Abstract

DNA methylation is essential for gene regulation, transposon silencing and imprinting. Although the generation of specific DNA methylation patterns is critical for these processes, how methylation is regulated at individual loci remains unclear.

DNA甲基化对于基因调控、转座子沉默和印迹至关重要。虽然特定的脱氧核糖核酸甲基化模式的产生对这些过程至关重要，但甲基化如何在单个位点受到调节仍不清楚。

Here we show that a family of four putative chromatin remodeling factors, CLASSY (CLSY) 1–4, are required for both locus-specific and global regulation of DNA methylation in Arabidopsis thaliana. Mechanistically, these factors act in connection with RNA polymerase-IV (Pol-IV) to control the production of 24-nucleotide small interfering RNAs (24nt-siRNAs), which guide DNA methylation. Individually, the CLSYs regulate Pol-IV–chromatin association and 24nt-siRNA production at thousands of distinct loci, and together, they regulate essentially all 24nt-siRNAs. Depending on the CLSYs involved, this regulation relies on different repressive chromatin modifications to facilitate locus-specific control of DNA methylation. Given the conservation between methylation systems in plants and mammals, analogous pathways may operate in a broad range of organisms.

在这里，我们表明一个由四个假定的染色质重塑因子组成的家族，GENEST(CLSY)1–4，是拟南芥中基因座特异性和全局调控DNA甲基化所必需的。从机制上讲，这些因子与核糖核酸聚合酶-ⅳ(Pol-ⅳ)一起控制24核苷酸小干扰核糖核酸(24nt-siRNAs)的产生，这些小干扰核糖核酸指导脱氧核糖核酸甲基化。单个来说，CLSYs在数千个不同的位点上调节Pol-ⅳ-染色质关联和24nt-siRNA的产生，它们一起调节基本上所有的24nt-SiRNA。根据所涉及的CLSYs，这种调节依赖于不同的抑制性染色质修饰，以促进对脱氧核糖核酸甲基化的位点特异性控制。鉴于植物和哺乳动物甲基化系统之间的保守性，类似的途径可能在广泛的生物体中运作。 

Results

1、The CLSY family controls 24nt-siRNA levels in a locus-specific manner

为了检验CLSY家族在RdDM途径中的作用，获得了每个CLSY基因的T-DNA插入突变体。

这些突变体中的基因表达谱证实了相应转录物的破坏，并证明在家族成员之间没有观察到明显的补偿性基因表达效应(Supplementary Fig. 1a and Supplementary Table 1)

(a) Genome browser screen shots depicting theexpression levels (reads per 10 million, rp10m) ofthe four CLSY genes in wild-type (WT) plants orthe indicated clsy mutant alleles. Theapproximate locations of the T-DNA insertions foreach allele are indicated by the hollow blacktriangles and the scale is indicated in brackets.描绘了野生型（WT）植物中四个CLSY基因或指示的clsy突变等位基因的表达水平（每1000万，rp10m的读数），每个等位基因的T-DNA插入的大致位置由空心的黑色三角形表示

看这些突变对24nt SiRNA的影响，通过 small RNA profiling (补充表2：Summary of the smRNA-seq data)，并与Pol-ⅳ突变体(nrpd1，以下称为Pol-ⅳ)和三个野生型对照中的结果进行比较。

在确定产生 small RNAs的基因座后，考虑到uniquely and multi-mapping reads (补充图1b和补充表3)，使用 ShortStack (补充表3和4a)鉴定了13，253个24nt-siRNA簇的核心组。ShortStack -一个小RNA基因的全面注释和定量的工具

b 条形图显示了在三个WT重复（WT_1、WT_2和WT_3）pol-iv突变体中的小型RNA-seq实验中鉴定出的21nt-、22nt-、23nt-和24nt-smRNA簇的数量

These core clusters were detected in all three wild-type replicates and account for more than 92% of the mapped 24nt-siRNAs in each experiment (Supplementary Fig. 1c). As expected from previous studies40,41, the expression of these 24nt-siRNA clusters is highly dependent on Pol-IV (Supplementary Fig. 1d,e).

这些核心簇在所有三个野生型重复中被检测到，并且在每个实验中占被作图的24nt-siRNAs的92%以上(补充图1c)。正如先前研究40，41所预期的，这些24nt-siRNA簇的表达高度依赖于Pol-IV(补充图1d，e)。

. (c) Bar graph showing the percent of 24nt-siRNAs in the three WT small RNA datasets that map to the core set of clusters (i.e. “covered” vs “uncovered”). For details regarding the generation of the core clusters, see Materials and Methods. (d) Pie chart showing the percent of core 24nt-siRNA clusters that are pol-iv-dependent. (e) Heatmap showing the levels of 24nt-siRNAs at pol-iv-dependent 24nt-siRNA clusters in the genotypes indicated below. The black triangle marks the midpoint of the 24nt-siRNA clusters and the flanking regions extend +/- 5kb.（c） 条形图，显示映射到核心簇集（即"覆盖"与"未覆盖"）的三个WT小RNA数据集中24nt-siRNA的百分比。（d） 饼图显示依赖于 pol-iv 的核心 24nt-siRNA 簇的百分比。（e） 热图显示以下基因型中pol-iv依赖性24nt-siRNA簇中24nt-siRNA的水平。黑色三角形标记24nt-siRNA簇的中点，侧翼区域延伸+/- 5kb。.

图e的看法：黑色三角形标记24nt-siRNA簇的中点，侧翼区域左右各延申延伸5kb；纵轴表示n=12771个clusters，颜色越红表示mapping到的位置pol-iv依赖性24nt-siRNA簇中24nt-siRNA的水平更高！能明显看出红色集中在24nt-siRNA簇的中点附近，由此可以看出这些24nt-siRNA簇的表达高度依赖于Pol-IV。

Further attesting 证明 to the robustness of these phenotypes, similar results were observed using only uniquely mapping reads (Supplementary Fig. 1h) or using data from an independent biological replicate (Supplementary Fig. 1i).

(h and i) Boxplots showing 24nt-siRNA levels at the indicated clsy-dependent clusters in the clsysingle mutants as compared to pol-iv. In h, only uniquely-mapped reads from the same data set used in Figure 1 are plotted and in i, both uniqueand multi-mapping reads from an independent, biological replicate (rep2) are plotted与 pol-iv 相比，箱线图显示了 clsy 单个突变体中指示的 clsy 依赖簇处的 24nt-siRNA 水平。在 h 中，仅绘制来自图 1 中使用的相同数据集的唯一映射读取，而在 i 中，绘制来自独立生物复制 （rep2） 的唯一和多映射读取

总的来说，这里鉴定的依赖于 clsy 的 24nt-siRNA 簇占全基因组 24nt-siRNA 产生基因座的大约 25%（图 1a），占野生型中存在的所有 24nt-siRNA 的 62.7%植物（补充图 1k）。 21nt- 和 22nt-siRNA 簇（包括 microRNA）的类似差异表达分析基本上没有鉴定出下调的簇（补充表 5）。综上所述，这些发现表明 CLSY 蛋白可作为 24nt-siRNA 表达的有效位点特异性调节因子。

a, Scaled Venn diagram based on the reduced 24nt-siRNA clusters provided in Supplementary Table 4 showing the relationships between loci with reduced 24nt-siRNA levels in the clsy single mutants. For readability, only overlaps >20 are labeled. A small number of overlaps between clsy2 and clsy3 are not shown due to spatial constraints, but an unscaled Venn diagram showing all overlaps is present in Supplementary Fig. 1f. a， 基于补充表4中提供的24nt-siRNA簇减少的缩放维恩图显示了clsy single mutants中位点与24nt-siRNA水平降低之间的关系。为了便于阅读，仅标记重叠>20。由于空间限制，没有显示clsy2和clsy3之间的少量重叠，但补充图中存在显示所有重叠的未缩放维恩图。1f.

为了确定由clsy单突变体调节的24nt-siRNA簇是否代表由这些因子控制的基因座的总数，我们产生了clsy双突变体的所有六个组合，并确定了它们的小RNA谱和减少的24nt-siRNA簇(图2a，b，补充图2a，b和补充表4)。

这确定了两个双突变体(clsy1,2 and clsy3,4) ，它们显示出明显的协同关系，影响更多的基因座(图2a)，并且相对于它们各自的单突变体显示出24nt-siRNA水平更强的降低(图2b)。 (clsy1,2 and clsy3,4)

