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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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Effects of elevated expression of HMGNs on transcription in MEFs. (A) Western blot analysis of stably infected MEFs. Shown are western analysis of MEFs stably expressing FLAG and HA tagged HMGN variants. Endogenous and exogenous HMGN proteins are indicated. C, control infection with empty virus; exp, experimental infection with indicated protein. Note comparable amounts of exogenous and endogenous proteins for all cell lines. (B) PCA of gene expression profiles in infected MEFs. Each sample was analyzed in triplicate. Stably expressed proteins are indicated. Each dot corresponds to individual pool of indicated HMGN variant. (C) The graph represents the number of genes changed following stable expression of an HMGN protein, compared with the control empty vector expression. Note the negligible effect of HMGN3a and HMGN5S17,21E on transcription. (D) Venn diagrams of down- and upregulated genes in infected MEFs. (E) The plot represents fold change in transcription for all affected genes following HMGN1, HMGN2 and HMGN5 overexpression. Note that most of the genes are affected up to 2-fold.
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Figure 3: Effects of elevated expression of HMGNs on transcription in MEFs. (A) Western blot analysis of stably infected MEFs. Shown are western analysis of MEFs stably expressing FLAG and HA tagged HMGN variants. Endogenous and exogenous HMGN proteins are indicated. C, control infection with empty virus; exp, experimental infection with indicated protein. Note comparable amounts of exogenous and endogenous proteins for all cell lines. (B) PCA of gene expression profiles in infected MEFs. Each sample was analyzed in triplicate. Stably expressed proteins are indicated. Each dot corresponds to individual pool of indicated HMGN variant. (C) The graph represents the number of genes changed following stable expression of an HMGN protein, compared with the control empty vector expression. Note the negligible effect of HMGN3a and HMGN5S17,21E on transcription. (D) Venn diagrams of down- and upregulated genes in infected MEFs. (E) The plot represents fold change in transcription for all affected genes following HMGN1, HMGN2 and HMGN5 overexpression. Note that most of the genes are affected up to 2-fold.

Mentions: Western blot analysis of the infected cells revealed that the level of expression of each exogenous protein was comparable to the level of its endogenous counterpart (Figure 3A). Thus, stably infected MEFs express ∼2-fold higher levels of a specific HMGN variant. The HMGN5-S17,21E protein contains mutations in two serine residues in the NBD which abolish its binding to nucleosomes (18) and thus served as a control for transcriptional effects due to chromatin binding. As an additional control, we infected MEFs with virus carrying an empty vector. For each variant, we analyzed the transcription profile of three independently infected pools of MEFs using mouse 430.2 Affymetrix expression arrays. We compared the transcription profile of MEFs overexpressing specific HMGN variants to the control cell lines transfected with empty vectors or with the HMGN5-S17,21E double-point mutant.Figure 3.


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)

Effects of elevated expression of HMGNs on transcription in MEFs. (A) Western blot analysis of stably infected MEFs. Shown are western analysis of MEFs stably expressing FLAG and HA tagged HMGN variants. Endogenous and exogenous HMGN proteins are indicated. C, control infection with empty virus; exp, experimental infection with indicated protein. Note comparable amounts of exogenous and endogenous proteins for all cell lines. (B) PCA of gene expression profiles in infected MEFs. Each sample was analyzed in triplicate. Stably expressed proteins are indicated. Each dot corresponds to individual pool of indicated HMGN variant. (C) The graph represents the number of genes changed following stable expression of an HMGN protein, compared with the control empty vector expression. Note the negligible effect of HMGN3a and HMGN5S17,21E on transcription. (D) Venn diagrams of down- and upregulated genes in infected MEFs. (E) The plot represents fold change in transcription for all affected genes following HMGN1, HMGN2 and HMGN5 overexpression. Note that most of the genes are affected up to 2-fold.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Effects of elevated expression of HMGNs on transcription in MEFs. (A) Western blot analysis of stably infected MEFs. Shown are western analysis of MEFs stably expressing FLAG and HA tagged HMGN variants. Endogenous and exogenous HMGN proteins are indicated. C, control infection with empty virus; exp, experimental infection with indicated protein. Note comparable amounts of exogenous and endogenous proteins for all cell lines. (B) PCA of gene expression profiles in infected MEFs. Each sample was analyzed in triplicate. Stably expressed proteins are indicated. Each dot corresponds to individual pool of indicated HMGN variant. (C) The graph represents the number of genes changed following stable expression of an HMGN protein, compared with the control empty vector expression. Note the negligible effect of HMGN3a and HMGN5S17,21E on transcription. (D) Venn diagrams of down- and upregulated genes in infected MEFs. (E) The plot represents fold change in transcription for all affected genes following HMGN1, HMGN2 and HMGN5 overexpression. Note that most of the genes are affected up to 2-fold.
Mentions: Western blot analysis of the infected cells revealed that the level of expression of each exogenous protein was comparable to the level of its endogenous counterpart (Figure 3A). Thus, stably infected MEFs express ∼2-fold higher levels of a specific HMGN variant. The HMGN5-S17,21E protein contains mutations in two serine residues in the NBD which abolish its binding to nucleosomes (18) and thus served as a control for transcriptional effects due to chromatin binding. As an additional control, we infected MEFs with virus carrying an empty vector. For each variant, we analyzed the transcription profile of three independently infected pools of MEFs using mouse 430.2 Affymetrix expression arrays. We compared the transcription profile of MEFs overexpressing specific HMGN variants to the control cell lines transfected with empty vectors or with the HMGN5-S17,21E double-point mutant.Figure 3.

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