The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis
The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis
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DOI：
10.1073/pnas.1433065100
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发表时间：
2003-07-22
影响因子：
11.1
通讯作者：
Ozato, K
中科院分区：
文献类型：
--
作者：
Dey, A;Chitsaz, F;Ozato, K
以前的体外研究表明，溴结构域结合组蛋白尾部的乙酰赖氨酸，导致该结构域参与破译组蛋白密码的建议。然而，几乎没有体内证据支持溴结构域在天然环境中与乙酰化染色质结合。Brd4是携带两个溴结构域的BET家族的成员。它与有丝分裂染色体有关，这是该科的特征。在这里，我们研究了Brd4与染色质在活细胞中的光漂白的相互作用。Brd 4是移动的，与染色质以快速的“开和关”结合模式相互作用。这种相互作用需要两个溴结构域。表明优先与乙酰化染色质的相互作用，Brd 4变得不那么移动的后，增加染色质乙酰化引起的组蛋白脱乙酰酶抑制剂。提供生化支持，盐溶解度的Brd4显着降低组蛋白乙酰化。这种变化也需要两个溴结构域。在肽结合试验中，Brd4与二乙酰化和四乙酰化的组蛋白H4和二乙酰化的H3强烈结合，但与单乙酰化和未乙酰化的H3和H4弱结合或根本不结合。相反，它不与未乙酰化的H4或H3结合。此外，Brd4与乙酰化的H4和H3共定位在有丝分裂染色体的非着丝粒区域。这种共定位也需要两个溴结构域。这些观察结果表明Brd4特异性识别乙酰化组蛋白密码，并且这种识别被传递到新分裂细胞的染色质上。
Previous in vitro studies showed that the bromodomain binds to acetyllysines on histone tails, leading to the proposal that the domain is involved in deciphering the histone code. However, there is little in vivo evidence supporting the binding of bromodomains to acetylated chromatin in the native environment. Brd4 is a member of the BET family that carries two bromodomains. It associates with mitotic chromosomes, a feature characteristic of the family. Here, we studied the interaction of Brd4 with chromatin in living cells by photobleaching. Brd4 was mobile and interacted with chromatin with a rapid "on and off" mode of binding. This interaction required both bromodomains. Indicating a preferential interaction with acetylated chromatin, Brd4 became less mobile upon increased chromatin acetylation caused by a histone deacetylase inhibitor. Providing biochemical support, salt solubility of Brd4 was markedly reduced upon increased histone acetylation. This change also required both bromodomains. In peptide binding assays, Brd4 avidly bound to di- and tetraacetylated histone H4 and diacetylated H3, but weakly or not at all to mono- and unacetylated H3 and H4. By contrast, it did not bind to unacetylated H4 or H3. Further, Brd4 colocalized with acetylated H4 and H3 in noncentromeric regions of mitotic chromosomes. This colocalization also required both bromodomains. These observations indicate that Brd4 specifically recognizes acetylated histone codes, and this recognition is passed onto the chromatin of newly divided cells.