Fig. 2 | Specific CLSY pairs regulate 24nt-siRNAs at non-overlapping and spatially distinct genomic loci. a,c,e, Scaled Venn diagrams showing the relationships between loci with reduced 24nt-siRNA levels in the indicated clsy single, double and quadruple mutants. For readability, only overlaps >20 are labeled, except in e, where the percentage of overlap between the two samples is shown instead. b,f, Box plots showing 24nt-siRNA levels in each clsy single, double or quadruple mutant compared to each other, WT controls and the pol-iv mutant, from a single experiment. Confirmatory data using additional alleles are presented in Supplementary Fig. 3. d, Chromosome 1 view of 24nt-siRNA clusters dependent on the genotypes indicated on the left, where the scale is the number of clusters per 100-kb bin. The red region corresponds to pericentromeric DNA56. The pie charts represent the genomewide (Chr1–5) distributions. Chromosomal views for Chr2–5 are present in Supplementary Fig. 2e

(a) Scaled Venn diagrams showing the relationships between loci with reduced 24nt-siRNA levels in the indicated clsy single and double mutants. (b) Boxplots showing 24nt-siRNA levels at the indicated clsy-dependent clusters in clsy single or double mutants as compared to WT controls and pol-iv.

值得注意的是，这些发现与以前的系统发育分析一致，因为CLSY1和CLSY2形成一个亚组，而CLSY3和CLSY4形成另一个亚组。

正如对依赖于单个CLSY蛋白的24nt-siRNA簇所观察到的，在clsy1，2-和clsy3，4-依赖簇处观察到的24nt-siRNAs的减少在很大程度上对相应的突变体是特异性的(图2b和补充图2c)。

补充图2(c) Proportional heatmaps showing the levels of 24nt-siRNAs at clsy-dependent 24nt-siRNA clusters in the genotypes indicated below. The black triangles mark the midpoints of the 24nt-siRNA clusters and the flanking regions extend +/- 5kb.

总的来说，这些clsy双突变体控制了所有24nt-siRNA簇的67%(图2c)，这相当于野生型植物中存在的所有24nt-siRNAs的88%(补充图2d)，突出了依赖于不同CLSY蛋白对的基因座特异性调节的第二层。

为了进一步研究clsy1，2-和clsy3，4-依赖的24nt-siRNA簇之间的关系，我们确定了它们彼此的重叠和它们的基因组分布。这些clsy对不仅调节互斥的24nt-siRNAs簇集(图2c)，它们还显示染色体臂(clsy1，2依赖簇)或着丝粒周围异染色质(clsy3，4依赖簇)的优先富集，indicating a striking distribution of labor amongst the CLSY family (图2d和补充图2e)。

值得注意的是，剩余的pol-iv依赖性24nt-siRNA簇在两个双突变体中都没有显著影响，在基因组中显示出甚至更极端的分配，78%位于着丝粒周围异染色质中(图2d和补充图2e)。这些集群是以低水平表达(补充图2c，d)，并且像依赖clsy3，4的24nt-siRNA簇一样，它们趋向于更大(补充图2f)。

为了确定这些剩余的基因座是否被所有四种clsy蛋白冗余控制，我们产生了一个CLSY四重突变体。在该突变体中，超过98%的所有pol-iv依赖性24nt-siRNA簇被减少(图2e)，并且这些簇中24nt-siRNAs的水平接近于零(图2f)。最后，我们证实了clsy单、双和四突变体对24ntsiRNA水平的影响和位点特异性，所有四个CLSY基因都有额外的突变等位基因(补充图3)。总之，这些发现表明，四种CLSY蛋白分别作为24ntsiRNA的高度位点特异性调节剂，并共同作为基本上所有Pol-IV依赖性24ntsiRNA的主要调节剂。

2、The CLSY family controls global DNA methylation patterns

为了评估clsy依赖性24nt-siRNA缺失对DNA甲基化模式的影响，我们进行了全基因组亚硫酸氢盐测序实验(补充表6 Summary of the MethylC-seq data )。

在拟南芥中，脱氧核糖核酸甲基化的模式可以大致分为两类:转座子和重复序列的甲基化methylation at transposons and repeats，这是通过RdDM途径建立的，发生在所有序列环境中(CG，CHG和CHH，其中H = A，T或C)，以及基因体甲基化，这仅限于CG环境，并通过仍然知之甚少的机制建立。

Thus, to best evaluate the roles of the clsy mutants, we determined differentially methylated regions (DMRs) 差异甲基化区域  for each genotype independently for the CG, CHG and CHH contexts (FC ≥ 40%, 20% or 10% for CG, CHG and CHH DMRs, respectively, relative to three wild-type controls with an FDR ≤ 0.01; Fig. 3a and Supplementary Table 7).因此，为了最好地评估clsy突变体的作用，我们在CG、CHG和CHH环境中独立地测定了每种基因型的差异甲基化区域(分别为CG、CHG和CHH的FC ≥ 40%、20%或10%，相对于三种野生型对照，FDR≤0.01；图3a和补充表7)。

3a, Table showing the numbers of hypomethylated DMRs in the genotypes and methylation contexts indicated, where H = A, T or C. The number of these DMRs that overlap (∩) with reduced 24nt-siRNA clusters (“DMR ∩ ↓ 24ntsiRNA clusters”) is also indicated and shaded from light blue to red according to the percentage of total DMRs represented 

Consistent with roles for the CLSY family in RdDM, this analysis showed a high degree of overlap between hypomethylated DMRs and reduced 24nt-siRNA clusters, especially for non-CG DMRs in the clsy double and quadruple mutants (Fig. 3a). Furthermore, even at DMRs that did not overlap with 24nt-siRNA clusters defined as being reduced, the 24nt-siRNA levels were still decreased (Supplementary Fig. 4).与clsy家族在RdDM中的作用一致，该分析显示低甲基化DMRs和减少的24nt-siRNA簇之间高度重叠，特别是对于CLSY双和四突变体中的非CG DMRs(图3a)。此外，即使在与被定义为减少的24nt-siRNA簇不重叠的dmr中，24nt-siRNA水平仍然降低(补充图4)。

因此，在非CG DMRs中，降低的DNA甲基化与24nt-siRNA损失高度相关。CG DMRs上的类似分析显示了与clsy突变体中减少的24nt-siRNA簇的最小重叠(图3a)，并揭示了这些区域中的绝大多数具有很少或没有24ntsiRNAs(补充图4)，这表明它们可能代表了体甲基化基因甲基化的自然变异，而不是靶向RdDM位点甲基化的缺陷。

尽管如此，与减少的24ntsiRNA簇重叠的CG DMRs的小子集(补充图4a)显示了明显的减少在24nt-siRNAs中， nearly phenocopying pol-iv mutants

总之，这些比较确定了基因座的子集，其中24nt-siRNA水平的降低导致每个序列背景下DNA甲基化的最显著变化。

正如预期的那样，鉴于在CG和CHG环境中存在控制DNA甲基化维持的途径5，在RdDM突变体中观察到的对DNA甲基化的最大影响是在CHH环境中。与它们的24ntsiRNA表型一致，每个clsy单一突变体在很大程度上不同的dmr组上影响DNA甲基化。同样，clsy1最强，有1，238个CHH DMRs，clsy3和clsy4分别有338个和161个，clsy2最弱，只有74个(图3a、b和补充图5a)。

a， 表格显示了所指示的基因型和甲基化环境中的次甲基化DMR的数量，其中H = A，T或C。还表示了与减少的24nt-siRNA簇重叠（∩）的这些DMR的数量（"DMR ∩↓ 24ntsiRNA簇"），并根据所代表的总DMR的百分比从浅蓝色着色为红色。b–d，低甲基化CHH DMR的鳞片维恩图，分别显示了由单突变体，双突变体和四突变体调节的位点之间的关系

进一步平行于24nt-siRNAs观察到的效应，clsy双突变体在互斥的CHH DMRs组中表现出相加效应(图3a，c)，四重突变体表现出最强的效应，与pol-iv突变体中鉴定的> 90%的CHH DMRs重叠(图3a，d)。对所有非CG DMRs(图3e和补充图5b)以及与减少的24nt-siRNA簇重叠的CG DMRs(补充图5c)的DNA甲基化的定量显示了在相应的突变背景中DNA甲基化的最强减少。此外，量化所有减少的24nt-siRNA簇的DNA甲基化水平，而不仅仅是对应于DMRs的那些，突出了类似的趋势:CG甲基化水平受影响最小，而在非CG环境中以基因型特异性的方式观察到更强的减少(补充图5d)。

总之，这些发现表明在clsy单突变体、双突变体和四突变体中观察到的24nt-siRNA水平的基因座特异性降低导致DNA甲基化的基因座特异性降低

3、The CLSY family is required for DNA methylation-mediated silencing

CLSY家族是DNA甲基化介导的沉默所必需的。

Given the known roles of DNA methylation in gene silencing, we conducted transcriptome profiling experiments to identify RdDM targets upregulated in pol-iv and clsy mutants (Supplementary Tables 1, 8 and 9). These analyses identified a total of 177 genes, repeats and unannotated transcripts upregulated at least twofold in pol-iv mutants.

 Although the clsy single mutants displayed weak expression phenotypes, at least one locus regulated predominantly by each mutant was identified (Fig. 4a, Supplementary Fig. 6a and Supplementary Table 9). Of these single mutants, clsy4 was by far the strongest. However, the vast majority of pol-iv loci were redundantly controlled by all four CLSY proteins, as the clsy quadruple mutant regulated approximately 50% of all pol-iv upregulated loci and nearly 80% of those were at least fivefold upregulated (Fig. 4a and Supplementary Table 9).

鉴于 DNA 甲基化在基因沉默中的已知作用，我们进行了转录组分析实验，以确定在 pol-iv 和 clsy 突变体中上调的 RdDM 靶标（补充表 1、8 和 9）。这些分析确定了总共 177 个基因、重复序列和未注释的转录本在 pol-iv 突变体中至少上调了两倍。

尽管 clsy 单突变体显示出弱表达表型，但至少鉴定了一个主要受每个突变体调节的基因座（图 4a、补充图 6a 和补充表 9）。在这些单一突变体中，clsy4 是迄今为止最强的。然而，绝大多数 pol-iv 基因座被所有四种 CLSY 蛋白冗余控制，因为 clsy 四重突变体调节了所有 pol-iv 上调基因座的大约 50%，其中近 80% 至少被上调了五倍（图 4a）和补充表 9)。

a, Plot showing the expression level of pol-iv-upregulated loci (represented as horizontal marks) in the clsy single, double and quadruple mutants. The marks in all genotypes are colored according to the expression level of upregulated loci in pol-iv and the number of upregulated loci in each mutant is indicated above.a， 图中显示了单突变体、双突变体和四突变体中 pol-iv 上调位点（表示为水平标记）的表达水平。所有基因型中的标记根据pol-iv中上调位点的表达水平进行着色，并且每个突变体中上调位点的数量如上所示。

To determine the extent to which the observed changes in gene expression correlate with altered 24nt-siRNA and DNA methylation profiles, we plotted these features side by side for all 177 loci (±2 kb) in the pol-iv and clsy quadruple mutants (Fig. 4b). On aggregate, these loci showed lower levels of 24nt-siRNAs and DNA methylation. For approximately half of the genes, as well as the majority of unannotated transcripts and repeats, discrete regions with more strongly reduced 24nt-siRNAs and DNA methylation levels were apparent either within the transcript itself or in the flanking 2-kb regions (Fig. 4b). Indeed, further characterization of these loci showed a high degree of overlap (80–100%) with the previously identified reduced 24nt-siRNA clusters and hypomethylated DMRs (Supplementary Fig. 6b,c and Supplementary Tables 4 and 7). In contrast, similar reductions were not observed in the clsy2 single mutant, which was the weakest mutant overall and thus served as a negative control (Supplementary Fig. 6d). Nonetheless, as in the pol-iv and clsy quadruple mutants, two of the three loci upregulated in the clsy2 mutant were associated with reduced 24nt-siRNA clusters and hypomethylated DMRs (Supplementary Fig. 6e). Together, these findings support the conclusion that these upregulated loci in the clsy mutants are normally silenced by DNA methylation that is controlled by the RdDM pathway.

为了确定观察到的基因表达变化在多大程度上与改变的 24nt-siRNA 和 DNA 甲基化谱相关，我们并排绘制了 pol-iv 和 clsy 四重突变体中所有 177 个基因座（±2 kb）的这些特征（图. 4b）。总的来说，这些基因座显示出较低水平的 24nt-siRNA 和 DNA 甲基化。对于大约一半的基因，以及大多数未注释的转录本和重复序列，24nt-siRNAs 和 DNA 甲基化水平降低更强烈的离散区域在转录本本身或侧翼 2-kb 区域中很明显（图 4b） ）。

