipitation. PTRs within HBB BACs bias a competition between peripheral versus pericentric heterochromatin (PCH) targeting towards peripheral targeting. Targeting to both heterochromatin compartments is dependent on H3K9 trimethylation. More generally, PTRs confer targeting from the PCH to the periphery, from the interior to the PCH, or have no targeting activity depending on the flanking DNA context, with peripheral targeting correlating with higher, domain-wide H3K9m3 levels. A combination of FISH, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over several hundred kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent Lamin Associated Domain. Our results demonstrate multiple cis elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.","abstract_html":"The mammalian cell nucleus is a complex yet highly organized structure. There is significant amount of knowledge about the organization of nucleus; however, the influence of such structural complexity of the nucleus in gene regulation is poorly understood. For better understanding of the effects of nuclear repositioning of a gene locus on gene expression, I investigated the physiological significance and mechanism of association of a gene locus with a nuclear sub-compartment. In chapter 1, I showed an actin dependent association of HSP70 transgenes with nuclear speckles after heat shock. I visualized HSP70 transgenes moving curvilinearly towards nuclear speckles over ~0.5-6 μm distances at velocities of 1-2 μm min-1. Observation of chromatin stretching in the direction of movement demonstrated a force generating mechanism. Transcription in nearly all cases increased noticeably only after initial contact with a nuclear speckle. The time lag between initial speckle contact and increased transcription was inversely proportional to the speckle size at first contact with the HSP70 transgene and after initial contact with small speckles an increase in speckle size typically preceded increased transcription. Our results demonstrate the existence of a still to be revealed machinery for moving chromatin in a direct path over long distances towards nuclear speckles in response to transcriptional activation. In Chapter 2, we used autonomous targeting of BAC transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs), including the 6 kb PTR1, target the ~100 kb beta-globin gene cluster and LCR region to the nuclear periphery and confer increased H3K9me3 modification, as assayed both by immunofluorescence and chromatin immunoprecipitation. PTRs within HBB BACs bias a competition between peripheral versus pericentric heterochromatin (PCH) targeting towards peripheral targeting. Targeting to both heterochromatin compartments is dependent on H3K9 trimethylation. More generally, PTRs confer targeting from the PCH to the periphery, from the interior to the PCH, or have no targeting activity depending on the flanking DNA context, with peripheral targeting correlating with higher, domain-wide H3K9m3 levels. A combination of FISH, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over several hundred kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent Lamin Associated Domain. Our results demonstrate multiple cis elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.","abstract_has_math":false,"creators":["Khanna, Nimish"],"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":["Belmont, Andrew S.","Freeman, Brian C.","Brieher, William M.","Prasanth, K V.","Selvin, Paul R."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-01-21T19:55:15Z","date_published":"2015-01-21T19:55:15Z","updated_at":"2026-07-22T22:26:07Z","subjects":["Nuclear organization","Chromatin dynamics"],"languages":["en"],"rights":["Copyright 2014 Nimish Khanna"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/72980","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Belmont, Andrew S.","Freeman, Brian C.","Brieher, William M.","Prasanth, K V.","Selvin, Paul R."]