Large Yellow Croaker ( Richardson) E2F4, a Cyclin-Dependent Transcription Factor, Forms a Heterodimer with DP1.

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1-8) of large yellow croaker and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1-6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7-8 only possess two duplicate DBDs but not DD, indicating that E2F7-8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1-6, the ORF of PcE2F1-6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1-6 were detected directly by yeast two-hybrid and BiFC. The , , , , , and genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1-5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1-6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1's subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1-6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The -, except for , mRNA levels were all detected higher in the liver. - were also highly specifically expressed in the kidney, , in the brain, and in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers.