Functional characterization of STATa/b genes encoding transcription factors from Branchiostoma belcheri.

The signal transducer and activator of transcription (STAT), as an important transcription factor of the Janus kinase (JAK)-STAT signaling pathway, is pivotal for development and immunity and well documented in vertebrates. However, the STAT gene has not been reported in chordate amphioxus (Branchiostoma belcheri). In this study, we firstly identify and characterize two STAT genes from Branchiostoma belcheri (designed as AmphiSTATa and AmphiSTATb). Secondly, our results reveal that AmphiSTATa is clustered with vertebrate STAT1, STAT2, STAT3 and STAT4, whereas AmphiSTATb is grouped with STAT5 and STAT6 based on phylogenetic analysis. Thirdly, AmphiSTATa and AmphiSTATb are found to widely express in five representative tissues of amphioxus (gill, hepatic cecum, intestine, muscle and notochord) by RT-qPCR analysis. Importantly, both AmphiSTATa and AmphiSTATb can be involved in innate immune responses to LPS stimulation. Fourthly, we demonstrate that AmphiSTATa and AmphiSTATb can form homodimers or heterodimers by Co-IP and Native-PAGE assay, and that AmphiSTATa and AmphiSTATb proteins can also distribute in cytoplasm and nucleus by the subcellular localization. Taken together, our findings not only reveal the roles of AmphiSTATa and AmphiSTATb in amphioxus innate immune responses to LPS stimulation, but provide a new insight into further elucidating the evolution and function of STATs in animals.

Phylogenetically conserved TAK1 participates in Branchiostoma belcheri innate immune response to LPS stimulus.

Transforming growth factor-ß activated kinase-1 (TAK1) is an important member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, which plays an important role in animal innate immune response. However, the TAK1 gene has yet not been reported in amphioxus to date. Here, we have identified and characterized a TAK1 gene from amphioxus (Branchiostoma belcheri) (named as AmphiTAK1) with the full-length cDNA of 3479 bp, including an ORF sequence of 1905 bp, a 5' UTR of 394 bp and a 3' UTR of 1180 bp. Moreover, the predicted AmphiTAK1 protein contains STKc_TAK1 domain, TAB1 and TAB2/3 binding domain which are conserved among chordate, and phylogenetic analysis also shows that the AmphiTAK1 is located at the bottom of the chordate, revealing AmphiTAK1 as a new member of the TAK1 gene family. The further qRT-PCR analysis has shown that AmphiTAK1 is widely expressed in six investigated tissues (gonad, gill, hepatic cecum, intestine, muscle and notochord) of Branchiostoma belcheri, with high expression in notochord and gonad, moderate in gill and hepatic cecum. Notably, the expression level of AmphiTAK1 is significantly up-regulated after LPS stimulation. Specially, we also find that AmphiTAK1 protein can interact with AmphiTAB1 by immunoprecipitation assay. These findings reveal that AmphiTAK1 might interact with AmphiTAB1 to involve in innate immune response of Branchiostoma belcheri. Taken together, our present works provide a new insight into evolution and innate immune response mechanism of AmphiTAK1 gene in Branchiostoma belcheri.

Genome-wide organization, evolutionary diversification of the COMMD family genes of amphioxus (Branchiostoma belcheri) with the possible role in innate immunity.

The COMMD (COpper Metabolism gene MURR1 Domain) gene family with ten members participates in various biological processes, such as the regulation of copper and sodium transport, NF-κB activity and cell cycle progression. However, studies on the COMMD gene family in amphioxus (Branchiostoma belcheri) are yet largely unknown. In this study, we have identified and characterized the ten COMMD family members from amphioxus (designated as AmphiCOMMDs). Firstly, we clone the full length of AmphiCOMMDs, and all AmphiCOMMD proteins contain the conserved COMM domain with two NES (Nuclear Export Signal) motifs. Secondly, the genomic structure analysis demonstrates that genes of the COMMD family have undergone intron loss and gain during the process of divergence from amphioxus to vertebrates. Thirdly, phylogenetic analysis indicates that AmphiCOMMDs are more closely related to vertebrates, implying the AmphiCOMMDs may be the ancestor of the vertebrate COMMDs. Fourthly, AmphiCOMMDs are ubiquitously and differentially expressed in five investigated tissues (muscles, gills, intestine, heaptic cecum and notochord). Finally, our results show that expression levels of AmphiCOMMD genes are fluctuating after LPS stimulation to some different extent. Taken together, our studies have elaborated the evolutionary dynamic and the innate immune role of the COMMD family genes in amphioxus.

Transcriptome-wide analysis of microRNAs in Branchiostoma belcheri upon Vibrio parahemolyticus infection.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in diverse biological processes via regulating expressions of target genes at post-transcriptional level. Amphioxus, as modern survivor of an ancient chordate lineage, is a model organism for comparative genomics study. However, miRNAs involved in regulating immune responses in Branchiostoma belcheri are largely unclear. Here, we systematically investigated the microRNAs (miRNAs) involved in regulating immune responses in the cephalochordate amphioxus (Branchiostoma belcheri) through next-generation deep sequencing of amphioxus samples infected with Vibrio parahemolyticus. We identified 198 novel amphioxus miRNAs, consisting of 12 conserved miRNAs, 33 candidate star miRNAs and 153 potential amphioxus-specific-miRNAs. Using microarray profiling, 14 miRNAs were differentially expressed post infection, suggesting they are immune-related miRNAs. Eight miRNAs (bbe-miR-92a-3p, bbe-miR-92c-3p, bbe-miR-210-5p, bbe-miR-22-3p, bbe-miR-1∼bbe-miR-133 and bbe-miR-217∼bbe-miR-216 clusters) were significantly increased at 12 h post-infection, while bbe-miR-2072-5p was downregulated at 6 h and 12 h. Three miRNAs, bbe-miR-1-3p, bbe-miR-22-3p and bbe-miR-92a-3p, were confirmed to be involved in immune responses to infection by qRT-PCR. Our findings further clarify important regulatory roles of miRNAs in the innate immune response to bacterial infection in amphioxus.