Givaty-Rapp Yemima, Yadav Narendra Singh, Khan Asif, Grafi Gideon
French Associates Institute of Agriculture and Biotechnology of Drylands, The Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.
PLoS One. 2017 Jan 9;12(1):e0170067. doi: 10.1371/journal.pone.0170067. eCollection 2017.
Cell dedifferentiation characterizes the transition of leaf cells to protoplasts and is accompanied by global chromatin decondensation. Here we show that in Arabidopsis, chromocentric chromatin undergoes prompt and gradual decondensation upon protoplasting. We hypothesized that prompt chromatin decondensation is unlikely to be driven solely by epigenetic means and other factors might be involved. We investigated the possibility that S1-type endonucleases are involved in prompt chromatin decondensation via their capability to target and cleave unpaired regions within superhelical DNA, leading to chromatin relaxation. We showed that the expression and activity of the S1-type endonuclease 2 (ENDO2) is upregulated in dedifferentiating protoplasts concomitantly with chromatin decondensation. Mutation of the ENDO2 gene did not block or delay chromocentric chromatin decondensation upon protoplasting. Further study showed that ENDO2 subcellular localization is essentially cytoplasmic (endoplasmic reticulum-associated) in healthy cells, but often localized to the nucleus and in senescing/dying cells it was associated with fragmented nuclei. Using in gel nuclease assays we identified two ENDO2 variants, designated N1 (cytoplasmic variant) and N2 (cytoplasmic and nuclear variant), and based on their capability to bind concanavalin A (ConA), they appear to be glycosylated and de-glycosylated (or decorated with ConA non-binding sugars), respectively. Our data showed that the genome is responding promptly to acute stress (protoplasting) by acquiring decondensation state, which is not dependent on ENDO2 activity. ENDO2 undergoes de-glycosylation and translocation to the nucleus where it is involved in early stages of cell death probably by introducing double strand DNA breaks into superhelical DNA leading to local chromatin relaxation and fragmentation of nuclei.
细胞去分化是叶细胞向原生质体转变的特征，同时伴随着整体染色质的解聚。在这里，我们表明在拟南芥中，着丝粒染色质在原生质体形成后会迅速且逐渐地解聚。我们推测，染色质的迅速解聚不太可能仅由表观遗传方式驱动，可能涉及其他因素。我们研究了S1型核酸内切酶通过靶向和切割超螺旋DNA内的未配对区域导致染色质松弛，从而参与染色质迅速解聚的可能性。我们发现，在去分化的原生质体中，S1型核酸内切酶2（ENDO2）的表达和活性伴随着染色质解聚而上调。ENDO2基因突变并未阻止或延迟原生质体形成时着丝粒染色质的解聚。进一步研究表明，在健康细胞中，ENDO2的亚细胞定位主要在细胞质（与内质网相关），但在衰老/死亡细胞中常定位于细胞核，且与细胞核碎片化有关。通过凝胶内核酸酶分析，我们鉴定出两种ENDO2变体，分别命名为N1（细胞质变体）和N2（细胞质和细胞核变体），基于它们与伴刀豆球蛋白A（ConA）结合的能力，它们似乎分别被糖基化和去糖基化（或被ConA非结合糖修饰）。我们的数据表明，基因组通过获得解聚状态迅速响应急性应激（原生质体形成），这并不依赖于ENDO2的活性。ENDO2发生去糖基化并转移到细胞核，在那里它可能通过在超螺旋DNA中引入双链DNA断裂导致局部染色质松弛和细胞核碎片化，从而参与细胞死亡的早期阶段。
