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Independent chromatin binding of ARGONAUTE4 and SPT5L/KTF1 mediates transcriptional gene silencing.

Rowley MJ, Avrutsky MI, Sifuentes CJ, Pereira L, Wierzbicki AT - PLoS Genet. (2011)

Bottom Line: Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism.As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci.We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism. Expression of these elements is suppressed by establishment of repressive chromatin modifications. In Arabidopsis thaliana, they are silenced by the siRNA-mediated transcriptional gene silencing pathway where long non-coding RNAs (lncRNAs) produced by RNA Polymerase V (Pol V) guide ARGONAUTE4 (AGO4) to chromatin and attract enzymes that establish repressive chromatin modifications. It is unknown how chromatin modifying enzymes are recruited to chromatin. We show through chromatin immunoprecipitation (ChIP) that SPT5L/KTF1, a silencing factor and a homolog of SPT5 elongation factors, binds chromatin at loci subject to transcriptional silencing. Chromatin binding of SPT5L/KTF1 occurs downstream of RNA Polymerase V, but independently from the presence of 24-nt siRNA. We also show that SPT5L/KTF1 and AGO4 are recruited to chromatin in parallel and independently of each other. As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.

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SPT5L and AGO4 are not required for Pol V binding to chromatin.(A–E) Pol V occupancy of Actin 2 (A) and Tubulin 8 (B) control loci, solo LTR (C), IGN5 (D) and AtSN1 (E) assayed by ChIP in Col-0 wild type, nrpe1, spt5l and ago4. No antibody controls (white bars) provide background level for ChIP samples (black bars). Bars represent mean value of ChIP signals normalized to Col-0 wild type. Error bars are standard deviations of three independent biological replicates.
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pgen-1002120-g002: SPT5L and AGO4 are not required for Pol V binding to chromatin.(A–E) Pol V occupancy of Actin 2 (A) and Tubulin 8 (B) control loci, solo LTR (C), IGN5 (D) and AtSN1 (E) assayed by ChIP in Col-0 wild type, nrpe1, spt5l and ago4. No antibody controls (white bars) provide background level for ChIP samples (black bars). Bars represent mean value of ChIP signals normalized to Col-0 wild type. Error bars are standard deviations of three independent biological replicates.

Mentions: SPT5L interacts with chromatin (Figure 1C–1I) as well as with AGO4, Pol V complex and Pol V transcripts [21]–[23]. SPT5L is also not required for the accumulation of Pol V-dependent transcripts at IGN5, IGN6 or AtSN1 [22]. This suggests that SPT5L should work downstream of Pol V. To test this prediction we assayed Pol V binding to chromatin by ChIP with antibody against NRPE1, the largest subunit of Pol V. Subsequent real-time PCR demonstrated recovery of DNA from Col-0 wild type at much higher level than from the nrpe1 mutant at IGN5, solo LTR and AtSN1 loci but not at Actin 2 or Tubulin 8 loci (Figure 2A–2E) demonstrating that Pol V binds chromatin at IGN5, solo LTR and AtSN1 loci. DNA recovery from spt5l mutant was comparable to Col-0 wild type (Figure 2A–2E) showing that SPT5L is not needed for Pol V binding to chromatin. Interestingly, Pol V binding to chromatin was reproducibly increased at solo LTR locus in ago4 mutant (Figure 2C), indicating that AGO4 may inhibit Pol V binding to chromatin possibly by affecting initiation and/or elongation of Pol V transcription. We conclude that SPT5L does not work upstream of Pol V in siRNA-mediated transcriptional gene silencing pathway.


Independent chromatin binding of ARGONAUTE4 and SPT5L/KTF1 mediates transcriptional gene silencing.

Rowley MJ, Avrutsky MI, Sifuentes CJ, Pereira L, Wierzbicki AT - PLoS Genet. (2011)

SPT5L and AGO4 are not required for Pol V binding to chromatin.(A–E) Pol V occupancy of Actin 2 (A) and Tubulin 8 (B) control loci, solo LTR (C), IGN5 (D) and AtSN1 (E) assayed by ChIP in Col-0 wild type, nrpe1, spt5l and ago4. No antibody controls (white bars) provide background level for ChIP samples (black bars). Bars represent mean value of ChIP signals normalized to Col-0 wild type. Error bars are standard deviations of three independent biological replicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111484&req=5

pgen-1002120-g002: SPT5L and AGO4 are not required for Pol V binding to chromatin.(A–E) Pol V occupancy of Actin 2 (A) and Tubulin 8 (B) control loci, solo LTR (C), IGN5 (D) and AtSN1 (E) assayed by ChIP in Col-0 wild type, nrpe1, spt5l and ago4. No antibody controls (white bars) provide background level for ChIP samples (black bars). Bars represent mean value of ChIP signals normalized to Col-0 wild type. Error bars are standard deviations of three independent biological replicates.
Mentions: SPT5L interacts with chromatin (Figure 1C–1I) as well as with AGO4, Pol V complex and Pol V transcripts [21]–[23]. SPT5L is also not required for the accumulation of Pol V-dependent transcripts at IGN5, IGN6 or AtSN1 [22]. This suggests that SPT5L should work downstream of Pol V. To test this prediction we assayed Pol V binding to chromatin by ChIP with antibody against NRPE1, the largest subunit of Pol V. Subsequent real-time PCR demonstrated recovery of DNA from Col-0 wild type at much higher level than from the nrpe1 mutant at IGN5, solo LTR and AtSN1 loci but not at Actin 2 or Tubulin 8 loci (Figure 2A–2E) demonstrating that Pol V binds chromatin at IGN5, solo LTR and AtSN1 loci. DNA recovery from spt5l mutant was comparable to Col-0 wild type (Figure 2A–2E) showing that SPT5L is not needed for Pol V binding to chromatin. Interestingly, Pol V binding to chromatin was reproducibly increased at solo LTR locus in ago4 mutant (Figure 2C), indicating that AGO4 may inhibit Pol V binding to chromatin possibly by affecting initiation and/or elongation of Pol V transcription. We conclude that SPT5L does not work upstream of Pol V in siRNA-mediated transcriptional gene silencing pathway.

Bottom Line: Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism.As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci.We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism. Expression of these elements is suppressed by establishment of repressive chromatin modifications. In Arabidopsis thaliana, they are silenced by the siRNA-mediated transcriptional gene silencing pathway where long non-coding RNAs (lncRNAs) produced by RNA Polymerase V (Pol V) guide ARGONAUTE4 (AGO4) to chromatin and attract enzymes that establish repressive chromatin modifications. It is unknown how chromatin modifying enzymes are recruited to chromatin. We show through chromatin immunoprecipitation (ChIP) that SPT5L/KTF1, a silencing factor and a homolog of SPT5 elongation factors, binds chromatin at loci subject to transcriptional silencing. Chromatin binding of SPT5L/KTF1 occurs downstream of RNA Polymerase V, but independently from the presence of 24-nt siRNA. We also show that SPT5L/KTF1 and AGO4 are recruited to chromatin in parallel and independently of each other. As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.

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