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Title: Independent Chromatin Binding of ARGONAUTE4 and SPT5L/KTF1 Mediates Transcriptional Gene Silencing}
Article:
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.
Introduction:
Eukaryotic genomes contain significant amounts of transposons and other repetitive DNA elements, which usually remain transcriptionally inactive. Efficient silencing of transposon transcription is essential for preventing their mobility and for maintaining genome integrity[1]. Transposon silencing has also been hypothesized to regulate expression of genes that contain transposable elements in their promoters and to facilitate the evolution of genomes[2]. Transposons are silenced at both transcriptional and post-transcriptional levels by mechanisms that involve small interfering RNAs( siRNAs)[3]. These 20–25-nt RNA molecules are generated by the RNase III enzyme Dicer and provide sequence specificity for effector complexes mediating RNA cleavage and/or the establishment of chromatin modifications that silence transcriptional activity[3]. In Arabidopsis thaliana, single-stranded RNA precursors for siRNA biogenesis are produced by RNA Polymerase II( Pol II) or RNA Polymerase IV( Pol IV), while the second strand is synthesized by RDR2( RNA-Dependent RNA Polymerase 2). DCL3( Dicer-like 3) cleaves double-stranded RNA into siRNAs that are then incorporated into ARGONAUTE4( AGO4)[4],[5]. This mechanism seems to be similar in maize where homologs of RDR2 and Pol IV have been shown to be involved in transcriptional gene silencing[6]–[8]. Recognition of target loci by AGO4-siRNA complexes requires sequence identity between siRNAs and the genomic loci. These loci, however, are often actively transcribed, and it is not clear if siRNAs base-pair interact with DNA or nascent RNA transcripts[3],[9]. The latter possibility is well supported in Schizosaccharomyces pombe where loci subject to siRNA-mediated transcriptional silencing are actively transcribed by RNA Polymerase II[10]–[12]. The central role of nascent transcripts in recognition of siRNA targets in S. pombe was observed by the ability of Argonaute proteins to cleave RNA. This ability is required for the establishment of repressive chromatin modifications[13]. Moreover, tethering Argonaute and siRNA-containing RITS( RNA-induced initiation of transcriptional gene silencing) complex to nascent transcripts is sufficient for the initiation of repressive chromatin modifications and transcriptional silencing[14]. This mechanism may be similar in Arabidopsis where transcriptional silencing requires a specialized RNA Polymerase complex known as RNA Polymerase V( Pol V)[15]–[17]. Pol V produces non-coding transcripts in otherwise silent chromatin, and its activity is required for the establishment and maintenance of repressive chromatin modifications[18]. Pol V-produced non-coding transcripts physically interact with AGO4 and recruit siRNA-AGO4 complexes to their targets[19]. Additionally, transcriptional silencing of several loci needs AGO4 slicer activity[20], suggesting that in plants siRNAs may recognize their targets by base-pair interactions with Pol V transcripts[19]. RNA Polymerases and AGO4 are assisted in their functions by several other known protein components of the plant silencing system, all of which are required for efficient establishment and maintenance of transcriptional silencing[5]. One of them is SPT5L( Suppressor of Ty insertion 5- like; also known as SPT5-like or KTF1), a homolog of SPT5 Pol II-associated elongation factor. It was shown to contain a domain rich in WG/GW repeats that facilitate physical interaction with AGO4[21]–[23]. Because SPT5L interacts with RNA but is not required for the accumulation of Pol V-dependent transcripts, it was hypothesized to work downstream of Pol V and recruit AGO4 to Pol V-transcribed loci[22],[24]. Despite the recent progress in understanding the mechanisms of transcriptional gene silencing, it is not known how siRNAs work with Pol V transcripts, AGO4 and other proteins to recruit chromatin modifying enzymes to their target loci in chromatin. It is unknown how chromatin-bound AGO4 recruits enzymes that establish repressive chromatin modifications. It is unknown if other protein components of the silencing system help AGO4 recruit chromatin modifying enzymes. It is also unknown in what order proteins involved in silencing are recruited to chromatin. Here we try to resolve the mechanism of siRNA-mediated recruitment of chromatin modifying enzymes to chromatin and the function of SPT5L in this process. We show that SPT5L physically interacts with chromatin and that SPT5L works downstream of Pol V but does not require 24-nt siRNA. SPT5L and AGO4 are recruited to chromatin in parallel and at least partially independently of each other and both are needed for DNA methylation and repressive histone modifications at several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.
