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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 interacts with chromatin in an siRNA–independent manner.(A–I) ChIP data showing SPT5L binding to chromatin in Col-0 wild type, rdr2 and spt5l mutants at loci transcribed by Pol V and silenced by siRNA-mediated transcriptional silencing: solo LTR(C), IGN5 (D), IGN20 (E), IGN22 (F), IGN23 (G), IGN25 (H) and IGN26 (I). Two loci transcribed by Pol II are shown as controls: Actin 2 (A) and Tubulin 8 (B). 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. (J) DNA methylation analysis of AtSN1, IGN5, IGN23 and IGN25 performed by digestion with HaeIII restriction endonuclease, IGN26 and solo LTR performed by digestion with AluI restriction endonuclease and IGN22 performed by digestion with AvaII restriction endonuclease. Digested genomic DNA was amplified by PCR. Sequences lacking HaeIII (Actin 2), AluI (IGN5) or AvaII (Actin 2) were used as loading controls.
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pgen-1002120-g004: SPT5L interacts with chromatin in an siRNA–independent manner.(A–I) ChIP data showing SPT5L binding to chromatin in Col-0 wild type, rdr2 and spt5l mutants at loci transcribed by Pol V and silenced by siRNA-mediated transcriptional silencing: solo LTR(C), IGN5 (D), IGN20 (E), IGN22 (F), IGN23 (G), IGN25 (H) and IGN26 (I). Two loci transcribed by Pol II are shown as controls: Actin 2 (A) and Tubulin 8 (B). 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. (J) DNA methylation analysis of AtSN1, IGN5, IGN23 and IGN25 performed by digestion with HaeIII restriction endonuclease, IGN26 and solo LTR performed by digestion with AluI restriction endonuclease and IGN22 performed by digestion with AvaII restriction endonuclease. Digested genomic DNA was amplified by PCR. Sequences lacking HaeIII (Actin 2), AluI (IGN5) or AvaII (Actin 2) were used as loading controls.

Mentions: The parallel and independent recruitment of SPT5L and AGO4 to chromatin suggests that they are both guided by the interactions with Pol V complex and/or Pol V transcripts. To test if SPT5L is also guided by siRNA we used ChIP to assay SPT5L binding to chromatin in rdr2, a mutant in an RNA-dependent RNA polymerase responsible for production of the majority of 24-nt siRNA [29]. The rdr2 mutation reduced the stability of SPT5L protein (Figure 1J, 1K) but did not cause reduction in DNA recovery of the tested loci after ChIP (Figure 4C–4I). This suggests that although RDR2 increases the amount of SPT5L protein, the chromatin-bound fraction of SPT5L is not affected by the rdr2 mutation. This also suggests the presence of siRNA-dependent pool of SPT5L that does not physically interact with assayed Pol V-transcribed loci.


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 interacts with chromatin in an siRNA–independent manner.(A–I) ChIP data showing SPT5L binding to chromatin in Col-0 wild type, rdr2 and spt5l mutants at loci transcribed by Pol V and silenced by siRNA-mediated transcriptional silencing: solo LTR(C), IGN5 (D), IGN20 (E), IGN22 (F), IGN23 (G), IGN25 (H) and IGN26 (I). Two loci transcribed by Pol II are shown as controls: Actin 2 (A) and Tubulin 8 (B). 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. (J) DNA methylation analysis of AtSN1, IGN5, IGN23 and IGN25 performed by digestion with HaeIII restriction endonuclease, IGN26 and solo LTR performed by digestion with AluI restriction endonuclease and IGN22 performed by digestion with AvaII restriction endonuclease. Digested genomic DNA was amplified by PCR. Sequences lacking HaeIII (Actin 2), AluI (IGN5) or AvaII (Actin 2) were used as loading controls.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111484&req=5

pgen-1002120-g004: SPT5L interacts with chromatin in an siRNA–independent manner.(A–I) ChIP data showing SPT5L binding to chromatin in Col-0 wild type, rdr2 and spt5l mutants at loci transcribed by Pol V and silenced by siRNA-mediated transcriptional silencing: solo LTR(C), IGN5 (D), IGN20 (E), IGN22 (F), IGN23 (G), IGN25 (H) and IGN26 (I). Two loci transcribed by Pol II are shown as controls: Actin 2 (A) and Tubulin 8 (B). 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. (J) DNA methylation analysis of AtSN1, IGN5, IGN23 and IGN25 performed by digestion with HaeIII restriction endonuclease, IGN26 and solo LTR performed by digestion with AluI restriction endonuclease and IGN22 performed by digestion with AvaII restriction endonuclease. Digested genomic DNA was amplified by PCR. Sequences lacking HaeIII (Actin 2), AluI (IGN5) or AvaII (Actin 2) were used as loading controls.
Mentions: The parallel and independent recruitment of SPT5L and AGO4 to chromatin suggests that they are both guided by the interactions with Pol V complex and/or Pol V transcripts. To test if SPT5L is also guided by siRNA we used ChIP to assay SPT5L binding to chromatin in rdr2, a mutant in an RNA-dependent RNA polymerase responsible for production of the majority of 24-nt siRNA [29]. The rdr2 mutation reduced the stability of SPT5L protein (Figure 1J, 1K) but did not cause reduction in DNA recovery of the tested loci after ChIP (Figure 4C–4I). This suggests that although RDR2 increases the amount of SPT5L protein, the chromatin-bound fraction of SPT5L is not affected by the rdr2 mutation. This also suggests the presence of siRNA-dependent pool of SPT5L that does not physically interact with assayed Pol V-transcribed loci.

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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