# Phylogeny, Functional Annotation, and Protein  Interaction Network Analyses of theXenopus tropicalis Basic Helix-Loop-Helix Transcription Factors

Authors: Wuyi Liu; Deyu Chen
Journal: BioMed Research International
 (2013)

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## Abstract

The previous survey identified 70 basic helix-loop-helix (bHLH) proteins, but it was proved to be incomplete, and the functional information and regulatory networks of frog bHLH transcription factors were not fully known. Therefore, we conducted an updated genome-wide survey in theXenopus tropicalis genome project databases and identified 105 bHLH sequences. Among the retrieved 105 sequences, phylogenetic analyses revealed that 103 bHLH proteins belonged to 43 families or subfamilies with 46, 26, 11, 3, 15, and 4 members in the corresponding supergroups. Next, gene ontology (GO) enrichment analyses showed 65 significant GO annotations of biological processes and molecular functions and KEGG pathways counted in frequency. To explore the functional pathways, regulatory gene networks, and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the genome annotation and pathway analysis using KOBAS, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified, and many hub proteins created a tight network or a functional module within the protein families.


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## Body

## 1. Introduction

Transcription factors are usually classified into different families based on their sequence of functional DNA-binding or protein-binding domains, which are highly conserved among many species and include many members mediating cell fate allocation during animal and plant development [1–11]. The expression and activity of basic helix-loop-helix (bHLH) transcription factors can be regulated in response to cell-cell signaling, leading to the transcription of specific sets of genes required for a cell to adopt particular fates. Due to their important functions found in various organisms, bHLH transcription factors have been the subject of many researches. The first report of bHLH transcription factors focused on the murine factors E12 and E47 [12]. Later, more and more bHLH proteins have been identified in living organisms. In 1997, Atchley and Fitch [1] proposed an organization for the classification of the bHLH proteins based on the phylogenetic analysis of the 122 bHLH domains combined with the presence or absence of another additional domain. Their analysis allowed for the defining of four different groups of bHLH protein families according to structural similarities [1]. This classification was performed using only the bHLH motif or domain, because the flanking regions for bHLH proteins are very divergent. Atchley and Fitch’s classification led to the postulation of four distinct groups based on amino acid patterns and E-box-binding specificity [1]. In 2002, Ledent et al. [4] defined 44 orthologous families or sub-families and 6 supergroups based on the DNA-binding activities of bHLH transcription factors after large-scale phylogenetic analyses. After the revision of Simionato et al. [6] in 2007, animal bHLH proteins are reclassified into 45 families. Among these 6 supergroups, members of groups A and B are common bHLH proteins [1, 3–6]. Group A proteins bind to CACCTG or CAGCTG, while group B proteins bind to CACGTG or CATGTTG. The consensus DNA binding sequences for these bHLH proteins form the typical E boxes (CANNTG). Group C proteins are complex molecules with one or two PAS domains following the bHLH domain, being inclined to bind the core sequence ACGTG or GCGTG. They are mainly responsible for the regulation of midline and tracheal development, circadian rhythms, and gene transcription in response to environmental toxins. Group D proteins correspond to bHLH proteins that are unable to bind to DNA due to lack of a basic domain. Both, group D and group F, are proteins that lack basic parts and act as antagonist partners of group A proteins in the heterodimers. Particularly, group F are a kind of COE proteins characterized by the presence of an additional COE domain involved in both dimerization and DNA binding. Group E proteins are another type of special transcription factors. They usually contain two additional domains named “Orange” and “WRPW” peptides in their carboxyl termini and they bind preferentially to sequences typical of N boxes (CACGCG or CACGAG). Generally speaking, all of the bHLH transcription factors share a common bHLH motif or domain of approximately 60 amino acids, which contains a basic region and two helices separated by a loop (HLH) region of variable length [3–5, 12]. The basic region is a DNA-binding domain, and the amphipathic α-helices of two bHLH proteins can interact with each other. The HLH domain promotes dimerization, and interaction between the helix regions of two different bHLH proteins leads to the formation of homodimeric or heterodimeric complexes, while the basic region of each partner recognizes and binds to a core hexanucleotide DNA sequence [2–4]. In a couple of reports [13, 14], Atchley et al. inferred a predictive motif for the bHLH domains based on 242 bHLH proteins, in which 19 conserved sites were found within the bHLH domains. It was found and proved that a sequence with no more than 9 mismatches could be a putative bHLH protein [15].Recently, in many organisms whose genomes have been released and are available, more and more bHLH proteins have been identified and bHLH transcription factor families have been analyzed due to their important and pivotal regulatory functions displayed in various organisms [3–25]. As well as Xenopus laevis the Xenopus tropicalis is a model organism for researches testing the developmental, behavioral, and neurological consequences of genetic variation [26–28]. The draft of Xenopus tropicalis genome assembly was submitted by American scientists at the Lawrence Berkeley National Laboratory in California [28], and the Xenopus tropicalis genome project is still underway. In previous work, the preliminary survey identified 70 bHLH transcription factors [16]. Recently, we found it was incomplete and the functional properties and regulatory networks of bHLH transcription factors were not fully analyzed. In this study, we used the criteria developed by Atchley et al. [13] and the 45 representative bHLH domains defined by Ledent et al. [4] and Simionato et al. [6] to do updated searches using BLAST search algorithms in the Xenopus tropicalis genomic database and identified 105 bHLH proteins. We next made large-scale phylogenetic analyses of the Xenopus tropicalis bHLH domains with the 118 human bHLH domains [6]; this allowed us to define the full set of bHLH orthologous genes and their related families. We further report the result of analyses of gene ontology (GO) annotations, functional pathways, and protein interaction networks based on the Xenopus tropicalis genomic databases.


## 2. Materials and Methods

### 2.1. BLAST Searches and Retrieval of bHLH Domains

At first, we followed the criteria developed by Atchley et al. [1, 13] to define a bHLH protein [13]. These searches initially yielded a few bHLH transcription factors (up to 20 protein sequences). The deduced predictive protein consensus motif of Atchley et al. [13] is “++X(3–6)E+XRX(3)αNX(2)ΦX(2)L+X(5–22)+X(2)KX(2)δLX(2)AδXYαX(2)L” where +=K, R; α=I, L, V; Φ=F, I, L; δ=I, V, T; E, R, K, A, and Y are as defined; X = any residue; X(i)=anyi residues; and X(i–j)=i to j of any residues. We also used the 45 representative bHLH domains from the tables provided by Ledent et al. [4] and Simionato et al. [6] to make multiple TBLASTN and BLASTP searches of bHLH domains against the Xenopus Genome Resources built by NCBI (http://www.ncbi.nlm.nih.gov/genome/guide/frog/) and Xenbase (http://www.xenbase.org/) for all putative bHLH proteins. Then, PSI-BLAST searches were conducted against the nonredundant database of Xenopus genomes at NCBI using the representative bHLH domain sequences. All of the TBLASTN, BLASTP, and PSI-BLAST searches were conducted with the methods and similar parameter setting-ups in the previous works [7, 16]. With these BLAST searches above, we obtained all of the putative bHLH proteins with no more than 9 mismatches among the 19 amino acids residues [15]. Moreover, we also did TBLASTN searches of frog EST data against the XenopusGenome EST databases with a stringency set as E≤0.0001 and an identity of 90% or higher as candidacy. The obtained EST data were translated into protein sequences using online analysis tools (http://www.genoscope.cns.fr/agc/tools/) to verify the putative bHLH sequences found.


### 2.2. Manual Improvement and Sequence Alignment

Protein sequence accession numbers and genomic contig numbers were finally obtained by BLASTP and TBLASTN searches against theXenopus tropicalis protein databases and genome sequence assembly (reference only) with the amino acid sequence of each identified bHLH domain. All of the obtained sequences were aligned using ClustalX 2.0 [29]. Redundant sequences of candidates were removed according to their corresponding serial numbers of the scaffold or clone or genomic contig, gene ID, protein ID, coding region, and alignment information. The, finaly, aligned bHLH domains were shaded using GeneDoc 2.6.02 [30] and copied into an RTF file for further annotation.


### 2.3. Analyses of Gene Ontology (GO) Annotations and Pathways

A functional annotation analysis ofXenopus tropicalis bHLH transcription factor genes was conducted. Gene ontology (GO) function enrichment was analyzed using DAVID Functional Annotation Bioinformatics Tools [31, 32], which use the ontology hierarchy tree and calculates and report statistical significance for GO term categories with a hypergeometric P value and enrichment scores. This approach directly scores predefined gene sets and/or pathways based on given gene lists.All of the bHLH transcription factor genes were also subjected to KOBAS analysis (http://kobas.cbi.pku.edu.cn/home.do), and significant pathways were retrieved at the default Pvalue≤0.5. We applied KOBAS vocabulary to first annotate all genes with corresponding KO and then identify both, the most frequent the statistically significantly enriched pathways. With rather strict cutoff of FDR ≤ 0.05, KOBAS found statistically significantly enriched pathways, as shown in Table 3.We could thus identify and select significantly enriched gene ontology terms and pathways using bioinformatics databases DAVID [31, 32] and KOBAS [33–35], respectively. We selected the functional categories that were more likely to be biologically meaningful by statistical significance of each functional category in the input set of genes versus all annotated genes in the Xenopus tropicalis genome.


### 2.4. Protein Interaction Network Analysis

To investigate possible interactions between the gene lists from our updated surveys, the STRING search tool was used for the creation of protein interaction network (PIN) files as previously described [36–38]. To increase the completeness of our results, this search was set to include full information extracted from the STRING biological interaction databases. The created networks were explored and compared based on their topological characteristics and gene products (proteins) by default with a confidence of score 0.15 [38].


### 2.5. Phylogenetic Analyses

The putativeXenopus tropicalis bHLH protein sequences, together with the human bHLH domains, were used to construct phylogenetic trees based on bayesian inference (BI) by MRBAYES 3.1.2 [37, 38] and maximum likelihood estimation (MLE) by PHYML 2.4.4 [44] with the JTT substitution frequency matrix [45], respectively. Phylogenetic analyses by BI and MLE were performed with the methods and similar parameter setting-ups in the previous works [7, 16]. Briefly, the BI analysis was performed with two independent Markov chains, each containing from 800 to 1100 million Monte Carlo steps until the standard deviation of split frequencies was below 0.01, with sample frequency saved every 1000 generations. Finally, all of the obtained trees were edited and displayed by means of the software package MEGA 4.0 [46].


