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Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes.

Mahapatra S, Dewari PS, Bhardwaj A, Bhargava P - Nucleic Acids Res. (2011)

Bottom Line: Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes.RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor.All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.

ABSTRACT
FACT complex is involved in elongation and ensures fidelity in the initiation step of transcription by RNA polymerase (pol) II. Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes. We report here H2A.Z-chaperone activity of the yeast FACT complex on the short, nucleosome-free, non-coding, pol III-transcribed yeast tRNA genes. On a prototype gene, yeast SUP4, chromatin remodeler RSC and FACT regulate its transcription through novel mechanisms, wherein the two gene-flanking nucleosomes containing H2A.Z, play different roles. Nhp6, which ensures transcription fidelity and helps load yFACT onto the gene flanking nucleosomes, has inhibitory role. RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor. All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

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RSC has a repressive role in SUP4 expression. (A) RNA was extracted from different mutants under active transcription conditions and SUP4 levels estimated as given under the ‘Material and Methods’ section. U4 was used as the internal control to normalize the SUP4 transcript levels. Average and standard deviations calculated from three independent isolations are plotted. (B) Relative enrichment of myc-tagged Sth1 subunit of RSC complex on SUP4 in both active and repressed conditions. (C) IEL analysis of the chromatin structure in RSC mutant and wild-type yeast cells. Ovals represent the nucleosomes; arrow and rectangle mark the gene position. Bent arrow marks the TSS, asterisk marks the MNase hypersensitive site on the gene region. Digestions with two MNase levels are shown for each strain. (D) Comparison of the digestion profiles of the similarly digested DNA in lanes 2 and 4 from the panel C. Profiles were generated using the PhosphorImager and the Image Gauge (Fuji) software. Asterisk marks the MNase hypersensitive site on the gene region while short arrow marks the direction of nucleosome shift. Box marks the gene region, bent arrow the TSS and Term the gene terminator. Light and dark ovals represent the positioned nucleosomes in wild-type and mutant cells, respectively. (E) Relative enrichment of myc-tagged Nhp6 in rsc4-Δ4 mutant in active condition. (F) High-resolution MNase footprinting. Samples were prepared and primer extension reactions carried out as described under the legends for the Figure 2D. The primer hybridizes to the bottom strand, 92 bp downstream of the TSS. Nucleosomes in rsc4-Δ4 mutant are restricted to downstream of the terminator. Three levels of MNase digestions are shown for wild type (lanes 1–3) and mutant (lanes 4–6) cells. Dark grey oval represents the downstream nucleosome in mutant while light grey ovals represent fuzzy nucleosomes in wild-type condition. Numbers in the right hand side denote bp with respect to TSS while short vertical bar marks the position of MNase hypersensitivity in lanes 4–6.
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Figure 3: RSC has a repressive role in SUP4 expression. (A) RNA was extracted from different mutants under active transcription conditions and SUP4 levels estimated as given under the ‘Material and Methods’ section. U4 was used as the internal control to normalize the SUP4 transcript levels. Average and standard deviations calculated from three independent isolations are plotted. (B) Relative enrichment of myc-tagged Sth1 subunit of RSC complex on SUP4 in both active and repressed conditions. (C) IEL analysis of the chromatin structure in RSC mutant and wild-type yeast cells. Ovals represent the nucleosomes; arrow and rectangle mark the gene position. Bent arrow marks the TSS, asterisk marks the MNase hypersensitive site on the gene region. Digestions with two MNase levels are shown for each strain. (D) Comparison of the digestion profiles of the similarly digested DNA in lanes 2 and 4 from the panel C. Profiles were generated using the PhosphorImager and the Image Gauge (Fuji) software. Asterisk marks the MNase hypersensitive site on the gene region while short arrow marks the direction of nucleosome shift. Box marks the gene region, bent arrow the TSS and Term the gene terminator. Light and dark ovals represent the positioned nucleosomes in wild-type and mutant cells, respectively. (E) Relative enrichment of myc-tagged Nhp6 in rsc4-Δ4 mutant in active condition. (F) High-resolution MNase footprinting. Samples were prepared and primer extension reactions carried out as described under the legends for the Figure 2D. The primer hybridizes to the bottom strand, 92 bp downstream of the TSS. Nucleosomes in rsc4-Δ4 mutant are restricted to downstream of the terminator. Three levels of MNase digestions are shown for wild type (lanes 1–3) and mutant (lanes 4–6) cells. Dark grey oval represents the downstream nucleosome in mutant while light grey ovals represent fuzzy nucleosomes in wild-type condition. Numbers in the right hand side denote bp with respect to TSS while short vertical bar marks the position of MNase hypersensitivity in lanes 4–6.