Discussion:
Our findings establish the order of events leading to siRNA-mediated establishment of transcriptional silencing. This process is initiated by recognition of silencing targets and production of two classes of non-coding RNA. The first class is siRNA which is produced from double-stranded RDR2 products by DCL3 and becomes incorporated into AGO4 and possibly also AGO6 and AGO9[4],[5],[24]. The second class is long non-coding RNA produced by Pol V and/or Pol II[18],[26]. Pol V transcription is initiated independently of siRNA and Pol V transcripts most likely are not precursors for siRNA biogenesis[18],[31]. Pol V recruitment to chromatin and transcription requires the presence of DMS3, DRD1 and RDM1, which either help initiate Pol V transcription or assist elongation of Pol V transcripts[18],[19],[32]. Pol V transcription is followed by association of two RNA-binding proteins with chromatin( Figure 7). First is AGO4 which is recruited to chromatin by Pol V transcripts and uses the incorporated siRNA to provide sequence-specificity of silencing[19]. The second is SPT5L( Figure 1, Figure 2), which is recruited to chromatin by an unknown mechanism, possibly involving interactions between SPT5L and Pol V complex and/or with Pol V transcripts[22],[23]. SPT5L binds chromatin independently of 24-nt siRNA( Figure 4) and is likely a general factor associated with transcribing Pol V and its transcripts[21]–[23]. Since SPT5L binds chromatin in the absence of AGO4( Figure 1), and the functional pool of AGO4 is able to bind chromatin in the absence of SPT5L( Figure 3), we concluded that they are recruited to chromatin in parallel and independently of each other. Both AGO4 and SPT5L are required for the establishment and/or maintenance of DNA methylation and repressive histone modifications at the majority of tested loci( Figure 5). This suggests that both are needed for the recruitment of enzymes establishing repressive chromatin modifications. Because AGO4 and SPT5L bind chromatin independently of each other, and, at the majority of tested loci both are required for establishment and maintenance of silencing, we propose that AGO4 and SPT5L create a binding platform for the recruitment of chromatin modifying proteins. One possibility is that both weakly interact with a downstream protein but the interaction becomes strong enough to recruit chromatin modifying enzymes only when both are present. Alternatively, AGO4 may be a sole interacting partner of downstream proteins but SPT5L, which has a C-terminal domain rich in WG/GW motifs, interacts with AGO4 and alters its conformation to facilitate the recruitment of chromatin modifying enzymes. Our results show that there are loci where DNA methylation is established in a Pol V, AGO4 and SPT5L-dependent manner( Figure 5A), but these loci have an overall low level of H3K9me2 and no change in the histone modifications in tested mutants( IGN23 in Figure 5E). It suggests that the de novo DNA methyltransferase DRM2 is likely the chromatin modifying enzyme directly recruited by the AGO4-SPT5L platform. It is also possible that DRM2 may be recruited indirectly by another protein that binds the AGO4-SPT5L platform. Binding of AGO4 and SPT5L to chromatin is mediated by multiple protein-protein and protein-RNA interactions. These interactions may mediate recruitment of proteins to specific genomic regions and/or stabilize binding after recruitment by an independent mechanism. SPT5L binding to chromatin occurs downstream of Pol V and is most likely mediated by protein-RNA interaction between SPT5L and Pol V transcripts[22]. Like canonical SPT5, SPT5L may also form a heterodimer with SPT4[33]. Alternatively, SPT5L may be recruited to chromatin by protein-protein interaction with Pol V complex as suggested by identification of SPT5L in Pol V holoenzyme[23] and interactions between yeast SPT5 as well as bacterial homolog of SPT5, nusG, with RNA polymerases[34],[35]. It is also possible that SPT5L is recruited to chromatin by interacting with both Pol V transcripts and Pol V complex. All these mechanisms explain the AGO4-independent binding of SPT5L to Pol V-transcribed loci. Interaction with Pol V transcripts seems to be the major factor recruiting AGO4-siRNA to chromatin[19]. AGO4 also interacts with WG/GW-rich C-terminal domains of Pol V and SPT5L[21],[22],[36]. Because Argonautes contain only one WG/GW binding pocket[37] these interactions may be employed sequentially. First, they help recruit AGO4 to chromatin by interaction with Pol V and then they stabilize the binding of AGO4 to chromatin on its target loci by interaction with SPT5L. It is consistent with our observation that AGO4 binding to chromatin is slightly reduced in the spt5l mutant( Figure 3). We show that SPT5L contributes to regulation of siRNA-mediated transcriptional silencing in a highly locus-specific manner. This is demonstrated by the observation that two of the tested loci require Pol V and AGO4 but not SPT5L for establishment and/or maintenance of repressive chromatin modifications( Figure 6). It could be explained by presence of the canonical SPT5 at a subset of silenced loci. However, both loci are occupied by SPT5L in wild type plants( Figure 1) suggesting that SPT5L is in fact involved in their silencing. Only when SPT5L is mutated, the canonical SPT5 is able to compensate the deficiency at these particular loci. Alternatively, it is possible that the observed locus-specificity of SPT5L is caused by the presence of both Pol V and Pol II at a subset of loci[26]. Pol II-bound canonical SPT5 may be able to compensate the lack of Pol V-bound SPT5L. The mechanism deciding locus specificity of the SPT5L function remains unknown. Our results also suggest the presence of two pools of AGO4: SPT5L-dependent and SPT5L-independent. Because both pools are detectable at loci that are silenced in a SPT5L-independent manner, the SPT5L-dependent pool of AGO4 is likely not required for silencing. It may be recruited independently of siRNA by direct interaction with SPT5L and may have some other, yet unknown and locus-specific functions.
Transposons and other repetitive elements occupy vast areas of the eukaryotic genomes . They pose a threat to genome integrity but at the same time regulate expression of many genes and have been proposed to be a major factor contributing to genome evolution . One of the processes responsible for controlling activity of transposons and other repetitive elements is transcriptional gene silencing . This process uses small interfering RNA and long non-coding RNA to recruit enzymes that establish repressive chromatin modifications . Several proteins have been identified to be needed for siRNA–mediated transcriptional silencing in Arabidopsis thaliana , however for many of them their position in the silencing pathway is unknown . One of those proteins is SPT5L/KTF1 , a homolog of an elongation factor associated with RNA Polymerase II . Here we establish the position of SPT5L in the silencing pathway and propose the molecular mechanism of its function . This gives further knowledge of the mechanism of transcriptional gene silencing and is important to understand how transposons are controlled .