},{"key":"dc:creator","label":"Author","values":["Khanna, Nimish"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-01-21T19:55:15Z","2017-01-22T10:15:27Z","2014-12","2015-01-21"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"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":["Nuclear organization","Chromatin dynamics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2014 Nimish Khanna"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/72980"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The mammalian cell nucleus is a complex yet highly organized structure. There is significant amount of knowledge about the organization of nucleus; however, the influence of such structural complexity of the nucleus in gene regulation is poorly understood. For better understanding of the effects of nuclear repositioning of a gene locus on gene expression, I investigated the physiological significance and mechanism of association of a gene locus with a nuclear sub-compartment. In chapter 1, I showed an actin dependent association of HSP70 transgenes with nuclear speckles after heat shock. I visualized HSP70 transgenes moving curvilinearly towards nuclear speckles over ~0.5-6 μm distances at velocities of 1-2 μm min-1. Observation of chromatin stretching in the direction of movement demonstrated a force generating mechanism. Transcription in nearly all cases increased noticeably only after initial contact with a nuclear speckle. The time lag between initial speckle contact and increased transcription was inversely proportional to the speckle size at first contact with the HSP70 transgene and after initial contact with small speckles an increase in speckle size typically preceded increased transcription. Our results demonstrate the existence of a still to be revealed machinery for moving chromatin in a direct path over long distances towards nuclear speckles in response to transcriptional activation. In Chapter 2, we used autonomous targeting of BAC transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs), including the 6 kb PTR1, target the ~100 kb beta-globin gene cluster and LCR region to the nuclear periphery and confer increased H3K9me3 modification, as assayed both by immunofluorescence and chromatin immunoprecipitation. PTRs within HBB BACs bias a competition between peripheral versus pericentric heterochromatin (PCH) targeting towards peripheral targeting. Targeting to both heterochromatin compartments is dependent on H3K9 trimethylation. More generally, PTRs confer targeting from the PCH to the periphery, from the interior to the PCH, or have no targeting activity depending on the flanking DNA context, with peripheral targeting correlating with higher, domain-wide H3K9m3 levels. A combination of FISH, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over several hundred kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent Lamin Associated Domain. Our results demonstrate multiple cis elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.","Item withdrawn by Laura Spradlin (lspradl2@illinois.edu) on 2014-10-30T17:54:38Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 8 Khanna_Nimish.docx: 45020873 bytes, checksum: e14b5c836b5a527526ae2897aa148ed3 (MD5) Khanna_Nimish.pdf: 16328076 bytes, checksum: 8b887674d4f15cc7a6030b7eab941887 (MD5) Video D6.mp4: 294695 bytes, checksum: 5cd58b955b6b8837f9a9e46fbe8ed5cc (MD5) Video D5.mp4: 241542 bytes, checksum: 8e6330e1714cb8f2f9ea4dd5e4cea292 (MD5) Video D4.mp4: 63287 bytes, checksum: b01373b185d3fed0a7564d2cd205912b (MD5) Video D3.mp4: 80391 bytes, checksum: 160353eabae12b8d13b4d07328d8171f (MD5) Video D2.mp4: 56724 bytes, checksum: 4b3a3c19febdfd7c1d21b10e1cb8d158 (MD5) Video D1.mp4: 417895 bytes, checksum: 75ededefdaed26251851e0df18c80622 (MD5)","Made available in DSpace on 2015-01-21T19:55:15Z (GMT). No. of bitstreams: 7 Nimish_Khanna.pdf: 16335604 bytes, checksum: ef59197f4b878b10ae9171c952a09b92 (MD5) Video E6.mp4: 294695 bytes, checksum: 5cd58b955b6b8837f9a9e46fbe8ed5cc (MD5) Video E5.mp4: 241542 bytes, checksum: 8e6330e1714cb8f2f9ea4dd5e4cea292 (MD5) Video E4.mp4: 63287 bytes, checksum: b01373b185d3fed0a7564d2cd205912b (MD5) Video E3.mp4: 80391 bytes, checksum: 160353eabae12b8d13b4d07328d8171f (MD5) Video E2.mp4: 56724 bytes, checksum: 4b3a3c19febdfd7c1d21b10e1cb8d158 (MD5) Video E1.mp4: 417895 bytes, checksum: 75ededefdaed26251851e0df18c80622 (MD5)","Embargo set by: Seth Robbins for item 73169 Lift date: 2017-01-21T19:56:18Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only Restriction Lifted for Item 73169 on 2017-01-22T10:15:27Z."]