POLY(ADP-RIBOSYLATION) OF HISTONES IN INTACT HUMAN KERATINOCYTES
POLY(ADP-RIBOSYLATION) OF HISTONES IN INTACT HUMAN KERATINOCYTES
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DOI：
10.1021/bi00435a063
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发表时间：
1989-05-02
期刊：
影响因子：
2.9
通讯作者：
CERUTTI, P
中科院分区：
文献类型：
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作者：
KRUPITZA, G;CERUTTI, P
染色体蛋白的多聚（adp -核糖基化）是致裂性DNA损伤剂影响染色质结构和功能的表观遗传结果。我们使用苯基硼酸树脂亲和层析和针对组蛋白H1、H2B、H2A、H3和H4的多克隆抗体免疫印迹相结合的方法，研究了在细胞外活性氧（AO）或烷基化剂n -甲基-n " -硝基-n -亚硝基胍（MNNG）诱导的永生化人角质形成细胞HaCaT中，主要类型组蛋白的聚（adp -核糖基化）反应。对聚ADPR反应的表征结果如下：(1)用蛇毒磷酸二酯酶对核提取物进行预处理，去除聚ADPR链，使苯硼酸盐保留的物质显著减少；(2) ADPR转移酶抑制剂苯甲酰胺（100 .mu）。M)抑制ao诱导的聚adp核糖基化；(3)聚（adp -核糖基化）降低了修饰组蛋白的电泳迁移率。在未处理的对照组中，有几个组蛋白是组成性多聚（adp -核糖基化）的：0.03%的H2A、0.04-0.06%的H2B和0.04%的H3.1携带至少一条长度未知的多聚（ADPR）链。AO瞬间增加了除H1外所有主要组蛋白的poly（ADPR）水平。50 .mu产生的AO暴露30 min后的替代程度。g/mL黄嘌呤和5 .mu。g/mL黄嘌呤氧化酶A24为0.8%，H4为0.3%,>为0.1%,H3.1为0.1%,H3.2为0.1%，H2B为0.1%。2 > 0.09%的H2A。在60分钟内，聚（ADPR）取代降低到H3和H4的控制水平，低于H2A和H2B的控制水平。添加苯甲酰胺（100 .mu）。M)阻止ao诱导的聚（adp -核糖基化）以及细胞NAD水平的下降（在30分钟）。MNNG在诱导组蛋白聚（adp -核糖基化）方面比AO更有效，且从电泳迁移率的显著降低判断，ADPR链的长度更长。5 μ m处理后20 min的替代程度依次递增。g/mL MNNG为H3 > 1.8% H1 > 1.2% H2B > 0.95% H4 0.36%。此外，变体形式H2B。x和未识别的低迁移率H2B-和h4相关抗原大量聚（adp -核糖基化）。我们的研究结果表明，AO和MNNG诱导不同模式的组蛋白聚（adp -核糖基化）。因此，不同的DNA链断裂剂预计会以一种特有的方式影响染色质的结构和功能。
The poly(ADP-ribosylation)of chromosomal proteins is an epigenetic consequence of clastogenic DNA damaging agents which affects chromatin structure and function. We studied the poly(ADP-ribosylation) of the major classes of histones in response to DNA breakage induced by an extracellular burst of active oxygen (AO) or the alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG) in the immortalized human keratinocytes HaCaT using a combination of affinity chromatography on phenylboronate resin and immunoblotting with polyclonal antibodies against histones H1, H2B, H2A, H3, and H4. The following findings characterized the poly(ADPR) reaction: (1) pretreatment of nuclear extracts with snake venom phosphodiesterase which removes poly(ADPR) chains strongly reduced the material which was retained by phenylboronate; (2) the ADPR transferase inhibitor benzamide (100 .mu.M) suppressed AO-induced poly(ADP-ribosylation); (3) poly(ADP-ribosylation) reduced the electrophoretic mobility of the modified histones. Several histones were constitutively poly(ADP-ribosylated) in untreated controls: 0.03% of H2A, 0.04-0.06% of H2B, and 0.04% of H3.1 carried at least one poly(ADPR) chain of undetermined length. AO transiently increased the poly(ADPR) levels of all major histones with the exception of H1. The extent of substitution 30 min after exposure to AO generated by 50 .mu.g/mL xanthine and 5 .mu.g/mL xanthine oxidase was 0.8% for A24 > 0.3% for H4 > 0.1% for H3.1 = 0.1% for H3.2 = 0.1% for H2B.2 > 0.09% for H2A. Within 60 min, poly(ADPR) substitution had decreased to control levels for H3 and H4 and below control levels for H2A and H2B. The addition of benzamide (100 .mu.M) prevented AO-induced poly(ADP-ribosylation) as well as the drop in cellular NAD levels (at 30 min). MNNG was more effective than AO in inducing histone poly(ADP-ribosylation), and to judge from the significant decreases in electrophoretic mobility, the lengths of the ADPR chains were longer. In increasing order, the extents of substitution 20 min after treatment with 5 .mu.g/mL MNNG were 1.8% for H3 > 1.2% for H1 > 0.95% for H2B > 0.36% for H4. In addition, the variant form H2B.x and unidentified low-mobility H2B- and H4-related antigens were substantially poly(ADP-ribosylated). Our results indicate that AO and MNNG induce distinct patterns of histone poly(ADP-ribosylation). Consequently, different DNA strand breaking agents are expected to affect chromatin structure and function in a characteristic fashion.