Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA.
BMC Mol Biol. 2018 Mar 27;19(1):5. doi: 10.1186/s12867-018-0106-7.
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.
BATF 家族转录因子（BATF、BATF2 和 BATF3）与 JUNB 形成异三聚体，并与 IRF4 或 IRF8 结合，从而调节体内 T 细胞和树突状细胞的细胞命运。虽然异三聚体的每种组合都具有独特的作用，但观察到一定程度的交叉补偿。IRF4 和 IRF8 与 BATF 因子和 JUNB 结合的差异作用的基础尚不清楚。我们假设这些异三聚体之间功能的差异可能是由于它们的 DNA 结合偏好不同所致。虽然当与 JUNB 形成异二聚体时，所有三种 BATF 家族转录因子的结合偏好都相似，但 IRF4 或 IRF8 对异二聚体/DNA 复合物的协同结合可能会改变这些偏好。我们使用 Spec-seq，它可以高效且准确地平行确定对大量序列的相对亲和力，来发现 IRF4、IRF8 和 BATF 家族成员的协同 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都表现出几乎相同的结合偏好，无论是预期的野生型结合位点 TRE（TGA(C/G)TCA）还是 CRE（TGACGTCA）。IRF4 和 IRF8 在与三种异二聚体中的任何一种结合时，表现出非常相似的 DNA 结合偏好。在不同异三聚体之间的半位点中没有发现结合偏好的重大变化。IRF 蛋白与单个核苷酸间隔或相反方向的替代结合模式结合时，与 BATF 结合位点的结合亲和力大大降低。此外，在所有 BATF-JUNB 组合中，IRF 结合都会降低对 CRE 结合位点的偏好。
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。与位点之间具有间隔的序列相比，IRF 蛋白与 BATF 位点相邻结合会大大增加亲和力，表明通过蛋白质-蛋白质相互作用进行协同结合。当 IRF 蛋白结合时，对 TRE 或 CRE 类型的 BATF 结合位点的偏好也会改变。这些体外偏好有助于理解体内结合活性。
