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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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Effect of Repression and H2A.Z deposition on the SUP4 gene locus in vivo. Chromatin organization at the SUP4 locus is probed by ChIP/Real Time PCR and footprinting methods. (A) Relative occupancy of FLAG-tagged H2B. (B) Relative occupancy of Myc-tagged H3. (C) Nucleosomes flank the histone-free SUP4 gene: Cartoon showing positions of the nucleosomes −1 and +1, relative to the cis elements of the gene, as marked in the panel 1C. (D) High-resolution MNase footprinting in vivo. MNase digested chromatin and naked DNA samples were used for extension with a primer which hybridizes to the bottom strand, 13 bp downstream of the TSS. Lane M shows a 50 bp ladder used as molecular size marker while GATC represent the sequencing reaction over genomic DNA. Two levels of MNase digestions are shown for SUP4 as naked DNA (lanes 1 and 2) and chromatin (lanes 3 and 4). Positions of the box B and terminator are marked while grey ovals represent nucleosomes. Position of MNase hypersensitivity immediate upstream of the terminator is marked with a short vertical bar in the left-hand side. (E) Low-resolution chromatin structure analysis by the IEL method. Grey ovals denote the nucleosomal size protections and arrows mark the MNase cut sites in the chromatin. Gene region is marked with a rectangle. All numbers represent bp with respect to TSS at +1. Numbers on the left-hand side mark the MNase cuts seen on the naked DNA in lanes 1 and 2. Numbers on the right-hand side mark the MNase cuts seen on chromatin and −1, +1 mark the gene flanking nucleosomes. MNase cleavage pattern of wild type (lanes 3–6) and Htz1Δ cells (lanes 7–10) without (lanes 3, 4 and 7, 8) or with nutritional stress (lanes 5, 6 and 9, 10) for 2 h is shown. (F) H2A.Z deposition in the nucleosomes around the SUP4 gene in vivo. Relative occupancies of FLAG-tagged H2A and H2A.Z on SUP4 against TELVIR region are shown. H2A.Z levels increase with repression.
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Figure 2: Effect of Repression and H2A.Z deposition on the SUP4 gene locus in vivo. Chromatin organization at the SUP4 locus is probed by ChIP/Real Time PCR and footprinting methods. (A) Relative occupancy of FLAG-tagged H2B. (B) Relative occupancy of Myc-tagged H3. (C) Nucleosomes flank the histone-free SUP4 gene: Cartoon showing positions of the nucleosomes −1 and +1, relative to the cis elements of the gene, as marked in the panel 1C. (D) High-resolution MNase footprinting in vivo. MNase digested chromatin and naked DNA samples were used for extension with a primer which hybridizes to the bottom strand, 13 bp downstream of the TSS. Lane M shows a 50 bp ladder used as molecular size marker while GATC represent the sequencing reaction over genomic DNA. Two levels of MNase digestions are shown for SUP4 as naked DNA (lanes 1 and 2) and chromatin (lanes 3 and 4). Positions of the box B and terminator are marked while grey ovals represent nucleosomes. Position of MNase hypersensitivity immediate upstream of the terminator is marked with a short vertical bar in the left-hand side. (E) Low-resolution chromatin structure analysis by the IEL method. Grey ovals denote the nucleosomal size protections and arrows mark the MNase cut sites in the chromatin. Gene region is marked with a rectangle. All numbers represent bp with respect to TSS at +1. Numbers on the left-hand side mark the MNase cuts seen on the naked DNA in lanes 1 and 2. Numbers on the right-hand side mark the MNase cuts seen on chromatin and −1, +1 mark the gene flanking nucleosomes. MNase cleavage pattern of wild type (lanes 3–6) and Htz1Δ cells (lanes 7–10) without (lanes 3, 4 and 7, 8) or with nutritional stress (lanes 5, 6 and 9, 10) for 2 h is shown. (F) H2A.Z deposition in the nucleosomes around the SUP4 gene in vivo. Relative occupancies of FLAG-tagged H2A and H2A.Z on SUP4 against TELVIR region are shown. H2A.Z levels increase with repression.

Mentions: Using micrococcal nuclease (MNase) digestion instead of sonication, we probed the SUP4 gene region (Figure 1C) for the presence of core histones by the ChIP and real time PCR method. As compared to nucleosome-dense TELVIR region, the gene region is found histone-depleted where levels appear close to background level (Figure 2A and B). Comparatively lower enrichment of H2B and H3 in downstream region as compared to upstream, suggests that the nucleosomes may be present on both sides of the SUP4 gene in vivo (−1 and +1, Figure 2C), but the +1 nucleosome is not well positioned. We confirmed the histone-depleted nature of the gene region in vivo by high-resolution MNase footprinting. As compared to naked DNA, chromatin shows MNase hypersensitivity at the position from +93 to +96 in the gene region (Figure 2D), while the region downstream of the terminator is protected (lanes 3, 4 versus 1, 2), suggesting presence of nucleosomes. The MNase digestion profile of rest of the gene sequence is same for both chromatin and naked DNA, suggesting the observed hypersensitivity probably represents the 5′-boundary of the +1 nucleosome, downstream of the gene.Figure 2.