## 2.1. BLAST Searches and Retrieval of bHLH Domains

At first, we followed the criteria developed by Atchley et al. [1, 13] to define a bHLH protein [13]. These searches initially yielded a few bHLH transcription factors (up to 20 protein sequences). The deduced predictive protein consensus motif of Atchley et al. [13] is “++X(3–6)E+XRX(3)αNX(2)ΦX(2)L+X(5–22)+X(2)KX(2)δLX(2)AδXYαX(2)L” where +=K, R; α=I, L, V; Φ=F, I, L; δ=I, V, T; E, R, K, A, and Y are as defined; X = any residue; X(i)=anyi residues; and X(i–j)=i to j of any residues. We also used the 45 representative bHLH domains from the tables provided by Ledent et al. [4] and Simionato et al. [6] to make multiple TBLASTN and BLASTP searches of bHLH domains against the Xenopus Genome Resources built by NCBI (http://www.ncbi.nlm.nih.gov/genome/guide/frog/) and Xenbase (http://www.xenbase.org/) for all putative bHLH proteins. Then, PSI-BLAST searches were conducted against the nonredundant database of Xenopus genomes at NCBI using the representative bHLH domain sequences. All of the TBLASTN, BLASTP, and PSI-BLAST searches were conducted with the methods and similar parameter setting-ups in the previous works [7, 16]. With these BLAST searches above, we obtained all of the putative bHLH proteins with no more than 9 mismatches among the 19 amino acids residues [15]. Moreover, we also did TBLASTN searches of frog EST data against the XenopusGenome EST databases with a stringency set as E≤0.0001 and an identity of 90% or higher as candidacy. The obtained EST data were translated into protein sequences using online analysis tools (http://www.genoscope.cns.fr/agc/tools/) to verify the putative bHLH sequences found.


## 2.2. Manual Improvement and Sequence Alignment

Protein sequence accession numbers and genomic contig numbers were finally obtained by BLASTP and TBLASTN searches against theXenopus tropicalis protein databases and genome sequence assembly (reference only) with the amino acid sequence of each identified bHLH domain. All of the obtained sequences were aligned using ClustalX 2.0 [29]. Redundant sequences of candidates were removed according to their corresponding serial numbers of the scaffold or clone or genomic contig, gene ID, protein ID, coding region, and alignment information. The, finaly, aligned bHLH domains were shaded using GeneDoc 2.6.02 [30] and copied into an RTF file for further annotation.


## 2.3. Analyses of Gene Ontology (GO) Annotations and Pathways

A functional annotation analysis ofXenopus tropicalis bHLH transcription factor genes was conducted. Gene ontology (GO) function enrichment was analyzed using DAVID Functional Annotation Bioinformatics Tools [31, 32], which use the ontology hierarchy tree and calculates and report statistical significance for GO term categories with a hypergeometric P value and enrichment scores. This approach directly scores predefined gene sets and/or pathways based on given gene lists.All of the bHLH transcription factor genes were also subjected to KOBAS analysis (http://kobas.cbi.pku.edu.cn/home.do), and significant pathways were retrieved at the default Pvalue≤0.5. We applied KOBAS vocabulary to first annotate all genes with corresponding KO and then identify both, the most frequent the statistically significantly enriched pathways. With rather strict cutoff of FDR ≤ 0.05, KOBAS found statistically significantly enriched pathways, as shown in Table 3.We could thus identify and select significantly enriched gene ontology terms and pathways using bioinformatics databases DAVID [31, 32] and KOBAS [33–35], respectively. We selected the functional categories that were more likely to be biologically meaningful by statistical significance of each functional category in the input set of genes versus all annotated genes in the Xenopus tropicalis genome.


## 2.4. Protein Interaction Network Analysis

To investigate possible interactions between the gene lists from our updated surveys, the STRING search tool was used for the creation of protein interaction network (PIN) files as previously described [36–38]. To increase the completeness of our results, this search was set to include full information extracted from the STRING biological interaction databases. The created networks were explored and compared based on their topological characteristics and gene products (proteins) by default with a confidence of score 0.15 [38].


## 2.5. Phylogenetic Analyses

The putativeXenopus tropicalis bHLH protein sequences, together with the human bHLH domains, were used to construct phylogenetic trees based on bayesian inference (BI) by MRBAYES 3.1.2 [37, 38] and maximum likelihood estimation (MLE) by PHYML 2.4.4 [44] with the JTT substitution frequency matrix [45], respectively. Phylogenetic analyses by BI and MLE were performed with the methods and similar parameter setting-ups in the previous works [7, 16]. Briefly, the BI analysis was performed with two independent Markov chains, each containing from 800 to 1100 million Monte Carlo steps until the standard deviation of split frequencies was below 0.01, with sample frequency saved every 1000 generations. Finally, all of the obtained trees were edited and displayed by means of the software package MEGA 4.0 [46].


