Noncanonical DNA motifs as transactivation targets by wild type and mutant p53.
Noncanonical DNA motifs as transactivation targets by wild type and mutant p53.
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DOI：
10.1371/journal.pgen.1000104
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发表时间：
2008-06-27
期刊：
影响因子：
4.5
通讯作者：
Resnick MA
中科院分区：
文献类型：
--
作者：
Jordan JJ;Menendez D;Inga A;Noureddine M;Bell DA;Resnick MA
人类p53主调控因子的序列特异性结合对其在环境应激下的肿瘤抑制活性至关重要。p53作为四聚体结合到两个相距0-13个核苷酸（nt）的十聚体半位点上，最初被共识定义为RRRCWWGYYY (n = 0-13) RRRCWWGYYY。为了更好地理解p53的序列、组织和水平在野生型（WT）和突变型p53在靶反应元件（REs）的转激活中的作用，我们利用出芽酵母和人类细胞系统解构了功能性p53的典型共识序列。与早期关于体外结合的报道相反，正如天然的TIGER RE所证明的那样，十聚体半位点之间距离的微小增加大大降低了p53的转激活。这一点通过一种新开发的半体外微球结合试验，在人类细胞提取物中得到了证实。这些结果与MDM2启动子中一对弱的全位点REs的协同激活增加形成对比，它们被一个进化保守的17bp间隔分隔开。令人惊讶的是，在非规范的1 / 2 -（单个十聚体）和3 / 4 -位点上可能存在大量的反激活，其中一些位点最初被归类为生物学相关的规范共识序列，包括PIDD和Apaf-1。P53家族成员p63和p73也产生了类似的结果。非规范元件的高效反激活需要四聚体p53，而羧基端、非特异性DNA结合域的存在增强了非规范序列的反激活。我们的研究结果表明，p53的RE序列、组织和水平可以强烈影响p53介导的转激活，从而改变了对功能p53靶点构成的看法。重要的是，包含1 / 2和3 / 4位点的REs极大地扩展了p53主调控网络。在人类细胞中，肿瘤抑制因子p53是引发多种生物反应所需的调控中心节点，包括应激或DNA损伤引起的细胞周期阻滞和死亡，其中p53突变是癌症的标志。作为一个主调控基因，p53通过直接与广泛接受的DNA结合序列相互作用来控制其网络中靶基因的作用，该序列由两个十聚体½位点组成，可以被多达13个碱基分开。虽然共识序列的不匹配是常见的，但规范的共识序列限制了p53转录网络中靶基因的组织和数量。利用酵母和人类细胞系统，我们的目标是进一步了解DNA序列、DNA组织和p53表达水平如何影响p53调控网络中基因的包含。我们发现间隔蛋白超过几个碱基的增加大大降低了对p53的反应性。重要的是，我们确定p53可以从仅包含十聚体1 / 2位点或3 / 4位点的非规范序列中起作用。这些发现进一步确定和扩大了可能受p53调控的潜在下游靶基因范围，使p53调控网络进一步多样化。
Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network. Within human cells, the tumor suppressor p53 is the central node of regulation required to elicit multiple biological responses that include cell cycle arrest and death in response to stress or DNA damage, where mutations in p53 are a hallmark of cancer. As a master regulatory gene, p53 controls the action of target genes within its network by directly interacting with a widely accepted consensus DNA binding sequence, composed of two decamer ½-sites that can be separated by up to 13 bases. While mismatches from consensus sequence are frequent, the canonical consensus sequence places a limitation upon the organization and number of target genes within the p53 transcriptional network. Using yeast and human cell systems, our goal was to further understand how the DNA sequence, DNA organization, and level of p53 expression might influence the inclusion of genes within the p53 regulatory network. We found that increases in spacer beyond a few bases greatly reduce responsiveness to p53. Importantly, we established that p53 can function from noncanonical sequences comprising only a decamer ½-site or a ¾-site. These findings further define and expand the universe of potential downstream target genes which may be regulated by p53 and bring further diversity into the p53 regulatory network.
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影响因子：
64.5
作者：
Cawley, S;Bekiranov, S;Gingeras, TR
通讯作者：
Gingeras, TR
DOI：
10.1073/pnas.92.19.8591
发表时间：
1995-09-12
影响因子：
11.1
作者：
BALAGURUMOORTHY, P;SAKAMOTO, H;HARRINGTON, RE
通讯作者：
HARRINGTON, RE
影响因子：
64.5
作者：
ELDEIRY, WS;TOKINO, T;VOGELSTEIN, B
通讯作者：
VOGELSTEIN, B
影响因子：
5.3
作者：
FUNK, WD;PAK, DT;SHAY, JW
通讯作者：
SHAY, JW
DOI：
10.1038/nsb730
发表时间：
2002-01-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
DiGiammarino, EL;Lee, AS;Kriwacki, RW
通讯作者：
Kriwacki, RW