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Effects of HMGN variants on the cellular transcription profile.

Rochman M, Taher L, Kurahashi T, Cherukuri S, Uversky VN, Landsman D, Ovcharenko I, Bustin M - Nucleic Acids Res. (2011)

Bottom Line: Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation.Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain.The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

View Article: PubMed Central - PubMed

Affiliation: Protein Section, Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20894, USA.

ABSTRACT
High mobility group N (HMGN) is a family of intrinsically disordered nuclear proteins that bind to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here, we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. We find that both up- and downregulation of HMGN expression altered the cellular transcription profile. Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation. Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain. Doubling the amount of HMGN had a significantly larger effect on the transcription profile than total deletion, suggesting that the intrinsically disordered structure of HMGN proteins plays an important role in their function. The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

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Effect of HMGN1 and HMGN3a on transcription in MIN6 cells. (A) Comparison of the protein levels of HMGN3 and HMGN1 in MIN6 and MEFs by western blot. CBB, Coomassie Blue staining. Western blot analysis of MIN6 cell lines stably expressing HMGN1 (B) or HMGN3a (C) proteins. Endogenous and exogenous FLAG and HA tagged (FLHA) proteins are indicated. c, control infection with empty virus; exp, experimental infection with indicated protein. The graph (D) represents the number of genes changed following overexpression of HMGN1 and HMGN3a proteins compared with the control empty vector expression. (E) Model for structural specificity of individual HMGN proteins. HMGN proteins consist of a conserved N-terminal region, which contains the NBD and the conserved octapeptide, RRSARLSA and a C-terminal region with a more variable sequence. The N- and C-terminal regions of each HMGN variant fit to give the specific property of each variant. Arrow marks the hypothetical connection between N- and C-regions; the geometry indicates unique combination of regions in each HMGN protein. Both the N- and the C-terminal regions interact with various protein partners. Some partners are shared between all HMGNs, whereas others are specific to individual proteins. Combinations of different interacting proteins will define the properties of each HMGN protein and its ability to affect chromatin architecture and transcription.
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Figure 5: Effect of HMGN1 and HMGN3a on transcription in MIN6 cells. (A) Comparison of the protein levels of HMGN3 and HMGN1 in MIN6 and MEFs by western blot. CBB, Coomassie Blue staining. Western blot analysis of MIN6 cell lines stably expressing HMGN1 (B) or HMGN3a (C) proteins. Endogenous and exogenous FLAG and HA tagged (FLHA) proteins are indicated. c, control infection with empty virus; exp, experimental infection with indicated protein. The graph (D) represents the number of genes changed following overexpression of HMGN1 and HMGN3a proteins compared with the control empty vector expression. (E) Model for structural specificity of individual HMGN proteins. HMGN proteins consist of a conserved N-terminal region, which contains the NBD and the conserved octapeptide, RRSARLSA and a C-terminal region with a more variable sequence. The N- and C-terminal regions of each HMGN variant fit to give the specific property of each variant. Arrow marks the hypothetical connection between N- and C-regions; the geometry indicates unique combination of regions in each HMGN protein. Both the N- and the C-terminal regions interact with various protein partners. Some partners are shared between all HMGNs, whereas others are specific to individual proteins. Combinations of different interacting proteins will define the properties of each HMGN protein and its ability to affect chromatin architecture and transcription.

Mentions: Surprisingly, our analysis revealed that overexpression of HMGN3a had no effect on transcription profile of the transfected MEFs. We previously reported that in MEFs the protein levels of HMGN3 are lower than those of HMGN1 and HMGN2 that are robustly expressed in most cells. However, HMGN3a is highly expressed in MIN6 cell, a mouse pancreatic cell line that secretes insulin (19). In these cells, small interfering RNA-mediated downregulation of HMGN3, but not that of HMGN1 or HMGN2, affects the transcription of genes involved in insulin secretion, suggesting that HMGN3a may affect transcription in a cell type-specific manner. To test this possibility, we first re-examined the relative amount of HMGN3 protein in MIN6 and MEF cells. Western blot analyses revealed that indeed the HMGN3 protein levels in MIN6 were significantly higher than in MEFs (Figure 5A). Next, we infected MIN6 cells with retroviral vectors expressing either HMGN1 or HMGN3a proteins and verified protein expression by western blot analysis (Figure 5B and C).Transcriptional array analysis revealed that in MIN6 cells, HMGN3a significantly changed the expression of 1429 genes; of these 471 were up-regulated and 958 genes were down-regulated (Figure 5D). In contrast, overexpression of HMGN1 in MIN6 cell line had no significant effect on the transcription profile. Because HMGN1 had significant effects on the transcription profile of MEFs (Figure 3), these results suggest cell type-specific transcription effects of HMGN variants.Figure 5.