## 3. Results and Discussion

### 3.1. Data Retrieval and Identification of bHLH Transcription Factors

The names and related information of the putativeXenopus tropicalis bHLH proteins are listed in Table 1. All of the bHLH domains obtained had more than 10 conserved amino acids [15]. The putative bHLH proteins were named according to their phylogenetic relationship with its corresponding human orthologs and paralogs. If a human bHLH sequence had two or more Xenopus tropicalis orthologous genes, we used “a,” “b,” and “c” or “1,” “2,” and “3” and so on, to number them. In the present work, 34 frog hypothetical and/or predicted proteins belonged to novel bHLH members and were reannotated in this study, that is, NP_001096226.2 (Genbank protein accession), NP_989390.1, NP_001096298.1, NP_001037951.1, NP_001107462.1, NP_001107508.1, NP_001120597.1, NP_001120597.1, XP_002931994.1, XP_002932187.1, XP_002933181.1, XP_002934026.1, XP_002934312.1, XP_002935013.1, XP_002935182.1, XP_002935886.1, XP_002935887.1, XP_002936042.1, XP_002937330.1, XP_002937913.1, XP_002938491.1, XP_002938497.1, XP_002938975.1, XP_002939165.1, XP_002940290.1, XP_002940370.1, XP_002941575.1, XP_002942929.1, XP_002943245.1, XP_002944430.1, XP_002944506.1, XP_002944648.1, XP_002944649.1, and XP_002939654.1.Table 1
Information of theXenopus tropicalis 105 bHLH transcription factors.
bHLHfamily
Gene name
Homo sapiens orthologous gene
Protein accessionc
Genome contigd
Name
MLE bootstrap value (%)a
BI posterior probability (%)b
ASCa
Xsash1
Hash1  (ASCL1)
89
99
XP_002944648.1
NW_003169609.1
ASCa
Xsash2
Hash2
n/m*
99
XP_002940290.1
NW_003163913.1
ASCb
Xsash3
Hash3  (ASCL3)
90
100
XP_002940370.1
NW_003163927.1
MyoD
Myf3
Myf3
96
94
NP_988972.1
NW_003166075.1
MyoD
Myf4
Myf4
94
100
NP_001016725.1
NW_003163495.1
MyoD
Myf5
Myf5
n/m
76
NP_988932.1
NW_003163331.1
MyoD
Myf6
Myf6
82
95
NP_001017160.1
NW_003163331.1
E12/E47
E2A
E2A
99
53
NP_001093743.1
NW_003163736.1
E12/E47
TCF3
TCF3
76
88
XP_002940299.1
NW_003163915.1
E12/E47
TCF4
TCF4
76
n/m*
NP_001096226.2
NW_003163423.1
Ngn
Xsath4c
Hath4c
83
78
NP_001116895.1
NW_003163503.1
NeuroD
NDF1  (neurod1)
NDF1  (NEUROD1)
n/m
n/m*
NP_001090868.1
NW_003163341.1
NeuroD
NDF2
NDF2  (NEUROD2)
65
63
NP_001072486.1
NW_003163936.1
NeuroD
Xsath2
Hath2
79
80
NP_001072273.1
NW_003163914.1
NeuroD
Xsath3
Hath3
97
99
NP_001124513.1
NW_003163487.1
Mist1
Mist1
Mist1
99
100
XP_002931994.1
NW_003163340.1
Beta3
Beta3a
Beta3a
70
53
XP_002944506.1
NW_003167409.1
Beta3
Beta3b
Beta3b
77
94
NP_001072933.1
NW_003163515.1
Oligo
Oligo1
Oligo1
97
100
XP_002938497.1
NW_003163700.1
Oligo
Oligo2
Oligo2
76
73
XP_002938491.1
NW_003163700.1
Oligo
Oligo3
Oligo3
83
90
NP_001008191.1
NW_003163713.1
Oligo
Oligo4
Oligo1  Oligo2
Oligo3
n/m
n/m
NP_001039180.1
NW_003163795.1
Net
Xsath6
Hath6
100
100
XP_002937330.1
NW_003163606.1
Mesp
Mesp1
Mesp1   Mesp2
pMesp1
n/m
n/m
NP_001039184.1
NW_003163348.1
Mesp
Mesp2
Mesp1   Mesp2
pMesp1
n/m
n/m
NP_001016653.1
NW_003163348.1
Mesp
pMespo
pMesp1
99
100
NP_001039104.1
NW_003163426.1
Twist
Twist1
Twist1
91
83
NP_989415.1
NW_003163378.1
Twist
Twist2
Twist2
98
100
NP_001096679.1
NW_003163487.1
Paraxis
Paraxis
Paraxis
62
83
NP_001016506.1
NW_003165117.1
Paraxis
Sclerax1
Sclerax
96
99
XP_002942929.1
NW_003164455.1
Paraxis
Sclerax2
Sclerax
74
59
XP_002937913.1
NW_003163647.1
MyoRa
MyoRa1
MyoRa1
63
60
NP_001096235.1
NW_003163586.1
MyoRa
MyoRa2
MyoRa2
n/m
62
NP_001103518.1
NW_003163498.1
MyoRb
MyoRb1
MyoRb1
78
94
GNOMON∣93674.pe  (ab initio protein)
NW_003164157.1
MyoRb
MyoRb2
MyoRb2
55
95
GNOMON∣522504.pe  (ab initio protein)
NW_003163470.1
Hand
Hand1
Hand1
94
100
NP_001016743.1
NW_003163350.1
Hand
Hand2
Hand2
99
55
NP_001093695.1
NW_003163380.1
PTFa
PTFa
PTFa
99
100
NP_001095279.1
NW_003163378.1
PTFb
PTFb
PTFb
91
100
XP_002933181.1
NW_003163373.1
SCL
Tal1
Tal1
77
62
NP_001135468.1
NW_003163327.1
SCL
Tal2
Tal2
72
76
XP_002934026.1
NW_003163404.1
SCL
Lyl1
Lyl1
86
97
XP_002939165.1
NW_003163774.1
NSCL
NSCL1
NSCL1
99
100
XP_002937307.1
NW_003163605.1
SRC
SRC1
SRC1
82
97
NP_001106383.1
NW_003163796.1
SRC
SRC2
SRC2
97
100
NP_001135631.1
NW_003163586.1
SRC
SRC3
SRC3
80
97
XP_002933204.1
NW_003163374.1
Figα
Figα
Figα
92
100
NP_001016342.1
NW_003163469.1
MYC
l-Myc
L-Myc
71
65
NP_001011144.1
NW_003164143.1
MYC
n-Myc
n-Myc
n/m
98
NP_989390.1
NW_003163721.1
MYC
v-Myc
v-Myc
91
99
NP_001006874.1
NW_003163866.1
Mad
Mxi1
Mxi1
85
97
NP_001008129
NW_003180496.1NW_003163820.1
Mad
Mad1
Mad1
n/m
88
NP_001072228.1
NW_003163469.1
Mad
Mad3
Mad3
99
100
NP_001017299.1
NW_003163577.1
Mad
Mad4
Mad4
89
100
NP_001096239.1
NW_003164437.1
Mnt
Mnt
Mnt
n/m
97
NP_001135494.1
NW_003163468.1
MAX
MAX
MAX
90
100
NP_001008208.1
NW_003163599.1
USF
USF1
USF1
92
99
NP_001096236.1
NW_003168160.1
USF
USF2
USF2
n/m
60
NP_001007857.1
NW_003163677.1
USF
USF3
USF3
85
99
NP_001120597.1
NW_003164188.1
MITF
MITF
MITF
n/m
n/m
NP_001093747.1
NW_003163951.1
MITF
TFEb
TFEb
84
100
NP_001072648.1
NW_003163367.1
MITF
TFEc
TFEc
66
99
XP_002935013.1
NW_003163447.1
MITF
TFE3
TFE3
85
78
XP_002944430.1
NW_003166883.1
SREBP
SREBP1a
SREBP1
88
99
XP_002935886.1
NW_003163500.1
SREBP
SREBP1b
SREBP1
88
99
XP_002935887.1
NW_003163500.1
SREBP
SREBP1c
SREBP1
88
99
XP_002944649.1
NW_003169615.1NW_003163500.1
SREBP
SREBP2
SREBP2
n/m
67
NP_001116910.1
NW_003163395.1
AP4
AP4
AP4
71
98
NP_001123841.1
NW_003163353.1
Mlx
MondoA
MondoA
89
100
NP_001090682.1
NW_003163637.1
TF4
TF4
TF4
88
100
GNOMON:712044.pe  (ab initio protein)
NW_003164277.1,NW_003164157.1
Clock
Clock
Clock
99
100
NP_001122127.1
NW_003163433.1
ARNT
ARNT1
ARNT1
n/m
n/m
NP_001116925.1
NW_003163477.1
ARNT
ARNT2
ARNT2
100
n/m
NP_001093686.1
NW_003163348.1
Bmal
Bmal2
Bmal2
63
100
NP_001096298.1
NW_003164805.1
AHR
AHR1
AHR1
92
99
XP_002933348.1
NW_003163378.1
AHR
AHR2
AHR2
91
100
XP_002935182.1
NW_003163457.1
Sim
Sim1
Sim1
n/m*
98
XP_002932187.1
NW_003163345.1
Sim
Sim2
Sim2
89
99
XP_002941575.1
NW_003164120.1
Trh
NPAS3
NPAS3
n/m
70
NP_001072647.1
NW_003163363.1
HIF
Hif1α
Hif1α
99
n/m
NP_001011165.1
NW_003163817.1
HIF
EPAS1
EPAS1
79
94
NP_001005647.1
NW_003163351.1
Emc
Id2
Id2
78
90
NP_988885.1
NW_003163451.1
Emc
Id3
Id3
79
98
NP_001016271.1
NW_003163432.1
Emc
Id4
Id4
86
54
NP_001004839.1
NW_003163385.1
Hey
Herp1
Herp1
83
97
NP_001007911.1
NW_003163551.1
Hey
Herp2
Herp2
86
92
XP_002936042.1
NW_003163507.1
Hey
HEYL
HEYL
98
100
XP_002934312.1
NW_003163416.1
H/E(spl)
Dec2
Dec2
99
n/m
NP_001027504.1
NW_003163993.1
H/E(spl)
Hes1a
Hes1
n/m
81
NP_001011194.1
NW_003163571.1
H/E(spl)
Hes1b
Hes1
n/m
81
NP_988870.1
NW_003163533.1
H/E(spl)
Hes5a
Hes5
n/m*
61
NP_001037880.1
NW_003163546.1
H/E(spl)
Hes5b
Hes5
n/m*
61
NP_001037974.1
NW_003163546.1
H/E(spl)
Hes5c
Hes5
n/m*
100
NP_001039178.1
NW_003163399.1
H/E(spl)
Hes5d
Hes5
n/m*
100
NP_001037951.1
NW_003163399.1
H/E(spl)
Hes5e
Hes5
n/m*
82
NP_001107462.1
No finding
H/E(spl)
Esr9
Hes5
n/m*
100
NP_001037989.1
NW_003163399.1
H/E(spl)
Hes6
Hes6
n/m
n/m
NP_001072210.1
NW_003163381.1
H/E(spl)
Hes7a
Hes7
73
97
NP_001039166.1
NW_003164377.1
H/E(spl)
Hes7b
Hes7
86
100
NP_001107508.1
NW_003164377.1
Coe
EBF1
EBF1
n/m
51
XP_002939654.1
NW_003163834.1
Coe
EBF2
EBF2
91
97
NP_989200.1
NW_003163356.1
Coe
EBF3
EBF3
91
66
XP_002932694.1
NW_003163358.1
Coe
EBF4
EBF4
91
66
XP_002932695.1
NW_003163358.1
Orphan
Orphan1
Orphan1
86
100
XP_002938975.1
NW_003163749.1
Orphan
Orphan4
Orphan4
94
100
XP_002943245.1
NW_003164609.1
Xenopus tropicalis bHLH genes were named according to their human orthologous genes’ names (or common abbreviations) and the referred nomenclature was mainly from the tables and additional tables provided by Ledent et al. [4] and Simionato et al. [6]. Bootstrap values were converted from phylogenetic analyses with human bHLH sequences using BI and MLE algorithm, respectively. MLE bootstrap valuea refers to the result from maximum likelihood estimate in phylogenetic analysis, and BI posterior probabilityb refers to the result from BI in phylogenetic analysis. The numbers in the phylogenetic trees are converted into percentages. cThe accession numbers were retrieved from the following resources; this sequence was verified by many EST TBLASTN search hits, such as EG651417.1 and CX503003.2 (EST accession number). These numbered as “NP” were from the RefSeq protein database and those numbered as “XP” were from the Build protein database. Notes in the brackets are also gene symbols according to records in NCBI and Xenbase. All of the bHLH genes are organized in the order of bHLH families manifested in Table 1 of Ledent et al. [4]. The question mark means no matching; mark n/m* means no monophyletic group with single particular orthologous gene sequences, but formed a monophyletic group with two or more orthologous gene sequences of the family; mark n/m denotes the case of lower bootstrap value estimated less than 50%.eThe accession numbers were retrieved from the ab initio protein database.In total, 105 putativeXenopus tropicalis bHLH protein sequences were identified with the BLASTP, TBLASTN, and PSI-BLAST searches and manual examination of the 19 conserved amino acid sites (Table 1, Figure 1). Among these putative bHLH protein sequences, most of these hypothetical proteins were newly produced in the Xenopus tropicalis genome project. We further identified and verified these hypothetical proteins with corresponding EST sequences obtained by TBLASTN searches against the expressed sequence database (data not shown).Figure 1
Alignment of 105Xenopus tropicalis bHLH domains. Designation of basic, helix 1, loop, and helix 2 follows Ferre-D’Amare et al. [39–43] and bHLH domains were shaded using GeneDoc. Family and bHLH protein names and high-order groups were organized according to Table 1 in the paper of Ledent et al. [4]. Highly conserved sites are shaded in black and indicated with asterisks on the top.In summary, two proteins identified belonging to none of these groups were classified as “orphans,” while the other 103 bHLH members belonged to 43 families with 46, 26, 11, 3, 15, and 4 bHLH members in the corresponding high-order groups A, B, C, D, E, and F, respectively. Figure1 showed the domain alignment of 105 Xenopus tropicalis bHLH proteins. In addition, the members of Delilah and Mist families were not found in this research.


### 3.2. Phylogenetic Analyses and Identification of Putative bHLH Proteins

Phylogenetic trees of MLE and BI showed the diversity of the frog bHLH transcription factor family. All of the data of phylogenetic trees forXenopus tropicalis bHLH proteins are available upon request. The topologies of these two inference methods agreed well with each other (Table 1). It was found that both human and frog proteomes have a number of lineage-specific bHLH families and their members. For example, in the Xenopus tropicalis proteomes, no orthologous genes for human TF12, Hath1, Hath4a,Hath4b, Hath5, andId1 could be found in the present research. However, the Xenopus tropicalis proteomes also have multiple orthologous genes corresponding to one human gene, such as SREBP1a, SREBP1b, andSREBP1c (orthologous genes of human SREBP1); Hes1a and Hes1b (orthologous genes of human Hes1); Hes6a and Hes6b (orthologous genes of human Hes6); Hes5a, Hes5b, Hes5c, Hes5d, Hes5e, andEsr9 (orthologous genes of human Hes5).


