Characterization of grass carp (Ctenopharyngodon idellus) Foxp1a/1b/2: Evidence for their involvement in the activation of peripheral blood lymphocyte subpopulations
Introduction
Foxp subfamily belongs to the forkhead-box (Fox) family of transcription factors with the sequence homology of their winged helix/forkhead DNA-binding domain [1]. Over 100 transcription factors with Fox domain have been found in species ranging from yeast to human with 17 subclasses, FoxA through FoxQ [2]. To date, four members (Foxp1–4) of Foxp subfamily have been functionally characterized in mammals [3], whereas only Foxp1 and Foxp2 were investigated in teleost fish [4], [5].
In mammals, Foxp1 and Foxp2 play critical roles in development, cancer and speech disorder. In human and mouse, Foxp1, firstly isolated from B lymphocyte, is widely expressed and has been implicated in cardiac [6], lung [7] and nervous system development [8]. Recent studies also indicated that Foxp1 as a key transcription factor is involved in B-cell development in mammal [9]. Moreover, Foxp1 has been identified as a tumor suppressor because of its abnormal expression patterns in solid tumors [10]. Further studies demonstrated that co-expression of Foxp1 and estrogen receptor (ER) in the nucleus of breast cancer cells was associated with improved patient survival, suggesting that Foxp1 may be useful as a potential therapeutic target in cancer [11], [12]. Foxp2 is coexpressed with Foxp1 in mouse lung and gut tissues [7], [13], and mutations in this gene are associated with familial language disorders in human, identifying it as a gene linked to speech and language development [14]. A recent study further confirmed that mutation of the DNA-binding region of the Foxp2 protein causes an inherited language disorder in mice [15].
Two members of the Foxp subfamily (Foxp1 and Foxp2) have been identified in fish (zebrafish and medaka) [4], [5]. The expression patterns of Foxp1 and Foxp2 in embryonic development have also been investigated in zebrafish [16], [17]. However, the functional roles of Foxp subfamily in fish immunity still remain unknown, and even the information about Foxp gene regulation in fish is limited. To increase knowledge of fish Foxp members and describe their roles in the immune response, we isolated and characterized three members of Foxp subfamily (Foxp1a/1b/2) from Chinese grass carp, and investigated the effects of LPS and PHA on their mRNA expression in grass carp PBLs. Furthermore, we defined the lymphocyte subpopulations correlated with the activation of these transcription factors.
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Animals
One-year-old Chinese grass carp (Ctenopharyngodon idellus), weighing 1000–1500 g, were obtained from Chengdu Tongwei Aquatic Science and Technology Company (China) and maintained in laboratory with natural temperature and photoperiod for 2 weeks prior to experimental processing. All experiments complied with the Regulation of Animal Experimentation of Sichuan province, China.
Cloning of grass carp Foxp1a, Foxp1b, Foxp2, CD4-like (CD4L), CD8α and CD8β cDNA
Total RNA was extracted from about 50 mg of different tissues individually using TRIzol Reagent (Invitrogen, Carlsbad,
Molecular cloning and sequence analysis of grass carp Foxp1a, Foxp1b, and Foxp2
We obtained three full-length cDNA sequences of grass carp Foxp1a, Foxp1b and Foxp2. Grass carp Foxp1a sequence consisted of 104 bp 5′UTR, 104 bp 3′UTR and 1278 bp ORF coding a 425-amino acid (aa) polypeptide (Supplementary Fig. S1A) (accession No: FJ415222). The ORF of grass carp Foxp1b with 1986 bp in length encoded a 661-aa polypeptide, with 5′UTR and 3′UTR are 86 bp and 201 bp, respectively (Supplementary Fig. S1B) (accession No: FJ415223). The cDNA of grass carp Foxp2 contained 146 bp 5′UTR, ORF
Discussion
To date, the studies regarding the Foxp1 and Foxp2 expression in fish species are very limited except some recent studies in zebrafish [4], [16], [17] and medaka [5]. They showed that fish Foxp1 and Foxp2 were expressed during embryonic development and neural development, which is consistent with the findings in mouse [20], frog [21], and bird [22]. In grass carp, qPCR revealed that Foxp1b transcript was detected in pituitary, brain, thymus, heart, head kidney, kidney, liver, spleen, muscle,
Acknowledgments
This work was sponsored by grants from National Natural Science Foundation of China (30972280) and Program for New Century Excellent Talents at University of China (NCEF-06-0814) to H.Z.
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