Limits...
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.

Show MeSH

Related in: MedlinePlus

Transcription of SUP4 gene and chromatin. (A) Transcription from chromatin is higher than naked DNA in vitro. TFIIIC-dependent transcription of naked DNA (cf. lanes 1 and 2) and chromatin (cf. lanes 3 and 4) produces two additional transcripts due to downstream initiations from the positions +4 and +8 with respect to TSS at +1. RM represents the position of recovery marker in the gel. Transcript from +1 site is marked. (B) SUP4 RNA synthesis under nutritional stress. The total SUP4 transcript levels at different time points of repression are normalized against the U4 transcript used as an internal control. Average and scatter of RNA levels estimated from three independent experiments are plotted. (C) Schematic diagram showing the amplicon sizes and primer pairs used in real time PCR and chromatin immunoprecipitation studies. Positions of the gene sequence elements with respect to the three amplicons representing the upstream, gene and downstream regions of SUP4 gene are shown. Bent arrow represents TSS. For all ChIP data, averages from three independent experiments with error bars, normalized against TELVIR region, are calculated. (D) Relative occupancy of FLAG-tagged C160 subunit of pol III on SUP4 in both active and repressed conditions. Nutrient deprivation with 0.15× YEP medium lacking glucose was used to repress the pol III transcription for 80 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105386&req=5

Figure 1: Transcription of SUP4 gene and chromatin. (A) Transcription from chromatin is higher than naked DNA in vitro. TFIIIC-dependent transcription of naked DNA (cf. lanes 1 and 2) and chromatin (cf. lanes 3 and 4) produces two additional transcripts due to downstream initiations from the positions +4 and +8 with respect to TSS at +1. RM represents the position of recovery marker in the gel. Transcript from +1 site is marked. (B) SUP4 RNA synthesis under nutritional stress. The total SUP4 transcript levels at different time points of repression are normalized against the U4 transcript used as an internal control. Average and scatter of RNA levels estimated from three independent experiments are plotted. (C) Schematic diagram showing the amplicon sizes and primer pairs used in real time PCR and chromatin immunoprecipitation studies. Positions of the gene sequence elements with respect to the three amplicons representing the upstream, gene and downstream regions of SUP4 gene are shown. Bent arrow represents TSS. For all ChIP data, averages from three independent experiments with error bars, normalized against TELVIR region, are calculated. (D) Relative occupancy of FLAG-tagged C160 subunit of pol III on SUP4 in both active and repressed conditions. Nutrient deprivation with 0.15× YEP medium lacking glucose was used to repress the pol III transcription for 80 min.

Mentions: Naked DNA transcription of SUP4 is known to be TFIIIC-dependent (Figure 1A, lanes 1 and 2). Transcription from SUP4 using pure TFIIIC, pol III and recombinant TFIIIB gives two additional transcripts, corresponding to downstream initiations from the positions +4 and +8 with respect to the TSS at +1. Similar to SNR6 (24), addition of TFIIIC to the chromatin assembled on the plasmid pLNwt yields 5-fold more transcript (Figure 1A, lane 2 versus 4). Chromatin formation suppresses the non-specific transcription from the plasmid backbone (data not shown) which may contribute to the observed higher than naked DNA (lane 2) level of transcription from chromatin (lane 4). Unlike SNR6, TFIIIC addition does not result in a nucleosome positioning on the transcribed region of the gene (Supplementary Figure S1) suggesting absence of nucleosome on the gene may be due to the ATP-dependent chromatin remodelling activities present in the S-190 extract, which probably mobilize the nucleosome away from the gene region, making it transcriptionally active in the presence of TFIIIC. These in vitro observations suggest that chromatin may influence SUP4 expression in vivo as well.Figure 1.


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)

Transcription of SUP4 gene and chromatin. (A) Transcription from chromatin is higher than naked DNA in vitro. TFIIIC-dependent transcription of naked DNA (cf. lanes 1 and 2) and chromatin (cf. lanes 3 and 4) produces two additional transcripts due to downstream initiations from the positions +4 and +8 with respect to TSS at +1. RM represents the position of recovery marker in the gel. Transcript from +1 site is marked. (B) SUP4 RNA synthesis under nutritional stress. The total SUP4 transcript levels at different time points of repression are normalized against the U4 transcript used as an internal control. Average and scatter of RNA levels estimated from three independent experiments are plotted. (C) Schematic diagram showing the amplicon sizes and primer pairs used in real time PCR and chromatin immunoprecipitation studies. Positions of the gene sequence elements with respect to the three amplicons representing the upstream, gene and downstream regions of SUP4 gene are shown. Bent arrow represents TSS. For all ChIP data, averages from three independent experiments with error bars, normalized against TELVIR region, are calculated. (D) Relative occupancy of FLAG-tagged C160 subunit of pol III on SUP4 in both active and repressed conditions. Nutrient deprivation with 0.15× YEP medium lacking glucose was used to repress the pol III transcription for 80 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105386&req=5

Figure 1: Transcription of SUP4 gene and chromatin. (A) Transcription from chromatin is higher than naked DNA in vitro. TFIIIC-dependent transcription of naked DNA (cf. lanes 1 and 2) and chromatin (cf. lanes 3 and 4) produces two additional transcripts due to downstream initiations from the positions +4 and +8 with respect to TSS at +1. RM represents the position of recovery marker in the gel. Transcript from +1 site is marked. (B) SUP4 RNA synthesis under nutritional stress. The total SUP4 transcript levels at different time points of repression are normalized against the U4 transcript used as an internal control. Average and scatter of RNA levels estimated from three independent experiments are plotted. (C) Schematic diagram showing the amplicon sizes and primer pairs used in real time PCR and chromatin immunoprecipitation studies. Positions of the gene sequence elements with respect to the three amplicons representing the upstream, gene and downstream regions of SUP4 gene are shown. Bent arrow represents TSS. For all ChIP data, averages from three independent experiments with error bars, normalized against TELVIR region, are calculated. (D) Relative occupancy of FLAG-tagged C160 subunit of pol III on SUP4 in both active and repressed conditions. Nutrient deprivation with 0.15× YEP medium lacking glucose was used to repress the pol III transcription for 80 min.
Mentions: Naked DNA transcription of SUP4 is known to be TFIIIC-dependent (Figure 1A, lanes 1 and 2). Transcription from SUP4 using pure TFIIIC, pol III and recombinant TFIIIB gives two additional transcripts, corresponding to downstream initiations from the positions +4 and +8 with respect to the TSS at +1. Similar to SNR6 (24), addition of TFIIIC to the chromatin assembled on the plasmid pLNwt yields 5-fold more transcript (Figure 1A, lane 2 versus 4). Chromatin formation suppresses the non-specific transcription from the plasmid backbone (data not shown) which may contribute to the observed higher than naked DNA (lane 2) level of transcription from chromatin (lane 4). Unlike SNR6, TFIIIC addition does not result in a nucleosome positioning on the transcribed region of the gene (Supplementary Figure S1) suggesting absence of nucleosome on the gene may be due to the ATP-dependent chromatin remodelling activities present in the S-190 extract, which probably mobilize the nucleosome away from the gene region, making it transcriptionally active in the presence of TFIIIC. These in vitro observations suggest that chromatin may influence SUP4 expression in vivo as well.Figure 1.

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