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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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HMGN proteins are intrinsically disordered. (A) Multiple sequence alignment of mouse HMGN1, HMGN2, HMGN3a and HMGN5 proteins by ClustalW. Only the first 94 amino acids of HMGN5 are aligned. The positively charged NBD, the hallmark of HMGN proteins, is shaded by a blue square. The core sequence of NBD that is conserved in all HMGN proteins is labeled in red. The exon structure of the HMGN genes is color-coded over the sequences; numbers over the exons correspond to the last amino acid encoded by the exons of the Hmgn2 gene because HMGN2 is the most evolutionarily conserved HMGN variant. Asterisks indicate identical amino acid, colon indicates conserved substitutions and dot indicates semi-conserved substitutions. The alignment of HMGN5 is separate from that of HMGN1-3. NLS, nuclear localization signal; RD, regulatory domain. Solid arrow indicates the position of the swap tail mutants (Figure 4). (B) Relative amino acid composition of various HMGN proteins in comparison with ordered proteins. Bars are calculated as C(x) − C(order)/C(order), where C(x) is the content of a given residue in HMGN and C(order) is its content in ordered proteins from Protein Data Bank (http://www.pdb.org/pdb/home/home.do). Negative bars correspond to residues underrepresented in HMGN, whereas positive bars correspond to residues overrepresented in HMGN. Data for typical intrinsically disordered proteins are shown for comparison (DisProt, http://www.disprot.org, black bars). Sets of bars correspond to mean values for all HMGNs (HMGN) as well as for individual HMGNs (HMGN1, HMGN2, HMGN3a and HMGN5). The graph demonstrates that potentially HMGNs are more disordered than the averaged disordered proteins. (C) PONDR VL-XT disorder prediction for mouse HMGNs. In PONDR plots, segments with scores >0.5 correspond to the disordered regions, whereas those <0.5 correspond to the ordered regions/binding sites. Note that disorder distribution in NBD (residues 18–42) is conserved for HMGN1, HGMN2 and HGMN3a. HGMN5 shows much less disorder conservation. (D) Predicting potential binding sites by ANCHOR algorithm. Potential binding sites are indicated by blue boxes.
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Figure 1: HMGN proteins are intrinsically disordered. (A) Multiple sequence alignment of mouse HMGN1, HMGN2, HMGN3a and HMGN5 proteins by ClustalW. Only the first 94 amino acids of HMGN5 are aligned. The positively charged NBD, the hallmark of HMGN proteins, is shaded by a blue square. The core sequence of NBD that is conserved in all HMGN proteins is labeled in red. The exon structure of the HMGN genes is color-coded over the sequences; numbers over the exons correspond to the last amino acid encoded by the exons of the Hmgn2 gene because HMGN2 is the most evolutionarily conserved HMGN variant. Asterisks indicate identical amino acid, colon indicates conserved substitutions and dot indicates semi-conserved substitutions. The alignment of HMGN5 is separate from that of HMGN1-3. NLS, nuclear localization signal; RD, regulatory domain. Solid arrow indicates the position of the swap tail mutants (Figure 4). (B) Relative amino acid composition of various HMGN proteins in comparison with ordered proteins. Bars are calculated as C(x) − C(order)/C(order), where C(x) is the content of a given residue in HMGN and C(order) is its content in ordered proteins from Protein Data Bank (http://www.pdb.org/pdb/home/home.do). Negative bars correspond to residues underrepresented in HMGN, whereas positive bars correspond to residues overrepresented in HMGN. Data for typical intrinsically disordered proteins are shown for comparison (DisProt, http://www.disprot.org, black bars). Sets of bars correspond to mean values for all HMGNs (HMGN) as well as for individual HMGNs (HMGN1, HMGN2, HMGN3a and HMGN5). The graph demonstrates that potentially HMGNs are more disordered than the averaged disordered proteins. (C) PONDR VL-XT disorder prediction for mouse HMGNs. In PONDR plots, segments with scores >0.5 correspond to the disordered regions, whereas those <0.5 correspond to the ordered regions/binding sites. Note that disorder distribution in NBD (residues 18–42) is conserved for HMGN1, HGMN2 and HGMN3a. HGMN5 shows much less disorder conservation. (D) Predicting potential binding sites by ANCHOR algorithm. Potential binding sites are indicated by blue boxes.

Mentions: Examination of the structure of genes coding for the various members of the HMGN family suggests that they originated from a common ancestor. All the genes contain relatively long 5′- and 3′-untranslated regions, six exons and the boundaries of the first four exons are highly conserved (Figure 1A). The gene coding for HMGN5 evolved recently because it is found only in mammals. All the proteins encoded by the genes contain a positively charged, highly conserved, NBD (Figure 1A) that serves as their main chromatin-binding site. Embedded in the NBD is the sequence RRSARLSA(K,M)P that has been shown to be the core sequence that specifically anchors HMGN proteins to the 147-bp nucleosome CP, the building block of the chromatin fiber (43). A NLS that is localized at the N-terminal part of the proteins is also highly conserved in all HMGN variants. The C-terminal region of the proteins, encoded by exons 5 and 6, differs significantly among the HMGN variants. The HMGN5 C-terminal domain is especially long and contains several repeats of a negatively charged sequence motif (18). The alignment shown in Figure 1A illustrates the major similarities and differences between the mouse HMGN variants. Mouse HMGN1, HMGN2 and HMGN3a are similar in size, ranging between 89 and 95 amino acids, and are more similar to each other than to HMGN5, which is 406 amino acid long. The alignment does not contain the splice variant HMGN3b, which lacks the 21 C-terminal residues of HMGN3a, nor the HMGN4 variant, which has not yet been investigated in detail.Figure 1.