### 3.3. Enriched Functional GO Annotations

Gene ontology (GO) annotations including biological process (BP), molecular function (MF), and cellular component (CC) were downloaded and investigated from the gene ontology database (http://www.geneontology.org/), and the genes were grouped according to their GO hierarchy annotations. To explore functional properties and identify groups of genes coding for proteins with similar function or with participation in common regulatory pathways, all of the retrieved putative bHLH genes were grouped and functionally classified and enriched according to available GO annotations, information from curated pathways, and known protein interactions. In the present work, the 105 frog bHLH genes were grouped into 7 supergroups according to Ledent et al. [4] and Simionato et al. [6] to get available GO annotations and their enrichment by categories (cutoff of P≤0.05). With gene accessions, protein accessions, and the other eligible sequence information in DAVID Bioinformatics Database [32] for Xenopus tropicalis bHLH transcription factors, we retrieved all of the significant GO annotations (cutoff of P≤0.05). There were 96 genes fitting the record of DAVID Bioinformatics Database [31, 32] and these genes obtained significant GO annotations, while the other nine genes did not get significant GO annotation and were discarded (mainly group D, F, and Orphans; Table 2).Table 2
GO enrichment by categories of super-groups by DAVID bioinformatics bases with 105Xenopus tropicalis bHLH transcription factors.
Group
Enriched genes
GO term ID
GO category
GO definition
Coherence (%)a
P value
A
43
GO:0030528
MF
Transcription regulator activity
100
2.50
E
-
27
GO:0045449
BP
Regulation of transcription
100
7.60
E
-
22
GO:0007517
BP
Muscle organ development
15.4
5.50
E
-
05
GO:0007519
BP
Skeletal muscle tissue development
7.7
1.30
E
-
02
GO:0055123
BP
Digestive system development
7.7
1.30
E
-
02
GO:0014706
BP
Striated muscle tissue development
7.7
1.30
E
-
02
GO:0043282
BP
Pharyngeal muscle development
7.7
1.30
E
-
02
GO:0002074
BP
Extraocular skeletal muscle development
7.7
1.30
E
-
02
GO:0048741
BP
Skeletal muscle fiber development
7.7
1.30
E
-
02
GO:0048747
BP
Muscle fiber development
7.7
1.30
E
-
02
GO:0060538
BP
Skeletal muscle organ development
7.7
1.30
E
-
02
GO:0060465
BP
Pharynx development
7.7
1.30
E
-
02
GO:0007423
BP
Sensory organ development
11.5
1.70
E
-
02
GO:0042692
BP
Muscle cell differentiation
7.7
2.00
E
-
02
GO:0060537
BP
Muscle tissue development
7.7
2.00
E
-
02
GO:0051146
BP
Striated muscle cell differentiation
7.7
2.00
E
-
02
GO:0055002
BP
Striated muscle cell development
7.7
2.00
E
-
02
GO:0055001
BP
Muscle cell development
7.7
2.00
E
-
02
GO:0003677
MF
DNA binding
26.9
8.70
E
-
02
B
267
GO:0030528
MF
Transcription regulator activity
100
8.10
E
-
20
GO:0045449
BP
Regulation of transcription
100
6.90
E
-
16
GO:0035257
MF
Nuclear hormone receptor binding
10.5
1.20
E
-
02
GO:0051427
MF
Hormone receptor binding
10.5
1.60
E
-
02
GO:0003713
MF
Transcription coactivator activity
10.5
2.00
E
-
02
GO:0003712
MF
Transcription cofactor activity
10.5
3.10
E
-
02
GO:0008134
MF
Transcription factor binding
10.5
6.90
E
-
02
GO:0006355
BP
Regulation of transcription, DNA-dependent
26.3
9.70
E
-
02
C
11
GO:0006350
BP
Transcription
100
1.40
E
-
07
GO:0030528
MF
Transcription regulator activity
100
4.70
E
-
07
GO:0006355
BP
Regulation of transcription, DNA-dependent
100
9.50
E
-
07
GO:0051252
BP
Regulation of RNA metabolic process
100
1.00
E
-
06
GO:0003677
MF
DNA binding
100
4.10
E
-
06
GO:0045449
BP
Regulation of transcription
100
9.30
E
-
06
GO:0003700
MF
Transcription factor activity
71.4
1.70
E
-
04
KEGG_Id:480089074
KEGG pathway
Circadian rhythm
28.6
3.90
E
-
03
D
3
None
None
None
None
None
E
15
GO:0030528
MF
Transcription regulator activity
100
1.30
E
-
16
GO:0045449
BP
Regulation of transcription
100
2.40
E
-
13
GO:0006350
BP
Transcription
68.8
7.90
E
-
09
GO:0003677
MF
DNA binding
75
1.10
E
-
07
GO:0006355
BP
Regulation of transcription, DNA-dependent
68.8
1.70
E
-
07
GO:0051252
BP
Regulation of RNA metabolic process
68.8
1.90
E
-
07
GO:0016564
MF
Transcription repressor activity
31.2
2.90
E
-
07
GO:0000122
BP
Negative regulation of transcription from RNA polymerase II promoter
25
1.80
E
-
05
GO:0009792
BP
Embryonic development ending in birth or egg hatching
25
7.50
E
-
05
GO:0043009
BP
Chordate embryonic development
25
7.50
E
-
05
GO:0045892
BP
Negative regulation of transcription, DNA-dependent
25
1.60
E
-
04
GO:0051253
BP
Negative regulation of RNA metabolic process
25
1.80
E
-
04
GO:0046982
MF
Protein heterodimerization activity
18.8
2.10
E
-
04
GO:0021915
BP
Neural tube development
18.8
2.20
E
-
04
GO:0016481
BP
Negative regulation of transcription
25
2.80
E
-
04
GO:0007219
BP
Notch signaling pathway
18.8
3.10
E
-
04
GO:0051172
BP
Negative regulation of nitrogen compound metabolic process
25
3.70
E
-
04
GO:0045934
BP
Negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
25
3.70
E
-
04
GO:0031327
BP
Negative regulation of cellular biosynthetic process
25
4.60
E
-
04
GO:0010558
BP
Negative regulation of macromolecule biosynthetic process
25
4.60
E
-
04
GO:0009890
BP
Negative regulation of biosynthetic process
25
5.00
E
-
04
GO:0010629
BP
Negative regulation of gene expression
25
5.70
E
-
04
GO:0006357
BP
Regulation of transcription from RNA polymerase II promoter
25
8.90
E
-
04
GO:0010605
BP
Negative regulation of macromolecule metabolic process
25
9.50
E
-
04
KEGG_Id:480089058
KEGG pathway
TGF-beta signaling pathway
18.8
2.40
E
-
03
GO:0033504
BP
Floor plate development
12.5
7.90
E
-
03
GO:0046983
MF
Protein dimerization activity
18.8
9.20
E
-
03
GO:0048635
BP
Negative regulation of muscle development
12.5
1.20
E
-
02
GO:0048634
BP
Regulation of muscle development
12.5
1.60
E
-
02
KEGG_Id:480089056
KEGG pathway
Notch signaling pathway
12.5
4.60
E
-
02
F
4
None
None
None
None
None
Orphan
2
None
None
None
None
None
All GO annotations terms in the table were from gene ontology database (http://www.geneontology.org/). GO annotations included every layer of biological process, molecular function, cellular component category, and KEGG pathway. When a GO term and its sublayer GO are both enriched in a group significantly, only deeper layer GO annotation is shown in the table. BP: biological process; MF: molecular function. The above table showed the GO annotations enriched significantly (P<0.05) in each group. aGO coherence of each group, measured as the percentage of genes in group covered by the GO category.Table 3
Significant pathways identified by KOBAS with 93Xenopus tropicalis bHLH transcription factors.
Term
Pathway database
Database ID
Sample gene number
Background number
P value
CorrectedP value
Genes
Circadian rhythm: mammal
KEGG
xtr04710
3
21
1.10
E
-
05
0.0001219
XSBmal2; XSDec2; XSClock
TGF-beta signaling pathway
KEGG
xtr04350
4
73
1.52
E
-
05
0.0001219
XSId3; XSId2; XSId4; XSnMyc
Notch signaling pathway
PANTHER
P00045
2
5
0.0004516
0.0024084
XSHes1a; XSHes1b; XSHerp1
Notch signaling pathway
KEGG
xtr04330
2
43
0.0037667
0.0150668
XSHes1a; XSHes1b; XSHes5a
Developmental biology
Reactome
None
2
106
0.0145401
0.0398896
XSHes1a; XSHes1b; XSNDF1; XSNDF2
Circadian clock
Reactome
None
1
8
0.0149586
0.0398896
XSClock
Herpes simplex infection
KEGG
xtr05168
2
128
0.0304156
0.0695215
XSBmal2; XSClock
MAPK signaling pathway
KEGG
xtr04010
2
220
0.0802242
0.1604484
XSMAX; XSnMyc
Fanconi anemia pathway
KEGG
xtr03460
1
51
0.1044307
0.1856547
XSHes1a; XSHes1b
ErbB signaling pathway
KEGG
xtr04012
1
70
0.1406256
0.2250009
XSnMyc
Melanogenesis
KEGG
xtr04916
1
86
0.1700261
0.2386888
XSMITF
Jak-STAT signaling pathway
KEGG
xtr04630
1
91
0.1790166
0.2386888
XSnMyc
Metabolism
Reactome
None
2
458
0.206835
0.2544026
XSSRC2; XSSRC1
Cell cycle
KEGG
xtr04110
1
116
0.2226023
0.2544026
XSnMyc
Wnt signaling pathway
KEGG
xtr04310
1
131
0.2476916
0.2642044
XSnMyc
Wnt signaling pathway
PANTHER
P00057
1
37
0.2756772
0.2756772
XSvMycAmong the genes, more than half were annotated as exhibiting “transcription regulator activity” and/or “regulation of transcription” or similar terms related to DNA-dependent regulation of transcription, DNA binding, or regulation of RNA metabolic process in the BP and MF categories. There were three significant KEEG pathways, that is, circadian rhythm (KEGG ID: 480089074,P value 0.0039), TGF-beta signaling pathway (KEGG Id: 480089058, P value 0.0024), and Notch signaling pathway (KEGG Id: 480089056, P value 0.046), and few significant GO terms for bHLH genes identified in the CC category for Xenopus tropicalis bHLH proteins in DAVID Bioinformatics Database [32]. In the BP category, a total of 47.83% of the significant GO annotations were annotated as transcription and transcription (factor) activity and/or regulation of transcription, while 28.26% of GO annotations were connected to muscle cell development or differentiation and 26.09% of GO annotations were related to negative regulation of cellular biosynthetic or macromolecular metabolic processes. Several genes in the BP category were associated with neural tube development, floor plate development, sensory organ development, chordate embryonic development, hormone receptor binding, and so forth. In the MF category, 56.25% of GO annotations were connected to transcription factor binding or transcription regulator activity, while 3 out of 16 of the GO annotations were related to DNA binding.DNA binding, protein dimerization, and transcription coactivator activity are important functional activities of bHLH domains. The DNA binding activity of bHLH proteins is mainly determined by the basic region [2]. Site-directed mutagenesis experiments and the crystal structure studies of bHLH proteins showed that the Glu-9/Arg-12 pair forms the CANNTG recognition motif, the critical Glu-9 contacts the first CA in the DNA-binding motif, and the role of Arg-12 is to fix and stabilize the position of the Glu-9 [35–38, 47]. To further understand the functions of Xenopus tropicalis bHLH genes as a whole, we collected GO enrichment data on the 105 Xenopus tropicalis bHLH genes with significant hypergeometric P values. Among all of the GO terms, 65 significant GO terms (P≤0.05) were identified showing key cellular components, molecular functions, biological processes, and KEGG pathways for the 105 Xenopus tropicalis bHLH genes (Table 2). Muscle organ development, embryonic development ending in birth or egg hatching, chordate embryonic development, sensory organ development, neural tube development, camera-type eye development and eye development, floor plate development, and muscle fiber and tissue development have high frequencies when taking no account of the frequent GO term categories of transcriptional factors such as (negative) regulation of transcription and regulation of metabolism and biosynthetic processes. It has been well known that the bHLH genes in various groups have special recognition motifs of DNA-binding sites such as E-box and G-box. So, how about the gene functions of each group? To explore these issues, we calculated the hypergeometric distribution enrichment score of gene molecular functions from group A to group F based on GO annotations of GO term categories including biological process, molecular function, cellular component, KEGG pathways, and other key words. However, only significant enriched annotations (cut off P≤0.05) in deeper layers (sublayers) are shown in Table 2. GO statistics analyzed with a brief summary of subtypes describing each subgroup are also listed in Table 2.Our analysis focused on significant GO terms for all of the wholeXenopus tropicalis bHLH gene family and for each subgroup (Table 2). We found that each subgroup (except for D and F with few members identified) of bHLH transcription factors has its own specific GO term categories (Table 2), when common GO terms of transcription such as transcription regulator activity, regulation of transcription, and DNA binding and protein dimerization activity are discounted. Group A is characterized with muscle organ development such as (striated) muscle cell differentiation and development, (skeletal) muscle fiber development, (extraocular) skeletal muscle tissue development, and striated muscle and pharyngeal muscle development. In addition, digestive system development, pharynx development, and sensory organ development are also included in this group (Table 2). The functions of bHLH members of group B and group C are mainly composed of transcription, transcription regulator activity, and regulation of transcription. However, group B is different from group C with some GO terms such as transcription coactivator activity, transcription cofactor activity, and (nuclear) hormone receptor binding (Table 2). Group E is composed of some functionally diversified transcription regulators whose GO terms are enriched in many aspects of transcription, such as transcription regulator activity, (negative) regulation of transcription, (negative) regulation of RNA metabolic process, (negative) regulation of transcription from RNA polymerase II promoter, (negative) regulation of nucleobase, nucleoside, and nucleotide and nucleic acid metabolic process, (negative) regulation of biosynthetic process, DNA binding, and protein heterodimerization activity. There are some special GO terms in group E, such as chordate embryonic development, floor plate development, neural tube development, anterior/posterior pattern formation, and (negative) regulation of muscle development (Table 2). KEGG terms, like TGF-beta signaling pathway and Notch signaling pathway, also provide key annotations and insights for bHLH members in group E.