 b, Heat maps and profile plots showing the expression levels of the upregulated TAIR10 genes (n = 115), unannotated transcripts (Un. txn; n = 26) and TAIR10 repeats (n = 36) shown in a, as well as the corresponding 24nt-siRNA and DNA methylation levels at these same loci. For the mRNA and 24nt-siRNA analyses, the log2 fold change in expression is plotted, and for the DNA methylation analysis, the percentage difference in methylation is plotted. Color bars indicating the scales are shown below. The heat maps include 2 kb flanking the transcription start site (S) and the transcription termination site (T) and were ranked according to the 24nt-siRNA and mCHH values in both mutants (pol-iv and clsy quadruple). The profiles of the genes, unannotated transcripts and repeats are in black, light blue and gray, respectively.b，热图和剖面图显示了a中显示的上调TAIR10基因（n = 115），未注释转录本（Un. txn; n = 26）和TAIR10重复序列（n = 36）的表达水平，以及这些相同位点上相应的24nt-siRNA和DNA甲基化水平。对于mRNA和24nt-siRNA分析，绘制了表达的log2倍变化，对于DNA甲基化分析，绘制了甲基化的百分比差异。指示刻度的色条如下所示。热图包括转录起始位点（S）和转录终止位点（T）两侧的2 kb，并根据两个突变体（pol-iv和clsy四元组）中的24nt-siRNA和mCHH值进行排名。基因图谱、未注释转录本和重复序列分别为黑色、浅蓝色和灰色。

事实上，这些基因座的进一步表征显示出与先前鉴定的减少的 24nt-siRNA 簇和低甲基化 DMR 高度重叠（80-100%）（补充图 6b、c 和补充表 4 和 7）。相比之下，在 clsy2 单突变体中没有观察到类似的减少，这是整体上最弱的突变体，因此作为阴性对照（补充图 6d）。尽管如此，与 pol-iv 和 clsy 四重突变体一样，在 clsy2 突变体中上调的三个基因座中的两个与减少的 24nt-siRNA 簇和低甲基化 DMR 相关（补充图 6e）。总之，这些发现支持这样的结论，即 clsy 突变体中的这些上调基因座通常被 RdDM 途径控制的 DNA 甲基化沉默。

As an additional test of the CLSY specificities, their roles in the establishment of DNA methylation were assessed using a well-vetted de novo methylation assay involving the transformation of an unmethylated FWA transgene into each mutant background46. In this assay, failure to methylate and silence the incoming transgene results in an increase in the number of leaves produced before flowering. Compared to the untransformed controls, several of the FWA-transformed clsy mutants showed delayed flowering (Fig.4c). In addition to the clsy1 mutant, which was previously shown to display a late-flowering phenotype in FWA assays37, clsy2 mutants also showed a slight delay, while theclsy3 and clsy4 mutants flowered at or near the normal number of leaves. This phenotype was enhanced in the clsy1,2 double mutant, which flowered nearly as late as the clsy quadruple and pol-iv mutants. Notably, the specificities observed for this de novo assay match those observed at the endogenous FWA gene, where 24nt-siRNA production depends on CLSY1 and CLSY2 (Fig.4d). These findings represent the first examples wherein bona fide components of the RdDM pathway (namely, CLSY3 and CLSY4) are not required to establish methylation in the FWA de novo assay and demonstrate that the locus specificity observed for the CLSY family extends to the establishment phase of the RdDM pathway.

作为对 CLSY 特异性的额外测试，使用经过充分审查的从头甲基化测定评估了它们在建立 DNA 甲基化中的作用，包括将未甲基化的 FWA 转基因转化为每个突变体背景 46。在该测定中，未能对传入的转基因进行甲基化和沉默会导致开花前产生的叶子数量增加。与未转化的对照相比，几个 FWA 转化的 clsy 突变体显示延迟开花（图 4c）。除了之前在 FWA 测定中显示出晚花表型的 clsy1 突变体 37 之外，clsy2 突变体也显示出轻微的延迟，而 clsy3 和 clsy4 突变体在正常叶数或接近正常叶数时开花。这种表型在 clsy1,2 双突变体中得到增强，其开花时间几乎与 clsy 四倍体和 pol-iv 突变体一样晚。值得注意的是，这种从头分析观察到的特异性与在内源性 FWA 基因中观察到的特异性相匹配，其中 24nt-siRNA 的产生取决于 CLSY1 和 CLSY2（图 4d）。这些发现代表了第一个例子，其中 RdDM 途径的真正成分（即 CLSY3 和 CLSY4）不需要在 FWA de novo 测定中建立甲基化，并证明对 CLSY 家族观察到的基因座特异性延伸到RdDM 途径。

