American Journal of Biochemistry and Biotechnology Original Research Paper Comparative Functional Analysis of the Basic Helix-Loop- Helix Proteins in the Clawed Frogs' Genomes With Common Essential Pathways and Enriched Gene Ontology Terms 1, 2 Wuyi Liu 1Biological and Food Engineering Faculty, Anhui Province Key Laboratory of Environmental Hormone and Reproduction, Fuyang Normal University, China 2 Department of Science and Technology, Fuyang Normal University, China Article history Abstract: The international Gene Ontology (GO) and pathway databases Received: 31-12-2018 were used to functionally analyze the clawed frogs' Basic Helix-Loop- Revised: 13-02-2019 Helix (bHLH) transcription factors of Xenopus tropicalis and Xenopus Accepted: 27-03-2019 laevis in a updated genome-wide survey. There were 41 GO terms and one pathway significantly enriched for Xenopus tropicalis , whereas there Corresponding Author: Wuyi Liu were 45 GO terms and 3 pathways significantly enriched for Xenopus Biological and Food laevis . Among those significantly enriched GO terms, the two clawed Engineering Faculty, Anhui frogs share 31 common functional GO annotations of these bHLH genes, Province Key Laboratory of including DNA-dependent transcription and (negative) transcription Environmental Hormone and regulation, DNA binding and bHLH binding, transcription factor complex Reproduction, Fuyang Normal and protein heterodimerization activity, (negative) regulation of RNA University, Fuyang City, China metabolic processes, nuclear translocator and repressor, myogenic basic Email: [email protected] muscle-specific protein, neurogenic differentiation factor and NeuroD. Furthermore, these frogs' bHLH genes were also found to play important roles in the regulation of gene expression in some important developmental or physiological processes, such as (skeletal) muscle cell differentiation, muscle organ development, biological rhythms and rhythmic process, hypoxia (adaption) and hypoxia-inducible factors, neurogenesis, neural tube development and neurogenic differentiation, whereas they were commonly significantly enriched in TGF-beta signaling pathway. These resulted data and information are very important for us to understand the functions, classification and evolution of frog bHLH genes. Keywords: Basic Helix-Loop-Helix Transcription Factor, Functional Annotation, Pathway, Gene Ontology, Clawed Frog Introduction bHLH transcription factor family are found to be crucial and they play many important roles in the cell The Basic Helix-Loop-Helix (bHLH) proteins are proliferation and differentiation, body immunity, currently recognized as the most important class of muscle tissue formation, neurons, resistance to stress, transcription factors. They can form specific development of the eye and intestine, hematopoietic interactions with the genetic cis-elements of function and coagulation function, adaptation to eukaryotes, thereby activating or inhibiting the hypoxic environment, sex determination and the process transcription and translation of the gene and they may of genetic development of animals and plants (Murre et also bind to the DNA binding proteins with activation or al ., 1989; Murre, 2019; Atchley and Fitch, 1997; Boggon inhibition activities (Murre et al ., 1989; Murre, 2019). et al ., 1999; Luscombe et al ., 2000: Riechmann et al ., They can combine with other transcription factors to form 2000; Stevens et al ., 2008; Wang et al ., 2010). The a complex genetic regulatory network too (Murre et al ., earliest reported bHLH protein in animals is the mouse 1989; Atchley and Fitch, 1997; Boggon et al ., 1999; transcription factors E12 and E47 (Murre et al ., 1989; Luscombe et al ., 2000; Riechmann et al ., 2000; Stevens Massari and Murre, 2000; Stevens et al ., 2008; Murre, et al ., 2008; Murre, 2019). At present, members of the 2019). Later studies suggested the animal bHLH proteins © 2019 Wuyi Liu. This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Wuyi Liu / American Journal of Biochemistry and Biotechnology 2019, 15 (1): 39.51 DOI: 10.3844/ajbbsp.2019.39.51 to be divided into six large categories of protein the way we perceive and understand the genomic subclasses that were subdivided as 45 sub-families datasets of genomes and/or the organization and heredity (Ledent and Vervoort, 2001; Ledent et al ., 2002; of biological organisms. Among them, the GO forum has Simionato et al ., 2007; Stevens et al ., 2008; Wang et al ., a database of dozens of animals, plants and 2010), whereas the plant bHLH proteins were divided into microorganisms. The GO forum and KEGG pathway more than 20 subclasses with conserved motifs or domains databases have constructed the relatively independent identified (Carretero-Paulet et al ., 2010; Yan et al ., 2015; ontological vocabularies (Kanehisa and Goto, 2000). For Liu et al ., 2018; Wei and Chen, 2018; Gao et al ., 2019), instance, the GO forum developed the standard varying from 21 to 32 subfamilies (Carretero-Paulet et al ., language "Ontology" with three levels of intrinsic 2010; Song et al ., 2014; Hudson and Hudson, 2014; Sun structure, namely Molecular Function (MF), et al ., 2015; Wang et al ., 2015; Yan et al ., 2015; Biological Process (BP) and Cellular Component Zhang et al ., 2015; Kavas et al ., 2016; Gao et al ., (CC). All the common attributes of those genes, 2017; Guo and Wang, 2017; Niu et al ., 2017; Liu et al ., transcripts and their products can be used to organize 2018; Lu et al ., 2018; Wei and Chen, 2018; Zhang et the different functional concepts and/or annotations of al ., 2018; Gao et al ., 2019). GO and KEGG pathways into the organic systems of Recently, as the whole genome genetic mapping of databases (Boggon et al ., 1999; Luscombe et al ., model animals and plants and the genome sequencing 2000; Riechmann et al ., 2000, Dennis et al ., 2003; projects of numerous species have been completed, Huang et al ., 2009). Therefore, the genomic databases more and more transcription factors are identified and of GO and KEGG pathways are the most basic utilized, which is important and feasible for studies on functional descriptions, structural composition the issues of functional characteristics and genetic descriptions, descriptions of synthesis and evolution of specific transcription factors. On the one decomposition and metabolic maps of specific genes hand, these inherent information stored in various and their expression and transcripts. genomes may be explored to rapidly elucidate the Both Xenopus tropicalis and Xenopus laevis are genetics and developmental mechanisms regulating the well-established biomedical model organisms for the processes of cell differentiation and organ the genetics and developmental research. In practice, development and organization growth of animal and Xenopus tropicalis and Xenopus laevis are common plant and fungi species with the great development of and yet important clawed frogs that are generally used in modern bioinformatics and genomics. On the other the laboratories of biomedical and developmental biology hand, the bHLH transcription factor families have (Bowes et al ., 2008; Hellsten et al ., 2010; Session et al ., been identified and analyzed in the currently available 2016; Elurbe et al ., 2017; Kamran et al ., 2018). Our group genomes of many metazoan species, such as human, previously identified some bHLH transcription factors orangutan, mouse, rat, giant panda, chicken, sparrow, both the genomes of Xenopus tropicalis and Xenopus pig, cow, dog, zebrafish, lizard, silkworm, bee and other laevis (Liu and Chen, 2013; Liu and Li, 2015), in which insects (Ledent and Vervoort, 2001; Ledent et al ., 2002; we used the predefined bHLH gene/protein Li et al ., 2006; Simionato et al ., 2007; Wang et al ., 2007; classification criteria (Atchley et al ., 1999; Atchley Wang et al ., 2008; Stevens et al ., 2008; Wang et al ., 2009; and Fitch, 1997) and the verified 45 representative Zheng et al ., 2009; Wang et al ., 2010; Liu and Zhao, bHLH proteins and 118 human bHLH protein motifs 2010; 2011; Dang et al ., 2011; Liu et al ., 2012; 2013; in search of novel bHLH sequence hints (Ledent and Liu and Chen, 2013; Wang et al ., 2015; Liu and Li, Vervoort, 2001; Ledent et al ., 2002; Simionato et al ., 2015; Liu, 2015; Li and Liu, 2017; Zhang et al ., 2017; 2007). In total, 105 bHLH proteins were identified Liu et al ., 2018; Murre, 2019). from the genomic databases of Xenopus tropicalis and On the other hand, the bioinformatics databases of 106 bHLH proteins were found from the genomic biological macromolecules and phenotypes and databases of Xenopus laevis (Liu and Chen, 2013; Liu genotypes of specific genes and/or traits, such as the and Li, 2015). All these bHLH proteins retrieved from annotations of Gene Ontology (GO) forum and the those clawed frogs' genomes are used in the biological pathway databases mainly including the preliminary classification and comparison of the frog pathway databases of Reactome and Kyoto bHLH transcription factors performed in the present Encyclopedia of Genes and Genomes (KEGG), have study. Furthermore, in view of the ongoing genome shown a leap forward development. These large sequencing projects of the clawed frogs' genomes, the bioinformatics databases have become an extremely unearthed new annotations and functional information important method and main analytical tool for and structural features of many bHLH proteins should studying the functional characteristics of genes and be identified