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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 has inhibitory effects on SUP4 transcription. (A) Effect of Nhp6 on SUP4 transcription in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery in the absence (lane1) and presence (lanes 2–10) of increasing amounts (36, 60, 72, 84, 99, 108, 120, 132 and 144 ng) of pure Nhp6A. Transcript and RM are as given for the Figure 1A. (B) Nhp6 confers the fidelity of transcription in vivo. Total RNA was isolated and used to generate extension products using the intron-specific primer of SUP4. The three products obtained from the two yeast strains using 5 or 10 µg total RNA are resolved on a gel as described under methods. Arrow marks the major SUP4 transcript initiating from the +1 bp position. Numbers on the right-hand side denote the positions of all the transcripts with respect to +1 site of SUP4, as explained in the text. (C) Relative enrichment of myc-tagged Nhp6A in both active and repressed states. (D) Relative enrichment of myc-tagged Spt16 in active and repressed states. (E) Relative enrichment of TAP-tagged Pob3 in both active and repressed states. (F) Effect of FACT on the chromatin transcription of SUP4 in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery. Pure FACT components in excess were added as indicated.
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Figure 4: FACT has inhibitory effects on SUP4 transcription. (A) Effect of Nhp6 on SUP4 transcription in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery in the absence (lane1) and presence (lanes 2–10) of increasing amounts (36, 60, 72, 84, 99, 108, 120, 132 and 144 ng) of pure Nhp6A. Transcript and RM are as given for the Figure 1A. (B) Nhp6 confers the fidelity of transcription in vivo. Total RNA was isolated and used to generate extension products using the intron-specific primer of SUP4. The three products obtained from the two yeast strains using 5 or 10 µg total RNA are resolved on a gel as described under methods. Arrow marks the major SUP4 transcript initiating from the +1 bp position. Numbers on the right-hand side denote the positions of all the transcripts with respect to +1 site of SUP4, as explained in the text. (C) Relative enrichment of myc-tagged Nhp6A in both active and repressed states. (D) Relative enrichment of myc-tagged Spt16 in active and repressed states. (E) Relative enrichment of TAP-tagged Pob3 in both active and repressed states. (F) Effect of FACT on the chromatin transcription of SUP4 in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery. Pure FACT components in excess were added as indicated.

Mentions: Nhp6 is reported to suppress the downstream initiations from naked SUP4 DNA in vitro (36). In a titration experiment (Figure 4A), addition of purified, recombinant Nhp6A to chromatin also abolishes downstream initiations and the right SUP4 transcript increases with increasing Nhp6 (lanes 1–3). However, in contrast to steady increase in naked DNA transcription with addition of upto ∼300 ng Nhp6 (data not shown), transcription from chromatin drops after the saturation levels of Nhp6 at 60 ng (lanes 4–10, Figure 4A). Expecting a similar effect of Nhp6 in vivo, we used an intron-specific primer which also detects transcripts from two isogenes of SUP4, all differing from each other by 1 or 2 bp in TSS selection (36). As marked in the Figure 4B, −1 represents barely visible SUP2 transcript; SUP3 and SUP4 appear together at +1; while +2 and +3 represent downstream initiated transcripts from SUP3 and SUP2, respectively. Deletion of Nhp6 is found to reduce transcription from the right positions in all three genes (−1 and +1 bands in lanes 3, 4 versus lanes 1, 2). In Nhp6ΔΔ cells, the downstream initiated transcripts from +2 and +3 (transcripts from SUP3 and SUP2 respectively), show an increase and a transcript initiated from +8 position, probably from SUP4, is also found, as seen in vitro (cf. Figures 1A and 4B).Figure 4.


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 has inhibitory effects on SUP4 transcription. (A) Effect of Nhp6 on SUP4 transcription in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery in the absence (lane1) and presence (lanes 2–10) of increasing amounts (36, 60, 72, 84, 99, 108, 120, 132 and 144 ng) of pure Nhp6A. Transcript and RM are as given for the Figure 1A. (B) Nhp6 confers the fidelity of transcription in vivo. Total RNA was isolated and used to generate extension products using the intron-specific primer of SUP4. The three products obtained from the two yeast strains using 5 or 10 µg total RNA are resolved on a gel as described under methods. Arrow marks the major SUP4 transcript initiating from the +1 bp position. Numbers on the right-hand side denote the positions of all the transcripts with respect to +1 site of SUP4, as explained in the text. (C) Relative enrichment of myc-tagged Nhp6A in both active and repressed states. (D) Relative enrichment of myc-tagged Spt16 in active and repressed states. (E) Relative enrichment of TAP-tagged Pob3 in both active and repressed states. (F) Effect of FACT on the chromatin transcription of SUP4 in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery. Pure FACT components in excess were added as indicated.
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Figure 4: FACT has inhibitory effects on SUP4 transcription. (A) Effect of Nhp6 on SUP4 transcription in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery in the absence (lane1) and presence (lanes 2–10) of increasing amounts (36, 60, 72, 84, 99, 108, 120, 132 and 144 ng) of pure Nhp6A. Transcript and RM are as given for the Figure 1A. (B) Nhp6 confers the fidelity of transcription in vivo. Total RNA was isolated and used to generate extension products using the intron-specific primer of SUP4. The three products obtained from the two yeast strains using 5 or 10 µg total RNA are resolved on a gel as described under methods. Arrow marks the major SUP4 transcript initiating from the +1 bp position. Numbers on the right-hand side denote the positions of all the transcripts with respect to +1 site of SUP4, as explained in the text. (C) Relative enrichment of myc-tagged Nhp6A in both active and repressed states. (D) Relative enrichment of myc-tagged Spt16 in active and repressed states. (E) Relative enrichment of TAP-tagged Pob3 in both active and repressed states. (F) Effect of FACT on the chromatin transcription of SUP4 in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery. Pure FACT components in excess were added as indicated.
Mentions: Nhp6 is reported to suppress the downstream initiations from naked SUP4 DNA in vitro (36). In a titration experiment (Figure 4A), addition of purified, recombinant Nhp6A to chromatin also abolishes downstream initiations and the right SUP4 transcript increases with increasing Nhp6 (lanes 1–3). However, in contrast to steady increase in naked DNA transcription with addition of upto ∼300 ng Nhp6 (data not shown), transcription from chromatin drops after the saturation levels of Nhp6 at 60 ng (lanes 4–10, Figure 4A). Expecting a similar effect of Nhp6 in vivo, we used an intron-specific primer which also detects transcripts from two isogenes of SUP4, all differing from each other by 1 or 2 bp in TSS selection (36). As marked in the Figure 4B, −1 represents barely visible SUP2 transcript; SUP3 and SUP4 appear together at +1; while +2 and +3 represent downstream initiated transcripts from SUP3 and SUP2, respectively. Deletion of Nhp6 is found to reduce transcription from the right positions in all three genes (−1 and +1 bands in lanes 3, 4 versus lanes 1, 2). In Nhp6ΔΔ cells, the downstream initiated transcripts from +2 and +3 (transcripts from SUP3 and SUP2 respectively), show an increase and a transcript initiated from +8 position, probably from SUP4, is also found, as seen in vitro (cf. Figures 1A and 4B).Figure 4.

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