Mentions: Nucleosomes flanking the tRNA genes (9) are reported to have H2A.Z. We looked for the presence of H2A and its only variant in the budding yeast, H2A.Z, in both the nucleosomes. As opposed to negligible H2A levels, H2A.Z shows significant enrichment in both, especially the −1 nucleosome (Figure 2F, Supplementary Figure S2). This profile of H2A.Z is similar to the general pattern of H2A.Z-containing nucleosomes flanking an NFR around the TSS, reported for active pol II-transcribed genes (10). However, IEL analysis shows that similar to SNR6 (17); the chromatin structure remains unperturbed in the absence of H2A.Z, even under repression (Figure 2D, lanes 7–10), supporting a previous report showing no role of H2A.Z in general nucleosome positioning or maintenance of nucleosomal organization (27). H2A.Z associates with both active and inactive genes (28). On SUP4, H3 levels do not change (Figure 2B) but H2A.Z (Figure 2F) levels increase under repression. As compared to wild-type cells, higher SUP4 RNA levels found in htz1Δ cells (Figure 3A), suggest its repressive role in SUP4 expression. Decrease in occupancy upon activation of SUP4 is consistent with the reports showing that H2A.Z is lost during the active transcription of a gene (29,30).Figure 3.


Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes.

Mahapatra S, Dewari PS, Bhardwaj A, Bhargava P - Nucleic Acids Res. (2011)