### 3.4. Pathways Analysis

We could identify and select significantly enriched gene ontology terms and pathways using DAVID [31, 32] and KOBAS [33–35] in the present study. We selected functional categories that were more likely to be biologically meaningful by calculating the statistical significance of each functional category in the input set of genes versus all annotated genes in theXenopus tropicalis genome. After the GO annotations of Xenopus tropicalis bHLH transcription factors with the DAVID Bioinformatics Tools, all of the bHLH transcription factor genes were also subjected to KOBAS analysis (http://kobas.cbi.pku.edu.cn/home.do) and significant pathways were retrieved at the default P values. We applied KOBAS to first annotate all of the genes with KO and to then identify both the most frequent and the statistically significantly enriched pathways. With the strict cutoff of FDR≤0.05, KOBAS found statistically significantly enriched pathways in public databases, such as KEEG, Reactome, and PANTHER, as shown in Table 3. Using this threshold, we identified 16 pathways as induced in the Xenopus tropicalis genomic gene samples (Table 3). Among these pathways, 11 pathways were from KEEG database, while six pathways were at the significant level of P≤0.05. Interestingly, four of the main central cell signaling systems, that is, Notch signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, and MAPK signaling pathway, were identified. There were two most significant components related to Notch signaling pathway (corrected P value 0.0024084 and 0.0150668) and circadian clock and/or circadian rhythm regulation (corrected P value 0.0001219 and 0.0398896), respectively. The Jak-STAT signaling pathway, which is regarded as one of the central cell signaling systemS for muscle development, was identified too. It was the same case that many bHLH proteins were enriched in TGF-beta signaling pathway and Notch signaling pathway as annotated using DAVID Bioinformatics Resources. Furthermore, many interesting pathways were also identified as significantly, such as ErbB signaling pathway, Fanconi anemia pathway, and herpes simplex infection.


### 3.5. Protein Interaction Network

To identify putative functional units that consist of proteins coded by the differentially expressed genes, direct and indirect interactions between these proteins were derived using the STRING search tool, which creates PIN files based on previously reported interactions between proteins. Based on 93 bHLH proteins and their 10 predicted functional partners (CARM1, INSIG2, MEF2C, VHL, INSIG1, MGC75596, NOTCH1, DLL1, and SCAP; relevant coefficient ≥ 0.967) inXenopus tropicalis genomic databases, large PIN files were derived and investigated for the presence of hub proteins defined as proteins with at least five interactions to other proteins (Figure 2). Altogether, 68 hub proteins were identified (i.e., MESPA, MESPB, MSGN1, EBF2, NEUROD6, NEUROD4, NEUROD2, NEUROD1, NEUROG1, NEUROG3, NHLH1, TCF21, TCF12, TCF4, TFEB, TFE3, HES4, HES5.1, HES7.1, HES1, DLL1, SIM1, SIM2, ID2, ID3, ID4, MYF6, MYF5, MYOG, MYOD1, NOTCH1, OLIG2, OLIG3, OLIG4, LYL1, HEY1, HEY2, TWIST1, HAND1, HAND2, MEF2C, MGC75596, MLX, MXI1, MAX, LMYC1, MNT, MYC, PTF1A, TAL1, MSC, TAL2, ATOH1, ATOH7, ARNT, ARNT2, AHR1, BHLHE40, BHLHE41, HIF1A, VHL, CLOCK, EPAS1, CARM1, NCOA1, NCOA2, NCOA3, and SREBF2; it should be noted that there are some aliases of bHLH proteins existing in the public databases). Among all proteins in the STRING databases, those were core-connected and had higher expression in many experimental data in the regulatory interaction network (Figure 2).Figure 2
STRING mapping profiles of protein interaction network (PIN) representing bHLH transcription factor protein interactions. Panel (a) showed the main figure of PIN profile and connectivity of hub proteins and the others. The protein interacting gene products are marked in blue and green lines. There are totally 68 hub proteins identified and many hub proteins created a tight network or a functional module within their protein families. Panel (b) magnified the implication of different connective lines with different data sources in the main figure.Interestingly, many hub proteins created a tight network or a functional module within their protein families, such as NEUROD6, NEUROD4, NEUROD2, NEUROD1, NEUROG1, NEUROG3, HES4, HES5.1, HES7.1, HES1, ID2, ID3, ID4, MYF6, MYF5, MYOG, MYOD1, MLX, MXI1, MAX, MITF, and MNT, which are all involved in the same or similar cellular machinery components and/or genetic functions (Figure2).


