A genome-wide survey on basic helix-loop-helix transcription factors in giant panda.

The giant panda (Ailuropoda melanoleuca) is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into "orphan". Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6), EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.

Introduction

The basic helix-loop-helix (bHLH) proteins form a large superfamily of transcription factors that play crucial roles in a wide range of developmental processes including neurogenesis, myogenesis, hematopoiesis, sex determination and gut development. The bHLH domain is approximately 60 amino acids long and comprises a DNA-binding basic region (b) and two helices separated by a variable loop region (HLH) [1]. The HLH domain promotes dimerization, allowing the formation of homodimeric or heterodimeric complexes between different bHLH family members. The two basic domains which are brought together through dimerization bind specific hexanucleotide sequences.

In the past two decades, protein functions of animal bHLH family members have been well characterized mainly through studies on bHLH proteins in model organisms including the nematode (Caenorhabditis elegans), fruit fly (Drosophila melanogaster) and mouse (Mus musculus). It has been established that animal bHLHs are classified into 45 families based on their different functions in the regulation of gene expression. In addition, they are divided into 6 groups according to target DNA elements they bind and their own structural characteristics. Specifically, group A consists of 22 families. They mainly regulate neurogenesis, myogenesis and mesoderm formation. Group B consists of 12 families. They mainly regulate cell proliferation and differentiation, sterol metabolism and adipocyte formation, and expression of glucose-responsive genes. Group C has 7 families. They are responsible for the regulation of midline and tracheal development, circadian rhythms, and for the activation of gene transcription in response to environmental toxins. Group D has only 1 family. It forms inactive heterodimers with group A bHLH proteins. Group E has 2 families, which regulate embryonic segmentation, somitogenesis and organogenesis etc. Group F also has 1 family. It regulates head development and formation of olfactory sensory neurons etc (reviewed in [2]).

With the completion of genome sequencing projects for an increased number of organisms, bHLH family members have been identified in genomes of over 20 organisms. These include 8 bHLH genes in Saccharomyces cerevisiae, 16 in Amphimedon queenslandica, 33 in Hydra magnipapillata, 42 in Caenorhabditis elegans, 46 in Ciona intestinalis, 50 in Strongylocentrotus purpuratus, 51 in Apis mellifera, 52 in Bombyx mori, 57 in Daphia pulex, 59 in Drosophila melanogaster, 63 in Lottia gigantea, 64 in Capitella sp 1, 68 in Nematodtella vectensis, 78 in Branchiostoma floridae, 87 in Tetraodon nigroviridis, 104 in Gallus gallus, 114 in Mus musculus, 114 in Rattus norvegicus, 118 in Homo sapiens, 139 in Brachydanio rerio, 147 in Arabidopsis, and 167 in Oryza sativa [3]–[12].

The giant panda, Ailuropoda melanoleuca, is a critically endangered mammal confined in six isolated mountain ranges of South-western China [13]. As one of the most primitive carnivores, giant panda not only has unique food habit, but also has highly specialized reproductive behavior and low fertility [14], all of which signify that the giant panda has considerably different regulatory mechanisms in growth and development. However, very little is known on structure and function of regulatory genes in the growth and development of giant panda [15], [16]. As bHLH proteins present great importance in the regulation of organismal development, in this study, we have made exhaustive effort to obtain the complete list of bHLH family members encoded in the genome of giant panda. As a result, 107 bHLH family members were identified. Phylogenetic analyses with their mouse bHLH homologues revealed that the 107 giant panda bHLH members belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while 3 members were assigned into “orphan”. The present study provides useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.

Materials and Methods

Blast Searches

The sets of 45 representative bHLH domains and 114 mouse bHLH motifs were from the additional files of previous reports [4], [17], respectively. Each sequence of both sets was used as query sequence to perform tblastn search against the giant panda genome sequences which were accessed through the hyperlink provided on GenBank's MapView webpage (http://www.ncbi.nlm.nih.gov/mapview/). The expect value (E) was set at 10 in order to obtain all bHLH related sequences. The obtained subject sequences were manually examined to keep only one sequence for those that have the same contig number, reading frame and coding regions, to add the missing amino acids to corresponding sites with the help of EditSeq program (version 5.01) of the DNAStar package, and to find introns within the bHLH motifs using NetGene2 application online (http://www.cbs.dtu.dk/services/NetGene2/). Sequence accession numbers of giant panda bHLH proteins were obtained by using amino acids of each identified bHLH motif to conduct blastp search against giant panda protein sequence databases which were also accessed through the hyperlink on GenBank's MapView webpage.