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)

HMGN proteins are intrinsically disordered. (A) Multiple sequence alignment of mouse HMGN1, HMGN2, HMGN3a and HMGN5 proteins by ClustalW. Only the first 94 amino acids of HMGN5 are aligned. The positively charged NBD, the hallmark of HMGN proteins, is shaded by a blue square. The core sequence of NBD that is conserved in all HMGN proteins is labeled in red. The exon structure of the HMGN genes is color-coded over the sequences; numbers over the exons correspond to the last amino acid encoded by the exons of the Hmgn2 gene because HMGN2 is the most evolutionarily conserved HMGN variant. Asterisks indicate identical amino acid, colon indicates conserved substitutions and dot indicates semi-conserved substitutions. The alignment of HMGN5 is separate from that of HMGN1-3. NLS, nuclear localization signal; RD, regulatory domain. Solid arrow indicates the position of the swap tail mutants (Figure 4). (B) Relative amino acid composition of various HMGN proteins in comparison with ordered proteins. Bars are calculated as C(x) − C(order)/C(order), where C(x) is the content of a given residue in HMGN and C(order) is its content in ordered proteins from Protein Data Bank (http://www.pdb.org/pdb/home/home.do). Negative bars correspond to residues underrepresented in HMGN, whereas positive bars correspond to residues overrepresented in HMGN. Data for typical intrinsically disordered proteins are shown for comparison (DisProt, http://www.disprot.org, black bars). Sets of bars correspond to mean values for all HMGNs (HMGN) as well as for individual HMGNs (HMGN1, HMGN2, HMGN3a and HMGN5). The graph demonstrates that potentially HMGNs are more disordered than the averaged disordered proteins. (C) PONDR VL-XT disorder prediction for mouse HMGNs. In PONDR plots, segments with scores >0.5 correspond to the disordered regions, whereas those <0.5 correspond to the ordered regions/binding sites. Note that disorder distribution in NBD (residues 18–42) is conserved for HMGN1, HGMN2 and HGMN3a. HGMN5 shows much less disorder conservation. (D) Predicting potential binding sites by ANCHOR algorithm. Potential binding sites are indicated by blue boxes.
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Figure 1: HMGN proteins are intrinsically disordered. (A) Multiple sequence alignment of mouse HMGN1, HMGN2, HMGN3a and HMGN5 proteins by ClustalW. Only the first 94 amino acids of HMGN5 are aligned. The positively charged NBD, the hallmark of HMGN proteins, is shaded by a blue square. The core sequence of NBD that is conserved in all HMGN proteins is labeled in red. The exon structure of the HMGN genes is color-coded over the sequences; numbers over the exons correspond to the last amino acid encoded by the exons of the Hmgn2 gene because HMGN2 is the most evolutionarily conserved HMGN variant. Asterisks indicate identical amino acid, colon indicates conserved substitutions and dot indicates semi-conserved substitutions. The alignment of HMGN5 is separate from that of HMGN1-3. NLS, nuclear localization signal; RD, regulatory domain. Solid arrow indicates the position of the swap tail mutants (Figure 4). (B) Relative amino acid composition of various HMGN proteins in comparison with ordered proteins. Bars are calculated as C(x) − C(order)/C(order), where C(x) is the content of a given residue in HMGN and C(order) is its content in ordered proteins from Protein Data Bank (http://www.pdb.org/pdb/home/home.do). Negative bars correspond to residues underrepresented in HMGN, whereas positive bars correspond to residues overrepresented in HMGN. Data for typical intrinsically disordered proteins are shown for comparison (DisProt, http://www.disprot.org, black bars). Sets of bars correspond to mean values for all HMGNs (HMGN) as well as for individual HMGNs (HMGN1, HMGN2, HMGN3a and HMGN5). The graph demonstrates that potentially HMGNs are more disordered than the averaged disordered proteins. (C) PONDR VL-XT disorder prediction for mouse HMGNs. In PONDR plots, segments with scores >0.5 correspond to the disordered regions, whereas those <0.5 correspond to the ordered regions/binding sites. Note that disorder distribution in NBD (residues 18–42) is conserved for HMGN1, HGMN2 and HGMN3a. HGMN5 shows much less disorder conservation. (D) Predicting potential binding sites by ANCHOR algorithm. Potential binding sites are indicated by blue boxes.
Mentions: Examination of the structure of genes coding for the various members of the HMGN family suggests that they originated from a common ancestor. All the genes contain relatively long 5′- and 3′-untranslated regions, six exons and the boundaries of the first four exons are highly conserved (Figure 1A). The gene coding for HMGN5 evolved recently because it is found only in mammals. All the proteins encoded by the genes contain a positively charged, highly conserved, NBD (Figure 1A) that serves as their main chromatin-binding site. Embedded in the NBD is the sequence RRSARLSA(K,M)P that has been shown to be the core sequence that specifically anchors HMGN proteins to the 147-bp nucleosome CP, the building block of the chromatin fiber (43). A NLS that is localized at the N-terminal part of the proteins is also highly conserved in all HMGN variants. The C-terminal region of the proteins, encoded by exons 5 and 6, differs significantly among the HMGN variants. The HMGN5 C-terminal domain is especially long and contains several repeats of a negatively charged sequence motif (18). The alignment shown in Figure 1A illustrates the major similarities and differences between the mouse HMGN variants. Mouse HMGN1, HMGN2 and HMGN3a are similar in size, ranging between 89 and 95 amino acids, and are more similar to each other than to HMGN5, which is 406 amino acid long. The alignment does not contain the splice variant HMGN3b, which lacks the 21 C-terminal residues of HMGN3a, nor the HMGN4 variant, which has not yet been investigated in detail.Figure 1.

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