## 3.1. Data Retrieval and Identification of bHLH Transcription Factors

The names and related information of the putativeXenopus tropicalis bHLH proteins are listed in Table 1. All of the bHLH domains obtained had more than 10 conserved amino acids [15]. The putative bHLH proteins were named according to their phylogenetic relationship with its corresponding human orthologs and paralogs. If a human bHLH sequence had two or more Xenopus tropicalis orthologous genes, we used “a,” “b,” and “c” or “1,” “2,” and “3” and so on, to number them. In the present work, 34 frog hypothetical and/or predicted proteins belonged to novel bHLH members and were reannotated in this study, that is, NP_001096226.2 (Genbank protein accession), NP_989390.1, NP_001096298.1, NP_001037951.1, NP_001107462.1, NP_001107508.1, NP_001120597.1, NP_001120597.1, XP_002931994.1, XP_002932187.1, XP_002933181.1, XP_002934026.1, XP_002934312.1, XP_002935013.1, XP_002935182.1, XP_002935886.1, XP_002935887.1, XP_002936042.1, XP_002937330.1, XP_002937913.1, XP_002938491.1, XP_002938497.1, XP_002938975.1, XP_002939165.1, XP_002940290.1, XP_002940370.1, XP_002941575.1, XP_002942929.1, XP_002943245.1, XP_002944430.1, XP_002944506.1, XP_002944648.1, XP_002944649.1, and XP_002939654.1.Table 1
Information of theXenopus tropicalis 105 bHLH transcription factors.
bHLHfamily
Gene name
Homo sapiens orthologous gene
Protein accessionc
Genome contigd
Name
MLE bootstrap value (%)a
BI posterior probability (%)b
ASCa
Xsash1
Hash1  (ASCL1)
89
99
XP_002944648.1
NW_003169609.1
ASCa
Xsash2
Hash2
n/m*
99
XP_002940290.1
NW_003163913.1
ASCb
Xsash3
Hash3  (ASCL3)
90
100
XP_002940370.1
NW_003163927.1
MyoD
Myf3
Myf3
96
94
NP_988972.1
NW_003166075.1
MyoD
Myf4
Myf4
94
100
NP_001016725.1
NW_003163495.1
MyoD
Myf5
Myf5
n/m
76
NP_988932.1
NW_003163331.1
MyoD
Myf6
Myf6
82
95
NP_001017160.1
NW_003163331.1
E12/E47
E2A
E2A
99
53
NP_001093743.1
NW_003163736.1
E12/E47
TCF3
TCF3
76
88
XP_002940299.1
NW_003163915.1
E12/E47
TCF4
TCF4
76
n/m*
NP_001096226.2
NW_003163423.1
Ngn
Xsath4c
Hath4c
83
78
NP_001116895.1
NW_003163503.1
NeuroD
NDF1  (neurod1)
NDF1  (NEUROD1)
n/m
n/m*
NP_001090868.1
NW_003163341.1
NeuroD
NDF2
NDF2  (NEUROD2)
65
63
NP_001072486.1
NW_003163936.1
NeuroD
Xsath2
Hath2
79
80
NP_001072273.1
NW_003163914.1
NeuroD
Xsath3
Hath3
97
99
NP_001124513.1
NW_003163487.1
Mist1
Mist1
Mist1
99
100
XP_002931994.1
NW_003163340.1
Beta3
Beta3a
Beta3a
70
53
XP_002944506.1
NW_003167409.1
Beta3
Beta3b
Beta3b
77
94
NP_001072933.1
NW_003163515.1
Oligo
Oligo1
Oligo1
97
100
XP_002938497.1
NW_003163700.1
Oligo
Oligo2
Oligo2
76
73
XP_002938491.1
NW_003163700.1
Oligo
Oligo3
Oligo3
83
90
NP_001008191.1
NW_003163713.1
Oligo
Oligo4
Oligo1  Oligo2
Oligo3
n/m
n/m
NP_001039180.1
NW_003163795.1
Net
Xsath6
Hath6
100
100
XP_002937330.1
NW_003163606.1
Mesp
Mesp1
Mesp1   Mesp2
pMesp1
n/m
n/m
NP_001039184.1
NW_003163348.1
Mesp
Mesp2
Mesp1   Mesp2
pMesp1
n/m
n/m
NP_001016653.1
NW_003163348.1
Mesp
pMespo
pMesp1
99
100
NP_001039104.1
NW_003163426.1
Twist
Twist1
Twist1
91
83
NP_989415.1
NW_003163378.1
Twist
Twist2
Twist2
98
100
NP_001096679.1
NW_003163487.1
Paraxis
Paraxis
Paraxis
62
83
NP_001016506.1
NW_003165117.1
Paraxis
Sclerax1
Sclerax
96
99
XP_002942929.1
NW_003164455.1
Paraxis
Sclerax2
Sclerax
74
59
XP_002937913.1
NW_003163647.1
MyoRa
MyoRa1
MyoRa1
63
60
NP_001096235.1
NW_003163586.1
MyoRa
MyoRa2
MyoRa2
n/m
62
NP_001103518.1
NW_003163498.1
MyoRb
MyoRb1
MyoRb1
78
94
GNOMON∣93674.pe  (ab initio protein)
NW_003164157.1
MyoRb
MyoRb2
MyoRb2
55
95
GNOMON∣522504.pe  (ab initio protein)
NW_003163470.1
Hand
Hand1
Hand1
94
100
NP_001016743.1
NW_003163350.1
Hand
Hand2
Hand2
99
55
NP_001093695.1
NW_003163380.1
PTFa
PTFa
PTFa
99
100
NP_001095279.1
NW_003163378.1
PTFb
PTFb
PTFb
91
100
XP_002933181.1
NW_003163373.1
SCL
Tal1
Tal1
77
62
NP_001135468.1
NW_003163327.1
SCL
Tal2
Tal2
72
76
XP_002934026.1
NW_003163404.1
SCL
Lyl1
Lyl1
86
97
XP_002939165.1
NW_003163774.1
NSCL
NSCL1
NSCL1
99
100
XP_002937307.1
NW_003163605.1
SRC
SRC1
SRC1
82
97
NP_001106383.1
NW_003163796.1
SRC
SRC2
SRC2
97
100
NP_001135631.1
NW_003163586.1
SRC
SRC3
SRC3
80
97
XP_002933204.1
NW_003163374.1
Figα
Figα
Figα
92
100
NP_001016342.1
NW_003163469.1
MYC
l-Myc
L-Myc
71
65
NP_001011144.1
NW_003164143.1
MYC
n-Myc
n-Myc
n/m
98
NP_989390.1
NW_003163721.1
MYC
v-Myc
v-Myc
91
99
NP_001006874.1
NW_003163866.1
Mad
Mxi1
Mxi1
85
97
NP_001008129
NW_003180496.1NW_003163820.1
Mad
Mad1
Mad1
n/m
88
NP_001072228.1
NW_003163469.1
Mad
Mad3
Mad3
99
100
NP_001017299.1
NW_003163577.1
Mad
Mad4
Mad4
89
100
NP_001096239.1
NW_003164437.1
Mnt
Mnt
Mnt
n/m
97
NP_001135494.1
NW_003163468.1
MAX
MAX
MAX
90
100
NP_001008208.1
NW_003163599.1
USF
USF1
USF1
92
99
NP_001096236.1
NW_003168160.1
USF
USF2
USF2
n/m
60
NP_001007857.1
NW_003163677.1
USF
USF3
USF3
85
99
NP_001120597.1
NW_003164188.1
MITF
MITF
MITF
n/m
n/m
NP_001093747.1
NW_003163951.1
MITF
TFEb
TFEb
84
100
NP_001072648.1
NW_003163367.1
MITF
TFEc
TFEc
66
99
XP_002935013.1
NW_003163447.1
MITF
TFE3
TFE3
85
78
XP_002944430.1
NW_003166883.1
SREBP
SREBP1a
SREBP1
88
99
XP_002935886.1
NW_003163500.1
SREBP
SREBP1b
SREBP1
88
99
XP_002935887.1
NW_003163500.1
SREBP
SREBP1c
SREBP1
88
99
XP_002944649.1
NW_003169615.1NW_003163500.1
SREBP
SREBP2
SREBP2
n/m
67
NP_001116910.1
NW_003163395.1
AP4
AP4
AP4
71
98
NP_001123841.1
NW_003163353.1
Mlx
MondoA
MondoA
89
100
NP_001090682.1
NW_003163637.1
TF4
TF4
TF4
88
100
GNOMON:712044.pe  (ab initio protein)
NW_003164277.1,NW_003164157.1
Clock
Clock
Clock
99
100
NP_001122127.1
NW_003163433.1
ARNT
ARNT1
ARNT1
n/m
n/m
NP_001116925.1
NW_003163477.1
ARNT
ARNT2
ARNT2
100
n/m
NP_001093686.1
NW_003163348.1
Bmal
Bmal2
Bmal2
63
100
NP_001096298.1
NW_003164805.1
AHR
AHR1
AHR1
92
99
XP_002933348.1
NW_003163378.1
AHR
AHR2
AHR2
91
100
XP_002935182.1
NW_003163457.1
Sim
Sim1
Sim1
n/m*
98
XP_002932187.1
NW_003163345.1
Sim
Sim2
Sim2
89
99
XP_002941575.1
NW_003164120.1
Trh
NPAS3
NPAS3
n/m
70
NP_001072647.1
NW_003163363.1
HIF
Hif1α
Hif1α
99
n/m
NP_001011165.1
NW_003163817.1
HIF
EPAS1
EPAS1
79
94
NP_001005647.1
NW_003163351.1
Emc
Id2
Id2
78
90
NP_988885.1
NW_003163451.1
Emc
Id3
Id3
79
98
NP_001016271.1
NW_003163432.1
Emc
Id4
Id4
86
54
NP_001004839.1
NW_003163385.1
Hey
Herp1
Herp1
83
97
NP_001007911.1
NW_003163551.1
Hey
Herp2
Herp2
86
92
XP_002936042.1
NW_003163507.1
Hey
HEYL
HEYL
98
100
XP_002934312.1
NW_003163416.1
H/E(spl)
Dec2
Dec2
99
n/m
NP_001027504.1
NW_003163993.1
H/E(spl)
Hes1a
Hes1
n/m
81
NP_001011194.1
NW_003163571.1
H/E(spl)
Hes1b
Hes1
n/m
81
NP_988870.1
NW_003163533.1
H/E(spl)
Hes5a
Hes5
n/m*
61
NP_001037880.1
NW_003163546.1
H/E(spl)
Hes5b
Hes5
n/m*
61
NP_001037974.1
NW_003163546.1
H/E(spl)
Hes5c
Hes5
n/m*
100
NP_001039178.1
NW_003163399.1
H/E(spl)
Hes5d
Hes5
n/m*
100
NP_001037951.1
NW_003163399.1
H/E(spl)
Hes5e
Hes5
n/m*
82
NP_001107462.1
No finding
H/E(spl)
Esr9
Hes5
n/m*
100
NP_001037989.1
NW_003163399.1
H/E(spl)
Hes6
Hes6
n/m
n/m
NP_001072210.1
NW_003163381.1
H/E(spl)
Hes7a
Hes7
73
97
NP_001039166.1
NW_003164377.1
H/E(spl)
Hes7b
Hes7
86
100
NP_001107508.1
NW_003164377.1
Coe
EBF1
EBF1
n/m
51
XP_002939654.1
NW_003163834.1
Coe
EBF2
EBF2
91
97
NP_989200.1
NW_003163356.1
Coe
EBF3
EBF3
91
66
XP_002932694.1
NW_003163358.1
Coe
EBF4
EBF4
91
66
XP_002932695.1
NW_003163358.1
Orphan
Orphan1
Orphan1
86
100
XP_002938975.1
NW_003163749.1
Orphan
Orphan4
Orphan4
94
100
XP_002943245.1
NW_003164609.1
Xenopus tropicalis bHLH genes were named according to their human orthologous genes’ names (or common abbreviations) and the referred nomenclature was mainly from the tables and additional tables provided by Ledent et al. [4] and Simionato et al. [6]. Bootstrap values were converted from phylogenetic analyses with human bHLH sequences using BI and MLE algorithm, respectively. MLE bootstrap valuea refers to the result from maximum likelihood estimate in phylogenetic analysis, and BI posterior probabilityb refers to the result from BI in phylogenetic analysis. The numbers in the phylogenetic trees are converted into percentages. cThe accession numbers were retrieved from the following resources; this sequence was verified by many EST TBLASTN search hits, such as EG651417.1 and CX503003.2 (EST accession number). These numbered as “NP” were from the RefSeq protein database and those numbered as “XP” were from the Build protein database. Notes in the brackets are also gene symbols according to records in NCBI and Xenbase. All of the bHLH genes are organized in the order of bHLH families manifested in Table 1 of Ledent et al. [4]. The question mark means no matching; mark n/m* means no monophyletic group with single particular orthologous gene sequences, but formed a monophyletic group with two or more orthologous gene sequences of the family; mark n/m denotes the case of lower bootstrap value estimated less than 50%.eThe accession numbers were retrieved from the ab initio protein database.In total, 105 putativeXenopus tropicalis bHLH protein sequences were identified with the BLASTP, TBLASTN, and PSI-BLAST searches and manual examination of the 19 conserved amino acid sites (Table 1, Figure 1). Among these putative bHLH protein sequences, most of these hypothetical proteins were newly produced in the Xenopus tropicalis genome project. We further identified and verified these hypothetical proteins with corresponding EST sequences obtained by TBLASTN searches against the expressed sequence database (data not shown).Figure 1
Alignment of 105Xenopus tropicalis bHLH domains. Designation of basic, helix 1, loop, and helix 2 follows Ferre-D’Amare et al. [39–43] and bHLH domains were shaded using GeneDoc. Family and bHLH protein names and high-order groups were organized according to Table 1 in the paper of Ledent et al. [4]. Highly conserved sites are shaded in black and indicated with asterisks on the top.In summary, two proteins identified belonging to none of these groups were classified as “orphans,” while the other 103 bHLH members belonged to 43 families with 46, 26, 11, 3, 15, and 4 bHLH members in the corresponding high-order groups A, B, C, D, E, and F, respectively. Figure1 showed the domain alignment of 105 Xenopus tropicalis bHLH proteins. In addition, the members of Delilah and Mist families were not found in this research.


