Protein partners of Kruppel-like factor 3

The aim of this study was to identify protein partners of the Kruppel-like factor 3 (KLF3) functional domain. We have previously shown that the non-DNA binding domain (the functional domain) of KLF3 play an important role in genomic localisation of this transcription factor (Burdach et al, 2014, Nucleic Acids Research, 42(1):276-89) and that the KLF3 functional domain is sufficient to direct an artificial zinc finger protein to new targets (Lim et al, 2016, Nucleic Acids Research, 44(7):3118-30). In this study we aimed to identify partner proteins of the KLF3 functional domain that might help to account for the role of this domain in genomic localisation. We had previously established HEK293 cell lines stably expressing either empty pMSCVpuro vector or pMSCVpuro-KLF3 functional domain (amino acids 1-262) with a C-terminal glycine-serine linker followed by a V5 epitope tag (Lim et al, 2016, Nucleic Acids Research, 44(7):3118-30). In this current study we performed two independent replicate experiments using the empty vector and KLF3 functional domain-V5 expressing HEK293 cells. Co-immunoprecipitation coupled with mass spectrometry was performed, using an antibody to the V5 tag, to identify endogenous HEK293 cell proteins that were bound by the KLF3 functional domain but not detected in the V5 co-immunoprecipitation samples from the empty vector cells. The mass spectrometry data was used to prioritise KLF3 functional domain partner proteins for further studies. In this experiment, WDR5 was identified as a partner protein of the KLF3 functional domain (in both independent replicates of the experiment) and the interaction of KLF3 and WDR5 was subsequently confirmed and the interaction interface of KLF3 that is bound by WDR5 mapped using co-immunoprecipitation experiments in COS cells.Sample description:- F002311 and F002312 are mass spec results from the empty transfected cell control cells- F002313 and F002314 are mass spec results from the cell stably expressing KLF3 functional domain

本研究旨在鉴定Kruppel样因子3（Kruppel-like factor 3, KLF3）功能结构域的蛋白质互作伴侣。
我们既往研究证实，KLF3的非DNA结合结构域（即其功能结构域）在该转录因子的基因组定位过程中发挥关键作用（Burdach等，2014，《核酸研究》，42(1):276-289）；同时证实KLF3功能结构域足以引导人工锌指蛋白靶向全新的基因组靶点（Lim等，2016，《核酸研究》，44(7):3118-30）。
本研究旨在鉴定KLF3功能结构域的互作伴侣蛋白，以阐明该结构域在基因组定位中的作用机制。
我们此前已构建稳定表达空pMSCVpuro载体，或携带C端甘氨酸-丝氨酸接头及V5表位标签（V5 epitope tag）的pMSCVpuro-KLF3功能结构域（氨基酸1-262）的HEK293细胞系（Lim等，2016，《核酸研究》，44(7):3118-30）。
本研究采用空载体组与KLF3功能结构域-V5表达组的HEK293细胞，开展两次独立重复实验。通过靶向V5标签的抗体开展免疫共沉淀（co-immunoprecipitation）结合质谱（mass spectrometry）分析，鉴定与KLF3功能结构域结合、且在空载体组的V5免疫共沉淀样本中未被检测到的内源性HEK293细胞蛋白。利用质谱数据筛选KLF3功能结构域的候选伴侣蛋白，以供后续研究使用。
本次实验中，WDR5被鉴定为KLF3功能结构域的互作伴侣蛋白（两次独立重复实验均成功检出）；后续通过COS细胞中的免疫共沉淀实验，证实了KLF3与WDR5的相互作用，并定位了WDR5所结合的KLF3互作界面。
样本说明：F002311与F002312为空转染细胞对照组的质谱检测结果；F002313与F002314为稳定表达KLF3功能结构域的细胞的质谱检测结果。