使用Spec-seq定量分析BATF家族蛋白/JUNB/IRF异源三聚体

IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology BMC Molecular Biology Pub Date : 2018-03-27 DOI:10.1186/s12867-018-0106-7
Yiming K. Chang, Zheng Zuo, Gary D. Stormo
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引用次数: 10

摘要

BATF家族转录因子(BATF, BATF2和BATF3)与JUNB和IRF4或IRF8形成异源三聚体,在体内调节T细胞和树突状细胞的细胞命运。虽然异源三聚体的每种组合都有不同的作用,但观察到一定程度的交叉补偿。IRF4和IRF8对BATF因子和JUNB差异作用的基础尚不清楚。我们认为这些异源三聚体的功能差异可能是由它们的DNA结合偏好的差异引起的。虽然所有三种BATF家族转录因子作为异二聚体与JUNB结合时具有相似的结合偏好,但IRF4或IRF8与异二聚体/DNA复合物的协同结合可能改变这种偏好。我们使用了Spec-seq,它允许高效和准确地测定大量序列的相对亲和力,以发现IRF4, IRF8和BATF家族成员的合作DNA结合之间的差异。我们发现,在没有IRF结合的情况下,所有三种异二聚体对预期的野生型结合位点TRE (TGA(C/G)TCA)和CRE (TGACGTCA)都表现出几乎相同的结合偏好。当IRF4和IRF8与三种异二聚体中的任何一种结合时,显示出非常相似的DNA结合偏好。在不同的异源三聚体之间的半位点上没有发现明显的结合偏好变化。IRF蛋白与IRF和BATF结合位点之间的单核苷酸间隔物或与相反方向的另一种结合方式结合的亲和力低得多。此外,在所有BATF-JUNB组合中,IRF结合时对CRE结合位点的偏好降低。BATF、BATF2和BATF3的特异性非常相似,它们与IRF4和IRF8的相互作用也非常相似。与BATF位点相邻结合的IRF蛋白相比,位点之间有间隔的序列亲和力显著增加,表明通过蛋白-蛋白相互作用协同结合。当IRF蛋白结合时,对BATF结合位点(TRE或CRE)类型的偏好也会发生改变。这些体外偏好有助于了解体内结合活性。
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Quantitative profiling of BATF family proteins/JUNB/IRF hetero-trimers using Spec-seq

BATF family transcription factors (BATF, BATF2 and BATF3) form hetero-trimers with JUNB and either IRF4 or IRF8 to regulate cell fate in T cells and dendritic cells in vivo. While each combination of the hetero-trimer has a distinct role, some degree of cross-compensation was observed. The basis for the differential actions of IRF4 and IRF8 with BATF factors and JUNB is still unknown. We propose that the differences in function between these hetero-trimers may be caused by differences in their DNA binding preferences. While all three BATF family transcription factors have similar binding preferences when binding as a hetero-dimer with JUNB, the cooperative binding of IRF4 or IRF8 to the hetero-dimer/DNA complex could change the preferences. We used Spec-seq, which allows for the efficient and accurate determination of relative affinity to a large collection of sequences in parallel, to find differences between cooperative DNA binding of IRF4, IRF8 and BATF family members.

We found that without IRF binding, all three hetero-dimer pairs exhibit nearly the same binding preferences to both expected wildtype binding sites TRE (TGA(C/G)TCA) and CRE (TGACGTCA). IRF4 and IRF8 show the very similar DNA binding preferences when binding with any of the three hetero-dimers. No major change of binding preferences was found in the half-sites between different hetero-trimers. IRF proteins bind with substantially lower affinity with either a single nucleotide spacer between IRF and BATF binding site or with an alternative mode of binding in the opposite orientation. In addition, the preference to CRE binding site was reduced with either IRF binding in all BATF–JUNB combinations.

The specificities of BATF, BATF2 and BATF3 are all very similar as are their interactions with IRF4 and IRF8. IRF proteins binding adjacent to BATF sites increases affinity substantially compared to sequences with spacings between the sites, indicating cooperative binding through protein–protein interactions. The preference for the type of BATF binding site, TRE or CRE, is also altered when IRF proteins bind. These in vitro preferences aid in the understanding of in vivo binding activities.

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来源期刊
BMC Molecular Biology
BMC Molecular Biology 生物-生化与分子生物学
CiteScore
4.80
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: BMC Molecular Biology is an open access journal publishing original peer-reviewed research articles in all aspects of DNA and RNA in a cellular context, encompassing investigations of chromatin, replication, recombination, mutation, repair, transcription, translation and RNA processing and function.
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