Project description:The Saccharomyces cerevisiae protein Nhp6A is a model for the abundant and multifunctional HMGB family of chromatin-associated proteins. Nhp6A binds DNA in vitro without sequence-specificity and bends DNA sharply, but its role in chromosome biology is poorly understood. We show by whole genome ChIP-chip that Nhp6A is localized to specific regions of chromosomes that include about 23% of RNA polymerase II promoters. Nhp6A binding functions to stabilize positioned nucleosomes within promoter and 5' coding regions of these genes. Both genomic binding and transcript expression studies point to functionally-related groups of genes that are specifically bound by Nhp6A and whose transcription is altered by the absence of Nhp6. Genomic analyses of Nhp6A mutants specifically defective in DNA bending reveals a critical role of DNA bending for co-regulation of transcription but not for targeted binding by Nhp6A. We conclude that the chromatin environment, not DNA sequence recognition, localizes Nhp6A binding and that Nhp6A stabilizes chromatin structure and co-regulates transcription. Two amino acid residues within the DNA binding interface of Nhp6A are critical for bending DNA . Substitution of methionine 29 to alanine (M29A) reduces the formation of 98 bp microcircles by 6-fold in vitro, but equilibrium binding to linear DNA is only reduced 2-fold. The severe phenylalanine 48 to alanine (F48A) mutation abolishes formation of 98 bp microcircles, while again only lowering equilibrium binding to linear DNA by 2-fold (Masse et al. 2002; Dai et al. 2005). We used these bending mutants to address if Nhp6A bending activity is required for transcriptional regulation? Specifically, genome-wide binding and transcript analysis was performed on nhp6A-M29A ∆nhp6B, nhp6A-F48A ∆nhp6B, and nhp6A-P18A ∆nhp6B cells. Nhp6A-P18A was included to control for the modest decrease in DNA binding observed in the bending mutants because it also exhibits a 2-fold reduction of DNA binding but without a significant change of DNA bending (Yen et al. 1998; Allain et al. 1999).

Project description:The Saccharomyces cerevisiae protein Nhp6A is a model for the abundant and multifunctional HMGB family of chromatin-associated proteins. Nhp6A binds DNA in vitro without sequence-specificity and bends DNA sharply, but its role in chromosome biology is poorly understood. We show by whole genome ChIP-chip that Nhp6A is localized to specific regions of chromosomes that include about 23% of RNA polymerase II promoters. Nhp6A binding functions to stabilize positioned nucleosomes within promoter and 5' coding regions of these genes. Both genomic binding and transcript expression studies point to functionally-related groups of genes that are specifically bound by Nhp6A and whose transcription is altered by the absence of Nhp6. Genomic analyses of Nhp6A mutants specifically defective in DNA bending reveals a critical role of DNA bending for co-regulation of transcription but not for targeted binding by Nhp6A. We conclude that the chromatin environment, not DNA sequence recognition, localizes Nhp6A binding and that Nhp6A stabilizes chromatin structure and co-regulates transcription. Two amino acid residues within the DNA binding interface of Nhp6A are critical for bending DNA . Substitution of methionine 29 to alanine (M29A) reduces the formation of 98 bp microcircles by 6-fold in vitro, but equilibrium binding to linear DNA is only reduced 2-fold. The severe phenylalanine 48 to alanine (F48A) mutation abolishes formation of 98 bp microcircles, while again only lowering equilibrium binding to linear DNA by 2-fold (Masse et al. 2002; Dai et al. 2005). We used these bending mutants to address if Nhp6A bending activity is required for transcriptional regulation? Specifically, genome-wide binding and transcript analysis was performed on nhp6A-M29A ∆nhp6B, nhp6A-F48A ∆nhp6B, and nhp6A-P18A ∆nhp6B cells. Nhp6A-P18A was included to control for the modest decrease in DNA binding observed in the bending mutants because it also exhibits a 2-fold reduction of DNA binding but without a significant change of DNA bending (Yen et al. 1998; Allain et al. 1999). Transcription in four different genotypes (∆nhp6A ∆nhp6B, nhp6A-P18A ∆nhp6B, nhp6A-F48A ∆nhp6B, nhp6A-M29A ∆nhp6B) was compared to the same reference genotype (Nhp6A ∆nhp6B). Biological replicates were performed for each comparison.

Dataset Information

Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

Publications

Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

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