Effects of HMGN variants on the cellular transcription profile.

Rochman M, Taher L, Kurahashi T, Cherukuri S, Uversky VN, Landsman D, Ovcharenko I, Bustin M - Nucleic Acids Res. (2011)

Effect of HMGN1 and HMGN3a on transcription in MIN6 cells. (A) Comparison of the protein levels of HMGN3 and HMGN1 in MIN6 and MEFs by western blot. CBB, Coomassie Blue staining. Western blot analysis of MIN6 cell lines stably expressing HMGN1 (B) or HMGN3a (C) proteins. Endogenous and exogenous FLAG and HA tagged (FLHA) proteins are indicated. c, control infection with empty virus; exp, experimental infection with indicated protein. The graph (D) represents the number of genes changed following overexpression of HMGN1 and HMGN3a proteins compared with the control empty vector expression. (E) Model for structural specificity of individual HMGN proteins. HMGN proteins consist of a conserved N-terminal region, which contains the NBD and the conserved octapeptide, RRSARLSA and a C-terminal region with a more variable sequence. The N- and C-terminal regions of each HMGN variant fit to give the specific property of each variant. Arrow marks the hypothetical connection between N- and C-regions; the geometry indicates unique combination of regions in each HMGN protein. Both the N- and the C-terminal regions interact with various protein partners. Some partners are shared between all HMGNs, whereas others are specific to individual proteins. Combinations of different interacting proteins will define the properties of each HMGN protein and its ability to affect chromatin architecture and transcription.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Effect of HMGN1 and HMGN3a on transcription in MIN6 cells. (A) Comparison of the protein levels of HMGN3 and HMGN1 in MIN6 and MEFs by western blot. CBB, Coomassie Blue staining. Western blot analysis of MIN6 cell lines stably expressing HMGN1 (B) or HMGN3a (C) proteins. Endogenous and exogenous FLAG and HA tagged (FLHA) proteins are indicated. c, control infection with empty virus; exp, experimental infection with indicated protein. The graph (D) represents the number of genes changed following overexpression of HMGN1 and HMGN3a proteins compared with the control empty vector expression. (E) Model for structural specificity of individual HMGN proteins. HMGN proteins consist of a conserved N-terminal region, which contains the NBD and the conserved octapeptide, RRSARLSA and a C-terminal region with a more variable sequence. The N- and C-terminal regions of each HMGN variant fit to give the specific property of each variant. Arrow marks the hypothetical connection between N- and C-regions; the geometry indicates unique combination of regions in each HMGN protein. Both the N- and the C-terminal regions interact with various protein partners. Some partners are shared between all HMGNs, whereas others are specific to individual proteins. Combinations of different interacting proteins will define the properties of each HMGN protein and its ability to affect chromatin architecture and transcription.
Mentions: Surprisingly, our analysis revealed that overexpression of HMGN3a had no effect on transcription profile of the transfected MEFs. We previously reported that in MEFs the protein levels of HMGN3 are lower than those of HMGN1 and HMGN2 that are robustly expressed in most cells. However, HMGN3a is highly expressed in MIN6 cell, a mouse pancreatic cell line that secretes insulin (19). In these cells, small interfering RNA-mediated downregulation of HMGN3, but not that of HMGN1 or HMGN2, affects the transcription of genes involved in insulin secretion, suggesting that HMGN3a may affect transcription in a cell type-specific manner. To test this possibility, we first re-examined the relative amount of HMGN3 protein in MIN6 and MEF cells. Western blot analyses revealed that indeed the HMGN3 protein levels in MIN6 were significantly higher than in MEFs (Figure 5A). Next, we infected MIN6 cells with retroviral vectors expressing either HMGN1 or HMGN3a proteins and verified protein expression by western blot analysis (Figure 5B and C).Transcriptional array analysis revealed that in MIN6 cells, HMGN3a significantly changed the expression of 1429 genes; of these 471 were up-regulated and 958 genes were down-regulated (Figure 5D). In contrast, overexpression of HMGN1 in MIN6 cell line had no significant effect on the transcription profile. Because HMGN1 had significant effects on the transcription profile of MEFs (Figure 3), these results suggest cell type-specific transcription effects of HMGN variants.Figure 5.

Bottom Line: Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation.Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain.The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

View Article: PubMed Central - PubMed

Affiliation: Protein Section, Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20894, USA.

ABSTRACT
High mobility group N (HMGN) is a family of intrinsically disordered nuclear proteins that bind to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here, we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. We find that both up- and downregulation of HMGN expression altered the cellular transcription profile. Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation. Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain. Doubling the amount of HMGN had a significantly larger effect on the transcription profile than total deletion, suggesting that the intrinsically disordered structure of HMGN proteins plays an important role in their function. The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

Show MeSH
Related in: MedlinePlus