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Miaochia Lo - One of the best experts on this subject based on the ideXlab platform.
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probing the interaction between the histone methyltransferase deacetylase subunit RBBP4 7 and the transcription factor bcl11a in epigenetic complexes
Journal of Biological Chemistry, 2017Co-Authors: Rebecca Moody, Miaochia Lo, Jennifer L Meagher, Nicholas O Stevers, Samantha Tinsley, Inkyung Jung, Aleksas Matvekas, Jeanne A StuckeyAbstract:: The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.
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Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes
Journal of Biological Chemistry, 2017Co-Authors: Rebecca Moody, Miaochia Lo, Jennifer L Meagher, Nicholas O Stevers, Samantha Tinsley, Inkyung Jung, Aleksas Matvekas, Jeanne A StuckeyAbstract:The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.
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abstract 3091 development and optimization of a fluorescence polarization based assay for the discovery of inhibitors of the RBBP4 7 histone h3 interaction
Cancer Research, 2016Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.
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Abstract 3091: Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction
Cancer Research, 2016Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.
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abstract 106 characterization of polycomb repressive complex 2 prc2 subunits retinoblastoma binding protein 4 and 7 RBBP4 7 in triple negative breast cancer
Cancer Research, 2015Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Around 15% of all breast cancers are classified as ER−/PR−/HER2−, or triple negative (TNBC). This subtype of breast cancer does not respond to traditional endocrine therapies or targeted agents against HER2 and is particularly invasive with a high rate of recurrence due to resistance of cancer stem cells against therapy. Currently no efficient treatments are available to target cancer stem cells in TNBC. With this unmet need, it is a necessity to determine the molecular mechanisms underlying stem cell biology in TNBC to develop better targeted therapies of this disease. Recent studies have shown that the subunits of the polycomb repressive complex 2 (PRC2), such as EZH2, have been linked to invasiveness, metastasis, and chemoresistance in TNBC. PRC2 consists of four core members: EZH2, SUZ12, EED and RBBP4/7 which together mediate H3K27 trimethylation to repress gene transcription. In addition to its role in gene regulation, PRC2 has been linked to the regulation of breast cancer stem cells. However, the function of RBBP4/7 in breast cancer and breast cancer stem cells is unknown. The purpose of this study is to examine the role of RBBP4/7 in PRC2 in maintenance of the H3K27me3 mark, regulation of cancer cell stemness, and breast tumorigenesis. Analyzing the expression of RBBP4/7 in publicly available data sets through ONCOMINE, we found that both RBBP4 and RbBP7 were overexpressed in breast cancers, especially TNBC, compared with normal breast tissues. Similar to EZH2, higher expression of RBBP4 and RbBP7 was correlated to higher incidence of metastasis and poor prognosis. Using RNA interference, we successfully knocked down RBBP4 and RbBP7 in TNBC cell lines SUM149 and MDA-MB-231. Our results show that knockdown of both RBBP4 and RbBP7 significantly decreases cell growth by 70%, reduces H3K27me3 levels and drastically inhibits the mammosphere forming capability of these breast cancer cell lines by 80%. In addition, there was an increase in apoptotic cells as assessed by Annexin V staining from 12% in untreated cells to 28% in the RBBP4/7 knockdown cells. Knockdown of either RBBP4 or RbBP7, on the other hand, only has a moderate effect on cell proliferation, H3K27me3 levels and mammosphere formation. Our studies indicate that together RBBP4 and RbBP7 help maintain PRC2 function and breast cancer stem cell phenotype and could be exploited as a novel therapeutic target for treating TNBC. The effects of RBBP4 and RbBP7 knockdown on target gene regulation and breast tumor initiation and progression using xenograft models are currently under investigation. Citation Format: Rebecca A. Reed, Miao-Chia Lo, Duxin Sun. Characterization of polycomb repressive complex 2 (PRC2) subunits retinoblastoma-binding protein 4 and 7 (RBBP4/7) in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 106. doi:10.1158/1538-7445.AM2015-106
Rebecca A Reed - One of the best experts on this subject based on the ideXlab platform.
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abstract 3091 development and optimization of a fluorescence polarization based assay for the discovery of inhibitors of the RBBP4 7 histone h3 interaction
Cancer Research, 2016Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.
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Abstract 3091: Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction
Cancer Research, 2016Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.
