Independent Chromatin Binding of ARGONAUTE4 and SPT5L/KTF1 Mediates Transcriptional Gene Silencing

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.

A Transcription Fork Model for Pol IV and Pol V-Dependent RNA-Directed DNA Methylation

“…Such a scenario might help to explain the Pol V-dependent chromatin association of AGO4 Rowley et al 2011) and the specificity of RNA-directed methylation, whereby RNA triggers as short as 30 nucleotides are capable of bringing about the methylation of the corresponding DNA sequence (Pelissierand Wassenegger2000). However, there is no definitive evidence for direct siRNA-DNA interactions.…”

RNA-directed DNA methylation in plants

“…SPT5-LIKE/KOW DOMAIN-CON-TAINING TRANSCRIPTION FAC-TOR 1 (SPT5L/KTF1) is recruited to Pol V transcripts independently of AGO4 and is thought to play a role in stabilizing the effector complex. 13,14 The IDN2-IDP protein complex also binds to Pol V transcripts 15 and recruits the SWI/SNF chromatin remodeling complex, whose activity is required for heterochromatin formation. 16 An important question is "What explains the target specificity of the RdDM pathway or of Pol IV and Pol V transcription?"…”

RNA-Directed DNA Methylation and Transcriptional Silencing in Arabidopsis

“…Moreover, KTF1/SPT5L is capable of binding to noncoding scaffold RNAs produced by Pol V [67]. Chromatin binding of AGO4 and KTF1 occurs downstream to Pol V [38,68]. KTF1 and AGO4 are recruited to chromatin in parallel and partially independently of each other, whereas KTF1 enhances AGO4 chromatin binding at a subset of RdDM sites [68].…”

“…KTF1 and AGO4 are recruited to chromatin in parallel and partially independently of each other, whereas KTF1 enhances AGO4 chromatin binding at a subset of RdDM sites [68]. The chromatin binding of KTF1/SPT5L and AGO4 may create a platform for the recruitment of DRM2 to chromatin at RdDM targets [68].…”