The CLSY family is required for Pol-IV chromatin association

To gain mechanistic insights into the roles of the CLSY proteins, 

we determined the enrichment of Pol-IV at 24nt-siRNA-producing loci by chromatin immunoprecipitation and sequencing (ChIP-seq) experiments using a previously characterized tagged Pol-IV line (pNRPD1::NRPD1-3xFLAG34) crossed into various clsy mutant backgrounds (Supplementary Table10).为了从机制上深入了解 CLSY 蛋白的作用，我们通过染色质免疫沉淀和测序 (ChIP-seq) 实验使用先前表征的标记 Pol-IV 系 (pNRPD1: :NRPD1-3xFLAG34) 进入各种 clsy 突变体背景（补充表 10）。

 In a wild-type background, Pol-IV was enriched at all classes of clsy-dependent 24nt-siRNA clusters and, consistent with previous Pol-IV ChIP-seq experiments34, Pol-IV was most enriched at highly expressed 24nt-siRNA clusters (e.g., clsy1-dependent loci) and less enriched at clusters with low expression (e.g., clsy4-dependent loci) (Fig.5a). In the clsy1,2 or clsy3,4 mutant backgrounds, Pol-IV enrichment was specifically reduced at the loci regulated by these factors, and in the clsy quadruple mutant, Pol-IV enrichment was depleted at all 24nt-siRNA loci (Fig.5b and Supplementary Fig.7a,b). In the clsy single mutants, reductions in Pol-IV enrichment were most clearly observed at clsy1- and clsy3-dependent loci (Supplementary Fig.7c). For the clsy2 mutant, for which only a few reduced 24nt-siRNA clusters were identified (n= 45), or the clsy4 mutant, for which the reduced 24nt-siRNA clusters are expressed at low levels even in wild-type plants (Fig.1b), global reductions were difficult to observe. However, individual examples of Pol-IV reduction in these mutants were identified (Supplementary Fig.7d), and in both cases these weaker mutants (clsy2 and clsy4) enhanced their stronger mutant counterparts (clsy1 and clsy3, respectively; Supplementary Fig.7a). 

Taken together, these findings demonstrate that the CLSY proteins are required for the locus-specific association of Pol-IV at chromatin.

在野生型背景中，Pol-IV 在所有类别的依赖于 clsy 的 24nt-siRNA 簇中都富集，并且与之前的 Pol-IV ChIP-seq 实验一致，Pol-IV 在高表达的 24nt-siRNA 簇中最为富集（例如，依赖于 clsy1 的基因座），而在低表达的簇（例如依赖于 clsy4 的基因座）富集较少（图 5a）。

在 clsy1,2 或 clsy3,4 突变体背景中，Pol-IV 富集在这些因子调控的基因座上特别减少，而在 clsy 四重突变体中，Pol-IV 富集在所有 24nt-siRNA 基因座上都被耗尽（图 5b）和补充图7a，b）。在 clsy 单突变体中，在 clsy1 和 clsy3 依赖性基因座上最清楚地观察到 Pol-IV 富集的减少（补充图 7c）。对于 clsy2 突变体，其仅鉴定了少数减少的 24nt-siRNA 簇（n = 45），或 clsy4 突变体，即使在野生型植物中，减少的 24nt-siRNA 簇也以低水平表达（图 2）。 1b)，很难观察到全球减少。然而，鉴定了这些突变体中 Pol-IV 减少的个别例子（补充图 7d），在这两种情况下，这些较弱的突变体（clsy2 和 clsy4）增强了它们更强的突变体对应物（分别为 clsy1 和 clsy3；补充图 7a） .

总之，这些发现表明 CLSY 蛋白是染色质上 Pol-IV 位点特异性关联所必需的。

Discussion

A major unanswered question in the field of epigenetics is how specific patterns of DNA methylation are generated and modulated—a critical step in deciphering  解密 epigenetic processes in both normal development and disease. As Pol-IV ‘kicks off’ the RdDM pathway by initiating the biogenesis of 24nt-siRNAs, which ultimately guide DNA methylation in a sequence-specific manner, understanding the regulation of this polymerase is essential to determining how specific DNA methylation patterns are generated. Previously, we identified the CLSY proteins as components of the Pol-IV complex(es)35, and here we show they act as locus-specific regulators of both 24nt-siRNA production and DNA methylation. This locus-specific behavior differs from previously characterized RdDM factors, as none rival the degree or comprehensive nature of the specificities displayed by the CLSY family. Overall, these findings not only shed light on the regulation of Pol-IV, but also uncover a long-sought layer of complexity within the RdDM pathway that enables the locus-specific control of DNA methylation patterns.

