Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, United States.
Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, United States.
Elife. 2024 May 30;12:RP89353. doi: 10.7554/eLife.89353.
Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in , suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in corresponded to the loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.
沉默途径可防止转座元件 (TE) 的增殖，并通过细胞分裂帮助维持基因组完整性。沉默的基因组区域可分为常染色质或异染色质，并可通过遗传上可分离的表观遗传途径靶向。在植物中，RNA 指导的 DNA 甲基化 (RdDM) 途径主要靶向常染色质区域，而 CMT DNA 甲基转移酶主要与异染色质相关。然而，许多表观遗传特征——包括 DNA 甲基化模式——在这些区域之间基本无法区分，因此功能分离是如何维持的尚不清楚。连接组蛋白 H1 优先定位于异染色质，并且已经提出它限制了 RdDM 的侵入。为了验证这一假设，我们通过对中央 RdDM 聚合酶 Pol V 的最大亚基 NRPE1 进行 ChIP-seq，在 突变体中跟踪 RdDM 基因组定位。H1 的缺失导致 NRPE1 主要在异染色质 TE 中富集。在 中，NRPE1 结合的增加与染色质可及性的增加相关，表明 H1 通过压缩染色质来限制 NRPE1 的占据。然而，RdDM 占据并没有影响 H1 的定位，这表明 H1 分层限制了 RdDM 的定位。H1 突变体经历主要的对称 (CG 和 CHG) DNA 甲基化增益，通过产生 双突变体，我们证明这些增益在很大程度上独立于 RdDM。然而，在 中，一些常染色质区域中 NRPE1 占据的丧失对应于所有序列背景下甲基化的丧失，而在异位结合的异染色质位点，NRPE1 的沉积与 CHH 背景下特异性增加的甲基化相关。此外，我们发现 H1 同样限制了甲基化读取器 SUVH1 和多梳介导的 H3K27me3 的占据。总之，这些结果支持了一种模型，即 H1 通过防止其他竞争途径的侵入来帮助维持异染色质的排他性。