## 3.2. Phylogenetic Analyses and Identification of Putative bHLH Proteins

Phylogenetic trees of MLE and BI showed the diversity of the frog bHLH transcription factor family. All of the data of phylogenetic trees forXenopus tropicalis bHLH proteins are available upon request. The topologies of these two inference methods agreed well with each other (Table 1). It was found that both human and frog proteomes have a number of lineage-specific bHLH families and their members. For example, in the Xenopus tropicalis proteomes, no orthologous genes for human TF12, Hath1, Hath4a,Hath4b, Hath5, andId1 could be found in the present research. However, the Xenopus tropicalis proteomes also have multiple orthologous genes corresponding to one human gene, such as SREBP1a, SREBP1b, andSREBP1c (orthologous genes of human SREBP1); Hes1a and Hes1b (orthologous genes of human Hes1); Hes6a and Hes6b (orthologous genes of human Hes6); Hes5a, Hes5b, Hes5c, Hes5d, Hes5e, andEsr9 (orthologous genes of human Hes5).


## 3.3. Enriched Functional GO Annotations

Gene ontology (GO) annotations including biological process (BP), molecular function (MF), and cellular component (CC) were downloaded and investigated from the gene ontology database (http://www.geneontology.org/), and the genes were grouped according to their GO hierarchy annotations. To explore functional properties and identify groups of genes coding for proteins with similar function or with participation in common regulatory pathways, all of the retrieved putative bHLH genes were grouped and functionally classified and enriched according to available GO annotations, information from curated pathways, and known protein interactions. In the present work, the 105 frog bHLH genes were grouped into 7 supergroups according to Ledent et al. [4] and Simionato et al. [6] to get available GO annotations and their enrichment by categories (cutoff of P≤0.05). With gene accessions, protein accessions, and the other eligible sequence information in DAVID Bioinformatics Database [32] for Xenopus tropicalis bHLH transcription factors, we retrieved all of the significant GO annotations (cutoff of P≤0.05). There were 96 genes fitting the record of DAVID Bioinformatics Database [31, 32] and these genes obtained significant GO annotations, while the other nine genes did not get significant GO annotation and were discarded (mainly group D, F, and Orphans; Table 2).Table 2
GO enrichment by categories of super-groups by DAVID bioinformatics bases with 105Xenopus tropicalis bHLH transcription factors.
Group
Enriched genes
GO term ID
GO category
GO definition
Coherence (%)a
P value
A
43
GO:0030528
MF
Transcription regulator activity
100
2.50
E
-
27
GO:0045449
BP
Regulation of transcription
100
7.60
E
-
22
GO:0007517
BP
Muscle organ development
15.4
5.50
E
-
05
GO:0007519
BP
Skeletal muscle tissue development
7.7
1.30
E
-
02
GO:0055123
BP
Digestive system development
7.7
1.30
E
-
02
GO:0014706
BP
Striated muscle tissue development
7.7
1.30
E
-
02
GO:0043282
BP
Pharyngeal muscle development
7.7
1.30
E
-
02
GO:0002074
BP
Extraocular skeletal muscle development
7.7
1.30
E
-
02
GO:0048741
BP
Skeletal muscle fiber development
7.7
1.30
E
-
02
GO:0048747
BP
Muscle fiber development
7.7
1.30
E
-
02
GO:0060538
BP
Skeletal muscle organ development
7.7
1.30
E
-
02
GO:0060465
BP
Pharynx development
7.7
1.30
E
-
02
GO:0007423
BP
Sensory organ development
11.5
1.70
E
-
02
GO:0042692
BP
Muscle cell differentiation
7.7
2.00
E
-
02
GO:0060537
BP
Muscle tissue development
7.7
2.00
E
-
02
GO:0051146
BP
Striated muscle cell differentiation
7.7
2.00
E
-
02
GO:0055002
BP
Striated muscle cell development
7.7
2.00
E
-
02
GO:0055001
BP
Muscle cell development
7.7
2.00
E
-
02
GO:0003677
MF
DNA binding
26.9
8.70
E
-
02
B
267
GO:0030528
MF
Transcription regulator activity
100
8.10
E
-
20
GO:0045449
BP
Regulation of transcription
100
6.90
E
-
16
GO:0035257
MF
Nuclear hormone receptor binding
10.5
1.20
E
-
02
GO:0051427
MF
Hormone receptor binding
10.5
1.60
E
-
02
GO:0003713
MF
Transcription coactivator activity
10.5
2.00
E
-
02
GO:0003712
MF
Transcription cofactor activity
10.5
3.10
E
-
02
GO:0008134
MF
Transcription factor binding
10.5
6.90
E
-
02
GO:0006355
BP
Regulation of transcription, DNA-dependent
26.3
9.70
E
-
02
C
11
GO:0006350
BP
Transcription
100
1.40
E
-
07
GO:0030528
MF
Transcription regulator activity
100
4.70
E
-
07
GO:0006355
BP
Regulation of transcription, DNA-dependent
100
9.50
E
-
07
GO:0051252
BP
Regulation of RNA metabolic process
100
1.00
E
-
06
GO:0003677
MF
DNA binding
100
4.10
E
-
06
GO:0045449
BP
Regulation of transcription
100
9.30
E
-
06
GO:0003700
MF
Transcription factor activity
71.4
1.70
E
-
04
KEGG_Id:480089074
KEGG pathway
Circadian rhythm
28.6
3.90
E
-
03
D
3
None
None
None
None
None
E
15
GO:0030528
MF
Transcription regulator activity
100
1.30
E
-
16
GO:0045449
BP
Regulation of transcription
100
2.40
E
-
13
GO:0006350
BP
Transcription
68.8
7.90
E
-
09
GO:0003677
MF
DNA binding
75
1.10
E
-
07
GO:0006355
BP
Regulation of transcription, DNA-dependent
68.8
1.70
E
-
07
GO:0051252
BP
Regulation of RNA metabolic process
68.8
1.90
E
-
07
GO:0016564
MF
Transcription repressor activity
31.2
2.90
E
-
07
GO:0000122
BP
Negative regulation of transcription from RNA polymerase II promoter
25
1.80
E
-
05
GO:0009792
BP
Embryonic development ending in birth or egg hatching
25
7.50
E
-
05
GO:0043009
BP
Chordate embryonic development
25
7.50
E
-
05
GO:0045892
BP
Negative regulation of transcription, DNA-dependent
25
1.60
E
-
04
GO:0051253
BP
Negative regulation of RNA metabolic process
25
1.80
E
-
04
GO:0046982
MF
Protein heterodimerization activity
18.8
2.10
E
-
04
GO:0021915
BP
Neural tube development
18.8
2.20
E
-
04
GO:0016481
BP
Negative regulation of transcription
25
2.80
E
-
04
GO:0007219
BP
Notch signaling pathway
18.8
3.10
E
-
04
GO:0051172
BP
Negative regulation of nitrogen compound metabolic process
25
3.70
E
-
04
GO:0045934
BP
Negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
25
3.70
E
-
04
GO:0031327
BP
Negative regulation of cellular biosynthetic process
25
4.60
E
-
04
GO:0010558
BP
Negative regulation of macromolecule biosynthetic process
25
4.60
E
-
04
GO:0009890
BP
Negative regulation of biosynthetic process
25
5.00
E
-
04
GO:0010629
BP
Negative regulation of gene expression
25
5.70
E
-
04
GO:0006357
BP
Regulation of transcription from RNA polymerase II promoter
25
8.90
E
-
04
GO:0010605
BP
Negative regulation of macromolecule metabolic process
25
9.50
E
-
04
KEGG_Id:480089058
KEGG pathway
TGF-beta signaling pathway
18.8
2.40
E
-
03
GO:0033504
BP
Floor plate development
12.5
7.90
E
-
03
GO:0046983
MF
Protein dimerization activity
18.8
9.20
E
-
03
GO:0048635
BP
Negative regulation of muscle development
12.5
1.20
E
-
02
GO:0048634
BP
Regulation of muscle development
12.5
1.60
E
-
02
KEGG_Id:480089056
KEGG pathway
Notch signaling pathway
12.5
4.60
E
-
02
F
4
None
None
None
None
None
Orphan
2
None
None
None
None
None
All GO annotations terms in the table were from gene ontology database (http://www.geneontology.org/). GO annotations included every layer of biological process, molecular function, cellular component category, and KEGG pathway. When a GO term and its sublayer GO are both enriched in a group significantly, only deeper layer GO annotation is shown in the table. BP: biological process; MF: molecular function. The above table showed the GO annotations enriched significantly (P<0.05) in each group. aGO coherence of each group, measured as the percentage of genes in group covered by the GO category.Table 3
Significant pathways identified by KOBAS with 93Xenopus tropicalis bHLH transcription factors.
Term
Pathway database
Database ID
Sample gene number
Background number
P value
CorrectedP value
Genes
Circadian rhythm: mammal
KEGG
xtr04710
3
21
1.10
E
-
05
0.0001219
XSBmal2; XSDec2; XSClock
TGF-beta signaling pathway
KEGG
xtr04350
4
73
1.52
E
-
05
0.0001219
XSId3; XSId2; XSId4; XSnMyc
Notch signaling pathway
PANTHER
P00045
2
5
0.0004516
0.0024084
XSHes1a; XSHes1b; XSHerp1
Notch signaling pathway
KEGG
xtr04330
2
43
0.0037667
0.0150668
XSHes1a; XSHes1b; XSHes5a
Developmental biology
Reactome
None
2
106
0.0145401
0.0398896
XSHes1a; XSHes1b; XSNDF1; XSNDF2
Circadian clock
Reactome
None
1
8
0.0149586
0.0398896
XSClock
Herpes simplex infection
KEGG
xtr05168
2
128
0.0304156
0.0695215
XSBmal2; XSClock
MAPK signaling pathway
KEGG
xtr04010
2
220
0.0802242
0.1604484
XSMAX; XSnMyc
Fanconi anemia pathway
KEGG
xtr03460
1
51
0.1044307
0.1856547
XSHes1a; XSHes1b
ErbB signaling pathway
KEGG
xtr04012
1
70
0.1406256
0.2250009
XSnMyc
Melanogenesis
KEGG
xtr04916
1
86
0.1700261
0.2386888
XSMITF
Jak-STAT signaling pathway
KEGG
xtr04630
1
91
0.1790166
0.2386888
XSnMyc
Metabolism
Reactome
None
2
458
0.206835
0.2544026
XSSRC2; XSSRC1
Cell cycle
KEGG
xtr04110
1
116
0.2226023
0.2544026
XSnMyc
Wnt signaling pathway
KEGG
xtr04310
1
131
0.2476916
0.2642044
XSnMyc
Wnt signaling pathway
PANTHER
P00057
1
37
0.2756772
0.2756772
XSvMycAmong the genes, more than half were annotated as exhibiting “transcription regulator activity” and/or “regulation of transcription” or similar terms related to DNA-dependent regulation of transcription, DNA binding, or regulation of RNA metabolic process in the BP and MF categories. There were three significant KEEG pathways, that is, circadian rhythm (KEGG ID: 480089074,P value 0.0039), TGF-beta signaling pathway (KEGG Id: 480089058, P value 0.0024), and Notch signaling pathway (KEGG Id: 480089056, P value 0.046), and few significant GO terms for bHLH genes identified in the CC category for Xenopus tropicalis bHLH proteins in DAVID Bioinformatics Database [32]. In the BP category, a total of 47.83% of the significant GO annotations were annotated as transcription and transcription (factor) activity and/or regulation of transcription, while 28.26% of GO annotations were connected to muscle cell development or differentiation and 26.09% of GO annotations were related to negative regulation of cellular biosynthetic or macromolecular metabolic processes. Several genes in the BP category were associated with neural tube development, floor plate development, sensory organ development, chordate embryonic development, hormone receptor binding, and so forth. In the MF category, 56.25% of GO annotations were connected to transcription factor binding or transcription regulator activity, while 3 out of 16 of the GO annotations were related to DNA binding.DNA binding, protein dimerization, and transcription coactivator activity are important functional activities of bHLH domains. The DNA binding activity of bHLH proteins is mainly determined by the basic region [2]. Site-directed mutagenesis experiments and the crystal structure studies of bHLH proteins showed that the Glu-9/Arg-12 pair forms the CANNTG recognition motif, the critical Glu-9 contacts the first CA in the DNA-binding motif, and the role of Arg-12 is to fix and stabilize the position of the Glu-9 [35–38, 47]. To further understand the functions of Xenopus tropicalis bHLH genes as a whole, we collected GO enrichment data on the 105 Xenopus tropicalis bHLH genes with significant hypergeometric P values. Among all of the GO terms, 65 significant GO terms (P≤0.05) were identified showing key cellular components, molecular functions, biological processes, and KEGG pathways for the 105 Xenopus tropicalis bHLH genes (Table 2). Muscle organ development, embryonic development ending in birth or egg hatching, chordate embryonic development, sensory organ development, neural tube development, camera-type eye development and eye development, floor plate development, and muscle fiber and tissue development have high frequencies when taking no account of the frequent GO term categories of transcriptional factors such as (negative) regulation of transcription and regulation of metabolism and biosynthetic processes. It has been well known that the bHLH genes in various groups have special recognition motifs of DNA-binding sites such as E-box and G-box. So, how about the gene functions of each group? To explore these issues, we calculated the hypergeometric distribution enrichment score of gene molecular functions from group A to group F based on GO annotations of GO term categories including biological process, molecular function, cellular component, KEGG pathways, and other key words. However, only significant enriched annotations (cut off P≤0.05) in deeper layers (sublayers) are shown in Table 2. GO statistics analyzed with a brief summary of subtypes describing each subgroup are also listed in Table 2.Our analysis focused on significant GO terms for all of the wholeXenopus tropicalis bHLH gene family and for each subgroup (Table 2). We found that each subgroup (except for D and F with few members identified) of bHLH transcription factors has its own specific GO term categories (Table 2), when common GO terms of transcription such as transcription regulator activity, regulation of transcription, and DNA binding and protein dimerization activity are discounted. Group A is characterized with muscle organ development such as (striated) muscle cell differentiation and development, (skeletal) muscle fiber development, (extraocular) skeletal muscle tissue development, and striated muscle and pharyngeal muscle development. In addition, digestive system development, pharynx development, and sensory organ development are also included in this group (Table 2). The functions of bHLH members of group B and group C are mainly composed of transcription, transcription regulator activity, and regulation of transcription. However, group B is different from group C with some GO terms such as transcription coactivator activity, transcription cofactor activity, and (nuclear) hormone receptor binding (Table 2). Group E is composed of some functionally diversified transcription regulators whose GO terms are enriched in many aspects of transcription, such as transcription regulator activity, (negative) regulation of transcription, (negative) regulation of RNA metabolic process, (negative) regulation of transcription from RNA polymerase II promoter, (negative) regulation of nucleobase, nucleoside, and nucleotide and nucleic acid metabolic process, (negative) regulation of biosynthetic process, DNA binding, and protein heterodimerization activity. There are some special GO terms in group E, such as chordate embryonic development, floor plate development, neural tube development, anterior/posterior pattern formation, and (negative) regulation of muscle development (Table 2). KEGG terms, like TGF-beta signaling pathway and Notch signaling pathway, also provide key annotations and insights for bHLH members in group E.


