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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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FACT is a H2A.Z chaperone. (A) In vivo analysis of SUP4 expression in mutants of Spt16 and Pob3. Total RNA was isolated and intron-specific primer extension products of SUP4 were visualized by phosphorImaging of the gel as described under methods. RNA levels were normalized against U4 and average from three independent experiments with scatter are shown. (B) Relative enrichment of HA-tagged H2A.Z in the absence of functional Spt16 in active condition. Comparative levels of H2A.Z are low in both the nucleosomes in Spt16 mutant cell. (C) HA-H2A.Z levels around the SUP4 gene in wild type cells change with time in parrallel to transcriptional repression under nutrient starvation. (D) Co-immunoprecipitation of H2A.Z and Spt16 in vivo and in vitro. Mock was as described under Supplementary Data. Input lanes are loaded with one-third (in vivo) or one-fourth (in vitro) of the sample as compared to IP (immunoprecipitate) lanes. Spt16 was at increasing amounts in IP1, 2 and 3. Upper panel: In vivo, H2A.Z-HA is immunoprecipitated with anti-HA antibody and IP is probed with anti-Spt16 antibody. Lanes 3 and 4 are the duplicates of the immunoprecipitation (IP). Lower panel: (i) and (ii) show in vitro pull-downs, using purified Spt16/Pob3 or 6XHis-tagged H2A.Z; probed with anti-H2A.Z or anti-Spt16 antibodies, respectively. Control shows the immobilized bait protein alone on beads while mock lanes show incubation of the prey with bare beads. (E) Relative enrichment of HA-tagged H2A.Z in rsc4-Δ4 mutant in active condition. (F) Relative enrichment of H2A.Z-HA on tDNA loci present on three different chromosomes.
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Figure 5: FACT is a H2A.Z chaperone. (A) In vivo analysis of SUP4 expression in mutants of Spt16 and Pob3. Total RNA was isolated and intron-specific primer extension products of SUP4 were visualized by phosphorImaging of the gel as described under methods. RNA levels were normalized against U4 and average from three independent experiments with scatter are shown. (B) Relative enrichment of HA-tagged H2A.Z in the absence of functional Spt16 in active condition. Comparative levels of H2A.Z are low in both the nucleosomes in Spt16 mutant cell. (C) HA-H2A.Z levels around the SUP4 gene in wild type cells change with time in parrallel to transcriptional repression under nutrient starvation. (D) Co-immunoprecipitation of H2A.Z and Spt16 in vivo and in vitro. Mock was as described under Supplementary Data. Input lanes are loaded with one-third (in vivo) or one-fourth (in vitro) of the sample as compared to IP (immunoprecipitate) lanes. Spt16 was at increasing amounts in IP1, 2 and 3. Upper panel: In vivo, H2A.Z-HA is immunoprecipitated with anti-HA antibody and IP is probed with anti-Spt16 antibody. Lanes 3 and 4 are the duplicates of the immunoprecipitation (IP). Lower panel: (i) and (ii) show in vitro pull-downs, using purified Spt16/Pob3 or 6XHis-tagged H2A.Z; probed with anti-H2A.Z or anti-Spt16 antibodies, respectively. Control shows the immobilized bait protein alone on beads while mock lanes show incubation of the prey with bare beads. (E) Relative enrichment of HA-tagged H2A.Z in rsc4-Δ4 mutant in active condition. (F) Relative enrichment of H2A.Z-HA on tDNA loci present on three different chromosomes.

Mentions: Spt16 is known to differentially affect transcription of the pol II-transcribed genes (38). The SUP4 RNA levels in cells defective in either Spt16 or Pob3 (Figure 5A) show an increase while pol II-transcribed genes CMD1 and U4 do not show change in their transcript levels in Spt16 mutant cells (Supplementary Figure S4). In contrast to roles of FACT subunits in pol II transcription activation (5,6,39,40), the higher SUP4 levels in Spt16 and Pob3 mutants suggest a repressive role for them in SUP4 expression.Figure 5.