在表观遗传学领域，一个尚未回答的主要问题是如何产生和调节特定的DNA甲基化模式——这是破译正常发育和疾病中表观遗传过程的关键一步。

由于Pol-IV通过启动24nt-siRNAs的生物发生来“启动”RdDM途径，最终以序列特异性的方式指导脱氧核糖核酸甲基化，理解这种聚合酶的调节对于确定特定的脱氧核糖核酸甲基化模式是如何产生的至关重要。

之前，我们将CLSY蛋白鉴定为Pol-IV复合物的成分，在这里我们表明它们作为24nt-siRNA产生和DNA甲基化的局部特异性调节因子。这种位点特异性行为不同于以前描述的RdDM因素，因为没有一个因素可以与CLSY家族所显示的特异性的程度或综合性质相媲美。总的来说，这些发现不仅阐明了Pol-IV的调节，而且揭示了RdDM途径中一个长期寻求的复杂层次，该层次能够对脱氧核糖核酸甲基化模式进行基因座特异性控制。

Investigation into the locus-specific behavior of the CLSYs indicated that different chromatin modifications are required for the production of 24nt-siRNAs depending on the CLSY proteins involved. For loci regulated by CLSY3 and CLSY4, CG methylation is required, but the connections (direct or indirect) between CG methylation and CLSY3 and CLSY4 remain to be elucidated. Perhaps further characterization of factors such as HISTONE DEACETYLASE 6, which participate in both the CG methylation and RdDM pathways51,52, will shed light on these connections.对CLSYs基因座特异性行为的研究表明，需要不同的染色质修饰24nt-siRNAs的产生依赖于所涉及的CLSY蛋白。对于由CLSY3和CLSY4调节的基因座，需要CG甲基化，但CG甲基化与CLSY3和CLSY4之间的联系(直接或间接)仍有待阐明。也许对参与CG甲基化和RdDM通路51，52的组蛋白去乙酰化酶6等因子的进一步表征将阐明这些联系。

For loci regulated by CLSY1 and CLSY2, our analyses provide a direct link to H3K9 methylation, as these two CLSY proteins are required for the association between the H3K9 methylation reader, SHH1, and the Pol-IV complex.

Finally, for the remaining loci that are redundantly controlled by all four CLSYs, it remains unclear whether different modes of regulation are employed, as these 24nt-siRNA clusters are expressed at low levels in all mutants tested (Fig. 6g). Together, these results show that specific chromatin features, including, but not limited to, CG and H3K9 methylation, can be leveraged to generate locus-specific control over DNA methylation.最后，对于所有四个CLSYs冗余控制的剩余基因座，仍不清楚是否采用了不同的调控模式，因为这些24ntsiRNA簇在所有测试的突变体中均以低水平表达(图6g)。总之，这些结果表明，特定的染色质特征，包括但不限于CG和H3K9甲基化，可以用来产生对脱氧核糖核酸甲基化的基因座特异性控制。

Indeed, such mechanisms appear to be conserved between plants and animals, as a similar, though less locus-specific, mechanism was recently identified in Drosophila wherein the core transcriptional machinery was shown to be linked to repressive histone marks in connection with the H3K9me3 reader, Rhino53.事实上，这种机制在植物和动物之间似乎是保守的，因为最近在果蝇中发现了一种类似的，尽管位点特异性较低的机制，其中核心转录机制被证明与H3K9me3读码器Rhino相关的抑制组蛋白标记相关。

Furthermore, given the widespread conservation of SNF2 chromatin remodeling factors in general and the specific conservation of the CLSY family in crops including rice54,55 and maize54, we anticipate that our findings will be informative for understanding the mechanisms governing the establishment of specific DNA methylation patterns in diverse organisms.

此外，鉴于SNF2染色质重塑因子的普遍保守性和CLSY家族在包括水稻54、55和玉米54在内的作物中的特异性保守性，我们预计我们的发现将有助于理解在不同生物体中建立特异性DNA甲基化模式的机制。