Currie Simon L, Doane Jedediah J, Evans Kathryn S, Bhachech Niraja, Madison Bethany J, Lau Desmond K W, McIntosh Lawrence P, Skalicky Jack J, Clark Kathleen A, Graves Barbara J
Department of Oncological Sciences, University of Utah School of Medicine, Salt Lake City, UT, 84112-5500, USA; Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112-5500, USA.
Departments of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
J Mol Biol. 2017 Oct 13;429(20):2975-2995. doi: 10.1016/j.jmb.2017.06.024. Epub 2017 Jul 17.
The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. High-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-motifs, which bind JUN and FOS transcription factor families, were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.
序列特异性转录因子对转录辅因子的招募对高亲和力和选择性相互作用的基础提出了挑战。扩展先前关于ETV5的N端激活域（AD）与中介体亚基25（MED25）相互作用的研究，我们发现相关ETS因子ETV4的AD和DNA结合域（DBD）中类似的富含芳香族的基序与MED25相互作用。这些ETV4区域独立结合MED25，表现出不同的动力学，并共同促成全长ETV4与MED25的高亲和力相互作用。与MED25的高亲和力相互作用对ETV1/4/5亚家族具有特异性，因为其他ETS因子的结合较弱。AD结合到MED25上的一个位点，DBD与三个MED25位点相互作用，使得全长ETV4的两个结构域能够同时结合。MED25还通过解除自身抑制来刺激ETV4的体外DNA结合活性。ETV1/4/5因子在前列腺癌中经常过度表达，在前列腺癌细胞系中的全基因组研究表明，ETV4和MED25占据富含ETS结合序列的增强子，并且对这些增强子调控的基因转录在功能上都很重要。在MED25占据的区域中观察到结合JUN和FOS转录因子家族的AP1基序，JUN/FOS也与MED25接触；FOS与ETV4 AD结合到相同的MED25位点，JUN与其他两个MED25位点相互作用。总之，我们描述了多价ETV4-和AP1-MED25相互作用的特征，从而表明这些因子参与将MED25招募到转录控制元件中。