RSC has a repressive role in SUP4 expression. (A) RNA was extracted from different mutants under active transcription conditions and SUP4 levels estimated as given under the ‘Material and Methods’ section. U4 was used as the internal control to normalize the SUP4 transcript levels. Average and standard deviations calculated from three independent isolations are plotted. (B) Relative enrichment of myc-tagged Sth1 subunit of RSC complex on SUP4 in both active and repressed conditions. (C) IEL analysis of the chromatin structure in RSC mutant and wild-type yeast cells. Ovals represent the nucleosomes; arrow and rectangle mark the gene position. Bent arrow marks the TSS, asterisk marks the MNase hypersensitive site on the gene region. Digestions with two MNase levels are shown for each strain. (D) Comparison of the digestion profiles of the similarly digested DNA in lanes 2 and 4 from the panel C. Profiles were generated using the PhosphorImager and the Image Gauge (Fuji) software. Asterisk marks the MNase hypersensitive site on the gene region while short arrow marks the direction of nucleosome shift. Box marks the gene region, bent arrow the TSS and Term the gene terminator. Light and dark ovals represent the positioned nucleosomes in wild-type and mutant cells, respectively. (E) Relative enrichment of myc-tagged Nhp6 in rsc4-Δ4 mutant in active condition. (F) High-resolution MNase footprinting. Samples were prepared and primer extension reactions carried out as described under the legends for the Figure 2D. The primer hybridizes to the bottom strand, 92 bp downstream of the TSS. Nucleosomes in rsc4-Δ4 mutant are restricted to downstream of the terminator. Three levels of MNase digestions are shown for wild type (lanes 1–3) and mutant (lanes 4–6) cells. Dark grey oval represents the downstream nucleosome in mutant while light grey ovals represent fuzzy nucleosomes in wild-type condition. Numbers in the right hand side denote bp with respect to TSS while short vertical bar marks the position of MNase hypersensitivity in lanes 4–6.
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Figure 3: RSC has a repressive role in SUP4 expression. (A) RNA was extracted from different mutants under active transcription conditions and SUP4 levels estimated as given under the ‘Material and Methods’ section. U4 was used as the internal control to normalize the SUP4 transcript levels. Average and standard deviations calculated from three independent isolations are plotted. (B) Relative enrichment of myc-tagged Sth1 subunit of RSC complex on SUP4 in both active and repressed conditions. (C) IEL analysis of the chromatin structure in RSC mutant and wild-type yeast cells. Ovals represent the nucleosomes; arrow and rectangle mark the gene position. Bent arrow marks the TSS, asterisk marks the MNase hypersensitive site on the gene region. Digestions with two MNase levels are shown for each strain. (D) Comparison of the digestion profiles of the similarly digested DNA in lanes 2 and 4 from the panel C. Profiles were generated using the PhosphorImager and the Image Gauge (Fuji) software. Asterisk marks the MNase hypersensitive site on the gene region while short arrow marks the direction of nucleosome shift. Box marks the gene region, bent arrow the TSS and Term the gene terminator. Light and dark ovals represent the positioned nucleosomes in wild-type and mutant cells, respectively. (E) Relative enrichment of myc-tagged Nhp6 in rsc4-Δ4 mutant in active condition. (F) High-resolution MNase footprinting. Samples were prepared and primer extension reactions carried out as described under the legends for the Figure 2D. The primer hybridizes to the bottom strand, 92 bp downstream of the TSS. Nucleosomes in rsc4-Δ4 mutant are restricted to downstream of the terminator. Three levels of MNase digestions are shown for wild type (lanes 1–3) and mutant (lanes 4–6) cells. Dark grey oval represents the downstream nucleosome in mutant while light grey ovals represent fuzzy nucleosomes in wild-type condition. Numbers in the right hand side denote bp with respect to TSS while short vertical bar marks the position of MNase hypersensitivity in lanes 4–6.
Mentions: Nucleosomes flanking the tRNA genes (9) are reported to have H2A.Z. We looked for the presence of H2A and its only variant in the budding yeast, H2A.Z, in both the nucleosomes. As opposed to negligible H2A levels, H2A.Z shows significant enrichment in both, especially the −1 nucleosome (Figure 2F, Supplementary Figure S2). This profile of H2A.Z is similar to the general pattern of H2A.Z-containing nucleosomes flanking an NFR around the TSS, reported for active pol II-transcribed genes (10). However, IEL analysis shows that similar to SNR6 (17); the chromatin structure remains unperturbed in the absence of H2A.Z, even under repression (Figure 2D, lanes 7–10), supporting a previous report showing no role of H2A.Z in general nucleosome positioning or maintenance of nucleosomal organization (27). H2A.Z associates with both active and inactive genes (28). On SUP4, H3 levels do not change (Figure 2B) but H2A.Z (Figure 2F) levels increase under repression. As compared to wild-type cells, higher SUP4 RNA levels found in htz1Δ cells (Figure 3A), suggest its repressive role in SUP4 expression. Decrease in occupancy upon activation of SUP4 is consistent with the reports showing that H2A.Z is lost during the active transcription of a gene (29,30).Figure 3.

Bottom Line: Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes.RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor.All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.

ABSTRACT
FACT complex is involved in elongation and ensures fidelity in the initiation step of transcription by RNA polymerase (pol) II. Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes. We report here H2A.Z-chaperone activity of the yeast FACT complex on the short, nucleosome-free, non-coding, pol III-transcribed yeast tRNA genes. On a prototype gene, yeast SUP4, chromatin remodeler RSC and FACT regulate its transcription through novel mechanisms, wherein the two gene-flanking nucleosomes containing H2A.Z, play different roles. Nhp6, which ensures transcription fidelity and helps load yFACT onto the gene flanking nucleosomes, has inhibitory role. RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor. All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

Show MeSH
Related in: MedlinePlus