Related Experiment Video
Updated: Jun 29, 2026
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
Published on: May 26, 2014
Large Yellow Croaker (Pseudosciaena crocea, Richardson) E2F4, a Cyclin-Dependent Transcription Factor, Forms a
Xiaohui Cai1, Honglin Chen2, Jing Fang1
1Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, College of Marine Sciences, Beibu Gulf University, Qinzhou 535011, China.
Abstract:
E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G1-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 Pseudosciaena crocea 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 PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 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 PcE2F1-6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1-4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 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.
Insights
Researchers identified eight E2F transcription factors and one dimerization partner (PcDP1) in large yellow croakers. They found that PcE2F4 interacts with PcDP1, suggesting a role in regulating cell cycles and immune pathways.
Area of Science:
- Molecular Biology
- Genomics
- Fish Biology
Background:
- E2F transcription factors are crucial regulators of cell cycle progression, particularly the G1-S phase transition and DNA synthesis.
- These factors function as heterodimers with DP proteins (DP1 or DP2), with the dimerization domain mediating protein-protein interactions.
Purpose of the Study:
- To identify and characterize E2F transcription factors and their dimerization partners in the large yellow croaker (Pseudosciaena crocea).
- To investigate the interaction between identified E2F proteins and the dimerization partner PcDP1.
- To explore the potential role of E2F/DP1 heterodimers in regulating cell cycle and immune pathways in this fish species.
Main Methods:
- Genome-wide identification of E2F transcription factors (PcE2F1-8) and a dimerization partner (PcDP1) in large yellow croaker.
- Bioinformatic analysis of conserved domains (DBD, DD) and prediction of functional domains like NLS and NES.
- Experimental validation of protein interactions using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays.
- Analysis of gene expression patterns using RT-qPCR in various tissues of healthy fish.
Main Results:
- Eight E2F genes (PcE2F1-8) and one PcDP1 gene were identified. PcE2F1-6 possess both DNA-binding and dimerization domains, while PcE2F7-8 lack dimerization domains.
- Sequence analysis revealed distinct domain characteristics among PcE2F proteins, including pocket protein-binding domains and nuclear localization signals (NLS).
- Yeast two-hybrid and BiFC assays confirmed a direct interaction between PcE2F4 and PcDP1. RT-qPCR showed tissue-specific expression patterns for PcE2F1-6, with high expression in liver, kidney, brain, and spleen.
Conclusions:
- The study identified key E2F transcription factors and a dimerization partner in large yellow croakers.
- A direct interaction between PcE2F4 and PcDP1 was experimentally validated, suggesting the formation of an E2F4/DP1 heterodimer.
- This interaction is likely involved in regulating cell cycle progression and immune-related pathways in large yellow croakers.
Related Concept Videos
01:51Lampbrush Chromosomes
LBCs are made up of two pairs of conjugating homologous chromatids. Each chromatid consists of alternatively positioned regions of condensed-inactive chromatin and loosely placed-active side loops, which can be contracted and extended. The loops resemble the...
02:04Polytene Chromosomes
02:02Cis-regulatory Sequences
02:41Lethal Alleles
Lucien Cuénot discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is responsible for the color of the coat in mice. This gene codes for an agouti-signaling protein, which is responsible for melanin distribution in mammals. The wild-type allele gives rise to gray-brown coat color in mice, while the mutant allele gives rise to yellow coat color. In addition to coat color, the agouti gene is associated with the yellow...