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abstract 106 characterization of polycomb repressive complex 2 prc2 subunits retinoblastoma binding protein 4 and 7 RBBP4 7 in triple negative breast cancer
Cancer Research, 2015Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Around 15% of all breast cancers are classified as ER−/PR−/HER2−, or triple negative (TNBC). This subtype of breast cancer does not respond to traditional endocrine therapies or targeted agents against HER2 and is particularly invasive with a high rate of recurrence due to resistance of cancer stem cells against therapy. Currently no efficient treatments are available to target cancer stem cells in TNBC. With this unmet need, it is a necessity to determine the molecular mechanisms underlying stem cell biology in TNBC to develop better targeted therapies of this disease. Recent studies have shown that the subunits of the polycomb repressive complex 2 (PRC2), such as EZH2, have been linked to invasiveness, metastasis, and chemoresistance in TNBC. PRC2 consists of four core members: EZH2, SUZ12, EED and RBBP4/7 which together mediate H3K27 trimethylation to repress gene transcription. In addition to its role in gene regulation, PRC2 has been linked to the regulation of breast cancer stem cells. However, the function of RBBP4/7 in breast cancer and breast cancer stem cells is unknown. The purpose of this study is to examine the role of RBBP4/7 in PRC2 in maintenance of the H3K27me3 mark, regulation of cancer cell stemness, and breast tumorigenesis. Analyzing the expression of RBBP4/7 in publicly available data sets through ONCOMINE, we found that both RBBP4 and RbBP7 were overexpressed in breast cancers, especially TNBC, compared with normal breast tissues. Similar to EZH2, higher expression of RBBP4 and RbBP7 was correlated to higher incidence of metastasis and poor prognosis. Using RNA interference, we successfully knocked down RBBP4 and RbBP7 in TNBC cell lines SUM149 and MDA-MB-231. Our results show that knockdown of both RBBP4 and RbBP7 significantly decreases cell growth by 70%, reduces H3K27me3 levels and drastically inhibits the mammosphere forming capability of these breast cancer cell lines by 80%. In addition, there was an increase in apoptotic cells as assessed by Annexin V staining from 12% in untreated cells to 28% in the RBBP4/7 knockdown cells. Knockdown of either RBBP4 or RbBP7, on the other hand, only has a moderate effect on cell proliferation, H3K27me3 levels and mammosphere formation. Our studies indicate that together RBBP4 and RbBP7 help maintain PRC2 function and breast cancer stem cell phenotype and could be exploited as a novel therapeutic target for treating TNBC. The effects of RBBP4 and RbBP7 knockdown on target gene regulation and breast tumor initiation and progression using xenograft models are currently under investigation. Citation Format: Rebecca A. Reed, Miao-Chia Lo, Duxin Sun. Characterization of polycomb repressive complex 2 (PRC2) subunits retinoblastoma-binding protein 4 and 7 (RBBP4/7) in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 106. doi:10.1158/1538-7445.AM2015-106
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Abstract 106: Characterization of polycomb repressive complex 2 (PRC2) subunits retinoblastoma-binding protein 4 and 7 (RBBP4/7) in triple-negative breast cancer
Cancer Research, 2015Co-Authors: Rebecca A Reed, Miaochia LoAbstract:Around 15% of all breast cancers are classified as ER−/PR−/HER2−, or triple negative (TNBC). This subtype of breast cancer does not respond to traditional endocrine therapies or targeted agents against HER2 and is particularly invasive with a high rate of recurrence due to resistance of cancer stem cells against therapy. Currently no efficient treatments are available to target cancer stem cells in TNBC. With this unmet need, it is a necessity to determine the molecular mechanisms underlying stem cell biology in TNBC to develop better targeted therapies of this disease. Recent studies have shown that the subunits of the polycomb repressive complex 2 (PRC2), such as EZH2, have been linked to invasiveness, metastasis, and chemoresistance in TNBC. PRC2 consists of four core members: EZH2, SUZ12, EED and RBBP4/7 which together mediate H3K27 trimethylation to repress gene transcription. In addition to its role in gene regulation, PRC2 has been linked to the regulation of breast cancer stem cells. However, the function of RBBP4/7 in breast cancer and breast cancer stem cells is unknown. The purpose of this study is to examine the role of RBBP4/7 in PRC2 in maintenance of the H3K27me3 mark, regulation of cancer cell stemness, and breast tumorigenesis. Analyzing the expression of RBBP4/7 in publicly available data sets through ONCOMINE, we found that both RBBP4 and RbBP7 were overexpressed in breast cancers, especially TNBC, compared with normal breast tissues. Similar to EZH2, higher expression of RBBP4 and RbBP7 was correlated to higher incidence of metastasis and poor prognosis. Using RNA interference, we successfully knocked down RBBP4 and RbBP7 in TNBC cell lines SUM149 and MDA-MB-231. Our results show that knockdown of both RBBP4 and RbBP7 significantly decreases cell growth by 70%, reduces H3K27me3 levels and drastically inhibits the mammosphere forming capability of these breast cancer cell lines by 80%. In addition, there was an increase in apoptotic cells as assessed by Annexin V staining from 12% in untreated cells to 28% in the RBBP4/7 knockdown cells. Knockdown of either RBBP4 or RbBP7, on the other hand, only has a moderate effect on cell proliferation, H3K27me3 levels and mammosphere formation. Our studies indicate that together RBBP4 and RbBP7 help maintain PRC2 function and breast cancer stem cell phenotype and could be exploited as a novel therapeutic target for treating TNBC. The effects of RBBP4 and RbBP7 knockdown on target gene regulation and breast tumor initiation and progression using xenograft models are currently under investigation. Citation Format: Rebecca A. Reed, Miao-Chia Lo, Duxin Sun. Characterization of polycomb repressive complex 2 (PRC2) subunits retinoblastoma-binding protein 4 and 7 (RBBP4/7) in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 106. doi:10.1158/1538-7445.AM2015-106
Jeanne A Stuckey - One of the best experts on this subject based on the ideXlab platform.
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probing the interaction between the histone methyltransferase deacetylase subunit RBBP4 7 and the transcription factor bcl11a in epigenetic complexes
Journal of Biological Chemistry, 2017Co-Authors: Rebecca Moody, Miaochia Lo, Jennifer L Meagher, Nicholas O Stevers, Samantha Tinsley, Inkyung Jung, Aleksas Matvekas, Jeanne A StuckeyAbstract:: The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.
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Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes
Journal of Biological Chemistry, 2017Co-Authors: Rebecca Moody, Miaochia Lo, Jennifer L Meagher, Nicholas O Stevers, Samantha Tinsley, Inkyung Jung, Aleksas Matvekas, Jeanne A StuckeyAbstract:The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.
Mario Torrado - One of the best experts on this subject based on the ideXlab platform.
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PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex
Nature Communications, 2018Co-Authors: Stephanie Link, Mario Torrado, Ramona M. M. Spitzer, Maryam Sana, Moritz Carl Völker-albert, Eva C. Keilhauer, Thomas Burgold, Sebastian Pünzeler, Ida LindströmAbstract:Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial–facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
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PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex
Nature Communications, 2018Co-Authors: Stephanie Link, Mario Torrado, Ramona M. M. Spitzer, Maryam Sana, Moritz Carl Völker-albert, Eva C. Keilhauer, Thomas Burgold, Sebastian Pünzeler, Ida Lindström, Andrea NistAbstract:PWWP2A is a chromatin-binding transcriptional regulator that mediates mitosis-progression. Here, the authors provide evidence that PWWP2A directly interacts with H2A.Z nucleosomes, DNA and H3K36me3, binds to an MTA1-specific subcomplex of the NuRD complex (M1HR) and promotes changes to histone acetylation. Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial–facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
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the mta1 subunit of the nucleosome remodeling and deacetylase complex can recruit two copies of RBBP4 7
Protein Science, 2016Co-Authors: Jason W Schmidberger, Mehdi Sharifi Tabar, Mario Torrado, Ana P G Silva, Michael J Landsberg, Lou Brillault, Saad Alqarni, Yi Cheng Zeng, Benjamin L Parker, Joel P MackayAbstract:The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.
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The MTA1 subunit of the nucleosome remodeling and deacetylase complex can recruit two copies of RBBP4/7
Protein Science, 2016Co-Authors: Jason W Schmidberger, Mehdi Sharifi Tabar, Mario Torrado, Ana P G Silva, Michael J Landsberg, Lou Brillault, Saad Alqarni, Yi Cheng Zeng, Benjamin L ParkerAbstract:The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.
Andrea Nist - One of the best experts on this subject based on the ideXlab platform.
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PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex
Nature Communications, 2018Co-Authors: Stephanie Link, Mario Torrado, Ramona M. M. Spitzer, Maryam Sana, Moritz Carl Völker-albert, Eva C. Keilhauer, Thomas Burgold, Sebastian Pünzeler, Ida Lindström, Andrea NistAbstract:PWWP2A is a chromatin-binding transcriptional regulator that mediates mitosis-progression. Here, the authors provide evidence that PWWP2A directly interacts with H2A.Z nucleosomes, DNA and H3K36me3, binds to an MTA1-specific subcomplex of the NuRD complex (M1HR) and promotes changes to histone acetylation. Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial–facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
