Evidence against a role for the JIL-1 kinase in H3S28 phosphorylation and 14-3-3 recruitment to active genes in Drosophila.
Evidence against a role for the JIL-1 kinase in H3S28 phosphorylation and 14-3-3 recruitment to active genes in Drosophila.
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DOI：
10.1371/journal.pone.0062484
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Johansen KM
中科院分区：
文献类型：
--
作者：
Wang C;Yao C;Li Y;Cai W;Bao X;Girton J;Johansen J;Johansen KM
JIL-1是控制组蛋白H3S10磷酸化的主要激酶，已被证明具有抵消异染色化和基因沉默的功能。然而，已经提出了另一种模型，在该模型中，转录需要JIL-1，此外还使H3S28磷酸化，并招募14-3-3到活性基因。由于这些发现与我们之前的研究结果不一致，即在完全缺乏JIL-1的情况下存在强劲的转录水平，并且在发育或热休克诱导的多丝染色体泡沫中不存在JIL-1，因此我们重新研究了JIL-1‘S在H3S28磷酸化和14-3-3募集中的可能作用。利用两种不同的H3S28ph抗体，我们用免疫细胞化学和免疫印迹法证明，在果蝇中，H3S28ph标记不存在于间期多线染色体上的背景以上水平，而仅存在于S2细胞和三龄幼虫神经母细胞分裂前期、中期和后期的染色体上。此外，这个有丝分裂的H3S28ph信号也存在于JIL-1缺失的突变背景中，这表明JIL-1不是有丝分裂的H3S28ph激酶。我们还证明了H3S28ph在热激喷发时不会被浓缩。使用两种不同的泛特异性14-3-3抗体以及一个增强子陷阱14-3-3ε-gfp系，我们发现14-3-3虽然存在于唾液腺细胞核中，但不定位于染色体，而仅定位于染色体周围的核基质。在我们的手中，14-3-3不会被招募到发育或热休克喷雾中。此外，使用LACO重复系链系统将Laci-JIL-1靶向多线染色体上的异位位点，我们发现只有H3S10ph存在并在这些位点上调，而不是H3S28ph或14-3-3。因此，我们的结果强烈反对H3S28磷酸化和14-3-3活性基因招募需要JIL-1的模型。
JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1’s possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes.
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影响因子：
7
作者：
Kellner WA;Ramos E;Van Bortle K;Takenaka N;Corces VG
通讯作者：
Corces VG
影响因子：
4.8
作者：
Bao, Xiaomin;Cai, Weili;Johansen, Kristen M.
通讯作者：
Johansen, Kristen M.
DOI：
10.3791/1748
发表时间：
2010-02-09
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Cai, Weili;Jin, Ye;Johansen, Kristen M
通讯作者：
Johansen, Kristen M
影响因子：
10.5
作者：
Ebert, A;Schotta, G;Reuter, G
通讯作者：
Reuter, G
影响因子：
4.6
作者：
Deng, Huai;Bao, Xiaomin;Johansen, Kristen M.
通讯作者：
Johansen, Kristen M.