## 3.4. Pathways Analysis

We could identify and select significantly enriched gene ontology terms and pathways using DAVID [31, 32] and KOBAS [33–35] in the present study. We selected functional categories that were more likely to be biologically meaningful by calculating the statistical significance of each functional category in the input set of genes versus all annotated genes in theXenopus tropicalis genome. After the GO annotations of Xenopus tropicalis bHLH transcription factors with the DAVID Bioinformatics Tools, all of the bHLH transcription factor genes were also subjected to KOBAS analysis (http://kobas.cbi.pku.edu.cn/home.do) and significant pathways were retrieved at the default P values. We applied KOBAS to first annotate all of the genes with KO and to then identify both the most frequent and the statistically significantly enriched pathways. With the strict cutoff of FDR≤0.05, KOBAS found statistically significantly enriched pathways in public databases, such as KEEG, Reactome, and PANTHER, as shown in Table 3. Using this threshold, we identified 16 pathways as induced in the Xenopus tropicalis genomic gene samples (Table 3). Among these pathways, 11 pathways were from KEEG database, while six pathways were at the significant level of P≤0.05. Interestingly, four of the main central cell signaling systems, that is, Notch signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, and MAPK signaling pathway, were identified. There were two most significant components related to Notch signaling pathway (corrected P value 0.0024084 and 0.0150668) and circadian clock and/or circadian rhythm regulation (corrected P value 0.0001219 and 0.0398896), respectively. The Jak-STAT signaling pathway, which is regarded as one of the central cell signaling systemS for muscle development, was identified too. It was the same case that many bHLH proteins were enriched in TGF-beta signaling pathway and Notch signaling pathway as annotated using DAVID Bioinformatics Resources. Furthermore, many interesting pathways were also identified as significantly, such as ErbB signaling pathway, Fanconi anemia pathway, and herpes simplex infection.


## 3.5. Protein Interaction Network

To identify putative functional units that consist of proteins coded by the differentially expressed genes, direct and indirect interactions between these proteins were derived using the STRING search tool, which creates PIN files based on previously reported interactions between proteins. Based on 93 bHLH proteins and their 10 predicted functional partners (CARM1, INSIG2, MEF2C, VHL, INSIG1, MGC75596, NOTCH1, DLL1, and SCAP; relevant coefficient ≥ 0.967) inXenopus tropicalis genomic databases, large PIN files were derived and investigated for the presence of hub proteins defined as proteins with at least five interactions to other proteins (Figure 2). Altogether, 68 hub proteins were identified (i.e., MESPA, MESPB, MSGN1, EBF2, NEUROD6, NEUROD4, NEUROD2, NEUROD1, NEUROG1, NEUROG3, NHLH1, TCF21, TCF12, TCF4, TFEB, TFE3, HES4, HES5.1, HES7.1, HES1, DLL1, SIM1, SIM2, ID2, ID3, ID4, MYF6, MYF5, MYOG, MYOD1, NOTCH1, OLIG2, OLIG3, OLIG4, LYL1, HEY1, HEY2, TWIST1, HAND1, HAND2, MEF2C, MGC75596, MLX, MXI1, MAX, LMYC1, MNT, MYC, PTF1A, TAL1, MSC, TAL2, ATOH1, ATOH7, ARNT, ARNT2, AHR1, BHLHE40, BHLHE41, HIF1A, VHL, CLOCK, EPAS1, CARM1, NCOA1, NCOA2, NCOA3, and SREBF2; it should be noted that there are some aliases of bHLH proteins existing in the public databases). Among all proteins in the STRING databases, those were core-connected and had higher expression in many experimental data in the regulatory interaction network (Figure 2).Figure 2
STRING mapping profiles of protein interaction network (PIN) representing bHLH transcription factor protein interactions. Panel (a) showed the main figure of PIN profile and connectivity of hub proteins and the others. The protein interacting gene products are marked in blue and green lines. There are totally 68 hub proteins identified and many hub proteins created a tight network or a functional module within their protein families. Panel (b) magnified the implication of different connective lines with different data sources in the main figure.Interestingly, many hub proteins created a tight network or a functional module within their protein families, such as NEUROD6, NEUROD4, NEUROD2, NEUROD1, NEUROG1, NEUROG3, HES4, HES5.1, HES7.1, HES1, ID2, ID3, ID4, MYF6, MYF5, MYOG, MYOD1, MLX, MXI1, MAX, MITF, and MNT, which are all involved in the same or similar cellular machinery components and/or genetic functions (Figure2).


## 4. Concluding Remarks

In this research, we have identified 105 bHLH domains and their protein sequences in theXenopus tropicalis genome databases by TBLASTN, BLASTP, and PSI-BLAST searches with the 45 representative bHLH domains as query sequences. Among these bHLH members, 34 hypothetical proteins, such as LOC100124777, were newly annotated by computational analysis and verified by EST searching in this research. These uncharacterized putative bHLH proteins may be novel transcription factors, which need further validation. The prediction of Xenopus tropicalis bHLH transcriptional factors will be very useful for the experiment identifying novel bHLH transcription factors and the construction of transcriptional regulatory network of Xenopus tropicalis. Through phylogenetic analyses of the Xenopus tropicalis bHLH protein domains with human bHLH orthologous protein sequences, we assigned the 105 Xenopus tropicalis bHLH genes to 43 families and two orphan genes according to the 45 defined bHLH families [3, 11]. Two families, for example, Mist and Delilah, were not found in the study.Further analysis of theXenopus tropicalis bHLH transcription factors and their functional properties showed that 96 out of 105 bHLH genes could be annotated and only four supergroups’ GO enrichment by categories were available [4]. GO enrichment statistics showed 65 significant GO annotations of biological processes and molecular functions counted in frequency. Besides common GO term categories of bHLH transcriptional factors, a large number of Xenopus tropicalis bHLH genes play significant role in muscle and organ development, chordate and neural development, floor plate and eye development, and so forth [39–43, 48–55]. Moreover, as the group analysis results described, different groups of proteins have their special gene functions when taking no account of the common GO term categories. The trends of the gene function enrichment may be led by their DNA-binding specificity [51–55]. Therefore, the biology function of the uncharacterized genes or proteins can be predicted through the function GO annotation of the group analysis. To explore the functional pathways, regulatory gene networks and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the KOBAS genomic annotation and pathway analysis, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified and many hub proteins created a tight network or a functional module within their protein families.The present research deepens our knowledge of frog bHLH transcription factors and provides a solid framework for further research on the functional and evolutionary aspects ofXenopus tropicalis bHLH transcription factors.



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*Source: 145037-2013-11-10.xml*