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)

FACT is a H2A.Z chaperone. (A) In vivo analysis of SUP4 expression in mutants of Spt16 and Pob3. Total RNA was isolated and intron-specific primer extension products of SUP4 were visualized by phosphorImaging of the gel as described under methods. RNA levels were normalized against U4 and average from three independent experiments with scatter are shown. (B) Relative enrichment of HA-tagged H2A.Z in the absence of functional Spt16 in active condition. Comparative levels of H2A.Z are low in both the nucleosomes in Spt16 mutant cell. (C) HA-H2A.Z levels around the SUP4 gene in wild type cells change with time in parrallel to transcriptional repression under nutrient starvation. (D) Co-immunoprecipitation of H2A.Z and Spt16 in vivo and in vitro. Mock was as described under Supplementary Data. Input lanes are loaded with one-third (in vivo) or one-fourth (in vitro) of the sample as compared to IP (immunoprecipitate) lanes. Spt16 was at increasing amounts in IP1, 2 and 3. Upper panel: In vivo, H2A.Z-HA is immunoprecipitated with anti-HA antibody and IP is probed with anti-Spt16 antibody. Lanes 3 and 4 are the duplicates of the immunoprecipitation (IP). Lower panel: (i) and (ii) show in vitro pull-downs, using purified Spt16/Pob3 or 6XHis-tagged H2A.Z; probed with anti-H2A.Z or anti-Spt16 antibodies, respectively. Control shows the immobilized bait protein alone on beads while mock lanes show incubation of the prey with bare beads. (E) Relative enrichment of HA-tagged H2A.Z in rsc4-Δ4 mutant in active condition. (F) Relative enrichment of H2A.Z-HA on tDNA loci present on three different chromosomes.
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Figure 5: FACT is a H2A.Z chaperone. (A) In vivo analysis of SUP4 expression in mutants of Spt16 and Pob3. Total RNA was isolated and intron-specific primer extension products of SUP4 were visualized by phosphorImaging of the gel as described under methods. RNA levels were normalized against U4 and average from three independent experiments with scatter are shown. (B) Relative enrichment of HA-tagged H2A.Z in the absence of functional Spt16 in active condition. Comparative levels of H2A.Z are low in both the nucleosomes in Spt16 mutant cell. (C) HA-H2A.Z levels around the SUP4 gene in wild type cells change with time in parrallel to transcriptional repression under nutrient starvation. (D) Co-immunoprecipitation of H2A.Z and Spt16 in vivo and in vitro. Mock was as described under Supplementary Data. Input lanes are loaded with one-third (in vivo) or one-fourth (in vitro) of the sample as compared to IP (immunoprecipitate) lanes. Spt16 was at increasing amounts in IP1, 2 and 3. Upper panel: In vivo, H2A.Z-HA is immunoprecipitated with anti-HA antibody and IP is probed with anti-Spt16 antibody. Lanes 3 and 4 are the duplicates of the immunoprecipitation (IP). Lower panel: (i) and (ii) show in vitro pull-downs, using purified Spt16/Pob3 or 6XHis-tagged H2A.Z; probed with anti-H2A.Z or anti-Spt16 antibodies, respectively. Control shows the immobilized bait protein alone on beads while mock lanes show incubation of the prey with bare beads. (E) Relative enrichment of HA-tagged H2A.Z in rsc4-Δ4 mutant in active condition. (F) Relative enrichment of H2A.Z-HA on tDNA loci present on three different chromosomes.
Mentions: Spt16 is known to differentially affect transcription of the pol II-transcribed genes (38). The SUP4 RNA levels in cells defective in either Spt16 or Pob3 (Figure 5A) show an increase while pol II-transcribed genes CMD1 and U4 do not show change in their transcript levels in Spt16 mutant cells (Supplementary Figure S4). In contrast to roles of FACT subunits in pol II transcription activation (5,6,39,40), the higher SUP4 levels in Spt16 and Pob3 mutants suggest a repressive role for them in SUP4 expression.Figure 5.

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