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)

Effect of Repression and H2A.Z deposition on the SUP4 gene locus in vivo. Chromatin organization at the SUP4 locus is probed by ChIP/Real Time PCR and footprinting methods. (A) Relative occupancy of FLAG-tagged H2B. (B) Relative occupancy of Myc-tagged H3. (C) Nucleosomes flank the histone-free SUP4 gene: Cartoon showing positions of the nucleosomes −1 and +1, relative to the cis elements of the gene, as marked in the panel 1C. (D) High-resolution MNase footprinting in vivo. MNase digested chromatin and naked DNA samples were used for extension with a primer which hybridizes to the bottom strand, 13 bp downstream of the TSS. Lane M shows a 50 bp ladder used as molecular size marker while GATC represent the sequencing reaction over genomic DNA. Two levels of MNase digestions are shown for SUP4 as naked DNA (lanes 1 and 2) and chromatin (lanes 3 and 4). Positions of the box B and terminator are marked while grey ovals represent nucleosomes. Position of MNase hypersensitivity immediate upstream of the terminator is marked with a short vertical bar in the left-hand side. (E) Low-resolution chromatin structure analysis by the IEL method. Grey ovals denote the nucleosomal size protections and arrows mark the MNase cut sites in the chromatin. Gene region is marked with a rectangle. All numbers represent bp with respect to TSS at +1. Numbers on the left-hand side mark the MNase cuts seen on the naked DNA in lanes 1 and 2. Numbers on the right-hand side mark the MNase cuts seen on chromatin and −1, +1 mark the gene flanking nucleosomes. MNase cleavage pattern of wild type (lanes 3–6) and Htz1Δ cells (lanes 7–10) without (lanes 3, 4 and 7, 8) or with nutritional stress (lanes 5, 6 and 9, 10) for 2 h is shown. (F) H2A.Z deposition in the nucleosomes around the SUP4 gene in vivo. Relative occupancies of FLAG-tagged H2A and H2A.Z on SUP4 against TELVIR region are shown. H2A.Z levels increase with repression.
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Figure 2: Effect of Repression and H2A.Z deposition on the SUP4 gene locus in vivo. Chromatin organization at the SUP4 locus is probed by ChIP/Real Time PCR and footprinting methods. (A) Relative occupancy of FLAG-tagged H2B. (B) Relative occupancy of Myc-tagged H3. (C) Nucleosomes flank the histone-free SUP4 gene: Cartoon showing positions of the nucleosomes −1 and +1, relative to the cis elements of the gene, as marked in the panel 1C. (D) High-resolution MNase footprinting in vivo. MNase digested chromatin and naked DNA samples were used for extension with a primer which hybridizes to the bottom strand, 13 bp downstream of the TSS. Lane M shows a 50 bp ladder used as molecular size marker while GATC represent the sequencing reaction over genomic DNA. Two levels of MNase digestions are shown for SUP4 as naked DNA (lanes 1 and 2) and chromatin (lanes 3 and 4). Positions of the box B and terminator are marked while grey ovals represent nucleosomes. Position of MNase hypersensitivity immediate upstream of the terminator is marked with a short vertical bar in the left-hand side. (E) Low-resolution chromatin structure analysis by the IEL method. Grey ovals denote the nucleosomal size protections and arrows mark the MNase cut sites in the chromatin. Gene region is marked with a rectangle. All numbers represent bp with respect to TSS at +1. Numbers on the left-hand side mark the MNase cuts seen on the naked DNA in lanes 1 and 2. Numbers on the right-hand side mark the MNase cuts seen on chromatin and −1, +1 mark the gene flanking nucleosomes. MNase cleavage pattern of wild type (lanes 3–6) and Htz1Δ cells (lanes 7–10) without (lanes 3, 4 and 7, 8) or with nutritional stress (lanes 5, 6 and 9, 10) for 2 h is shown. (F) H2A.Z deposition in the nucleosomes around the SUP4 gene in vivo. Relative occupancies of FLAG-tagged H2A and H2A.Z on SUP4 against TELVIR region are shown. H2A.Z levels increase with repression.
Mentions: Using micrococcal nuclease (MNase) digestion instead of sonication, we probed the SUP4 gene region (Figure 1C) for the presence of core histones by the ChIP and real time PCR method. As compared to nucleosome-dense TELVIR region, the gene region is found histone-depleted where levels appear close to background level (Figure 2A and B). Comparatively lower enrichment of H2B and H3 in downstream region as compared to upstream, suggests that the nucleosomes may be present on both sides of the SUP4 gene in vivo (−1 and +1, Figure 2C), but the +1 nucleosome is not well positioned. We confirmed the histone-depleted nature of the gene region in vivo by high-resolution MNase footprinting. As compared to naked DNA, chromatin shows MNase hypersensitivity at the position from +93 to +96 in the gene region (Figure 2D), while the region downstream of the terminator is protected (lanes 3, 4 versus 1, 2), suggesting presence of nucleosomes. The MNase digestion profile of rest of the gene sequence is same for both chromatin and naked DNA, suggesting the observed hypersensitivity probably represents the 5′-boundary of the +1 nucleosome, downstream of the gene.Figure 2.

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