},{"key":"dc:title","label":"Title","values":["Mechanisms and functional significance of nuclear compartmentalization"]}]}],"canonical_facts":{"dc:contributor":["Belmont, Andrew S.","Freeman, Brian C.","Brieher, William M.","Prasanth, K V.","Selvin, Paul R."],"dc:creator":["Khanna, Nimish"],"dc:date":["2015-01-21T19:55:15Z","2017-01-22T10:15:27Z","2014-12","2015-01-21"],"dc:description":["The mammalian cell nucleus is a complex yet highly organized structure. There is significant amount of knowledge about the organization of nucleus; however, the influence of such structural complexity of the nucleus in gene regulation is poorly understood. For better understanding of the effects of nuclear repositioning of a gene locus on gene expression, I investigated the physiological significance and mechanism of association of a gene locus with a nuclear sub-compartment. In chapter 1, I showed an actin dependent association of HSP70 transgenes with nuclear speckles after heat shock. I visualized HSP70 transgenes moving curvilinearly towards nuclear speckles over ~0.5-6 μm distances at velocities of 1-2 μm min-1. Observation of chromatin stretching in the direction of movement demonstrated a force generating mechanism. Transcription in nearly all cases increased noticeably only after initial contact with a nuclear speckle. The time lag between initial speckle contact and increased transcription was inversely proportional to the speckle size at first contact with the HSP70 transgene and after initial contact with small speckles an increase in speckle size typically preceded increased transcription. Our results demonstrate the existence of a still to be revealed machinery for moving chromatin in a direct path over long distances towards nuclear speckles in response to transcriptional activation. In Chapter 2, we used autonomous targeting of BAC transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs), including the 6 kb PTR1, target the ~100 kb beta-globin gene cluster and LCR region to the nuclear periphery and confer increased H3K9me3 modification, as assayed both by immunofluorescence and chromatin immunoprecipitation. PTRs within HBB BACs bias a competition between peripheral versus pericentric heterochromatin (PCH) targeting towards peripheral targeting. Targeting to both heterochromatin compartments is dependent on H3K9 trimethylation. More generally, PTRs confer targeting from the PCH to the periphery, from the interior to the PCH, or have no targeting activity depending on the flanking DNA context, with peripheral targeting correlating with higher, domain-wide H3K9m3 levels. A combination of FISH, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over several hundred kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent Lamin Associated Domain. Our results demonstrate multiple cis elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.","Item withdrawn by Laura Spradlin (lspradl2@illinois.edu) on 2014-10-30T17:54:38Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 8 Khanna_Nimish.docx: 45020873 bytes, checksum: e14b5c836b5a527526ae2897aa148ed3 (MD5) Khanna_Nimish.pdf: 16328076 bytes, checksum: 8b887674d4f15cc7a6030b7eab941887 (MD5) Video D6.mp4: 294695 bytes, checksum: 5cd58b955b6b8837f9a9e46fbe8ed5cc (MD5) Video D5.mp4: 241542 bytes, checksum: 8e6330e1714cb8f2f9ea4dd5e4cea292 (MD5) Video D4.mp4: 63287 bytes, checksum: b01373b185d3fed0a7564d2cd205912b (MD5) Video D3.mp4: 80391 bytes, checksum: 160353eabae12b8d13b4d07328d8171f (MD5) Video D2.mp4: 56724 bytes, checksum: 4b3a3c19febdfd7c1d21b10e1cb8d158 (MD5) Video D1.mp4: 417895 bytes, checksum: 75ededefdaed26251851e0df18c80622 (MD5)","Made available in DSpace on 2015-01-21T19:55:15Z (GMT). No. of bitstreams: 7 Nimish_Khanna.pdf: 16335604 bytes, checksum: ef59197f4b878b10ae9171c952a09b92 (MD5) Video E6.mp4: 294695 bytes, checksum: 5cd58b955b6b8837f9a9e46fbe8ed5cc (MD5) Video E5.mp4: 241542 bytes, checksum: 8e6330e1714cb8f2f9ea4dd5e4cea292 (MD5) Video E4.mp4: 63287 bytes, checksum: b01373b185d3fed0a7564d2cd205912b (MD5) Video E3.mp4: 80391 bytes, checksum: 160353eabae12b8d13b4d07328d8171f (MD5) Video E2.mp4: 56724 bytes, checksum: 4b3a3c19febdfd7c1d21b10e1cb8d158 (MD5) Video E1.mp4: 417895 bytes, checksum: 75ededefdaed26251851e0df18c80622 (MD5)","Embargo set by: Seth Robbins for item 73169 Lift date: 2017-01-21T19:56:18Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only Restriction Lifted for Item 73169 on 2017-01-22T10:15:27Z."],"dc:identifier":["http://hdl.handle.net/2142/72980"],"dc:language":["en"],"dc:rights":["Copyright 2014 Nimish Khanna"],"dc:subject":["Nuclear organization","Chromatin dynamics"],"dc:title":["Mechanisms and functional significance of nuclear compartmentalization"],"dc:type":["text"],"thesis:degree_discipline":["Cell and Developmental Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:07Z"}