Sequence Alignment

All sequences that had been improved by the above methods were aligned using ClustalW program embedded in MEGA4 [18] with default settings. Each sequence was examined for their amino acid residues at the 19 conserved sites by manual checking [19]. Sequences with less than nine variations were regarded as potential giant panda bHLH members. The sequences which have less than ten conserved amino acids were discarded and the rest sequences were aligned again using ClustalW. The aligned giant panda bHLH motifs were shaded in GeneDoc Multiple Sequence Alignment Editor and Shading Utility (Version 2.6.02) [20] and copied to rich text file for further annotation.

Phylogenetic Analyses

Phylogenetic analyses to all the identified giant panda bHLH members were carried out in two steps. First, all the obtained giant panda bHLH motif sequences were used to build neighbor-joining (NJ) distance tree with the 114 mouse bHLH motif sequences using PAUP 4.0 Beta 10 [21] based on a step matrix constructed from Dayhoff PAM 250 distance matrix by R. K. Kuzoff (http://paup.csit.fsu.edu/nfiles.html). Then, each giant panda bHLH motif sequence was used to conduct in-group phylogenetic analyses [9] with mouse bHLH motif sequences. That is, each amino acid sequence of giant panda bHLH motifs was used to construct NJ, maximum parsimony (MP), and maximum likelihood (ML) phylogenetic trees with mouse bHLH family members of the corresponding group, respectively. The NJ trees were bootstrapped with 1,000 replicates to provide information about their statistical reliability. MP analysis was performed using heuristic searches and bootstrapped with 100 replicates. ML trees were constructed using TreePuzzle 5.2 [22] with quartet-puzzling tree-search procedure and 25,000 puzzling steps. Model of substitution was set to the Jones-Taylor-Thornton [23]. Other parameters were set to default values.

Results and Discussion

Giant Panda bHLH Family Members

The tblastn searches, sequence alignment, and examination of the 19 conserved amino acid sites revealed that there were 107 bHLH genes encoded in giant panda genome. The names of all 107 giant panda bHLH members are listed in Table 1. Each identified giant panda bHLH (GpbHLH) gene was named according to nomenclature used by mouse bHLH sequences. The alignment of all 107 GpbHLH motifs is shown in Figure S1 and the phylogenetic tree constructed using amino acids from 107 GpbHLH motifs and 114 mouse bHLH motifs is shown in Figure S2. Figures S1 and S2 together show that there were 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively. And additional 3 members were assigned into “orphan”. We found that gene encoding for member of Delilah family was not found in the giant panda genome. In Figure S1, there are two most conserved sites located at sites 23 and 59 of the bHLH motif. Besides, there are other eight sites which are also conserved as indicated with asterisks on top of Figure S1 (amino acid sequences of all 107 giant panda bHLH motifs are available in file S1).

Table 1

A complete list of bHLH genes from giant panda.

Family	Gene name	Mouse homologue	Bootstrap values			Protein accession number	Annotation in GenBank
			NJ	MP	ML
ASCa	GpAsh1	Mash1	99	92	99	XP_002915515.1	Hypothetical protein
	GpAsh2	Mash2	92	91	97	XP_002920180.1	Ash2
ASCb	GpAsh3a	Mash3a	98	99	90	XP_002916197.1	Ash3a
	GpAsh3b	Mash3b	98	97	100	hmm367624.p	Hypothetical protein
	GpAsh3c	Mash3c	99	97	71	hmm285394.p	Hypothetical protein
MyoD	GpMyoD	MyoD	99	97	94	XP_002928807.1	MyoD
	GpMyoG	MyoG	100	98	96	XP_002925479.1	MyoG
	GpMyf5	Myf5	99	77	78	XP_002916822.1	Myf5
	GpMyf6	Myf6	99	89	78	XP_002916823.1	Myf6
E12/E47	GpTF12	TF12	82	n/m*	n/m*	XP_002920720.1	TF12
	GpE2A	E2A	100	97	98	XP_002923565.1	E2A
	GpKA1	KA1	65	87	57	Not available	/
	GpTCF4	TCF4	90	21	82	XP_002914713.1	TCF4
Ngn	GpAth4a	Math4a	99	95	97	XP_002926036.1	Neurogenin-2-like
	GpAth4b	Math4b	99	93	99	XP_002913770.1	Neurogenin-3-like
	GpAth4c	Math4c	99	85	99	XP_002913012.1	Neurogenin-1-like
NeuroD	GpNDF1	NDF1	89	27	80	XP_002922319.1	NDF1
	GpNDF2	NDF2	88	68	89	XP_002916875.1	NDF2
	GpAth2	Math2	93	77	89	XP_002919308.1	NDF6
	GpAth3	Math3	99	96	98	XP_002930692.1	NDF4
Atonal	GpAth1	Math1	100	99	100	XP_002915330.1	Ath1
	GpAth5	Math5	100	100	100	XP_002913786.1	Ath7
Mist	GpMist1	Mist1	99	97	n/m	Not available	/
Beta3	GpBeta3b a	Beta3b	100	59	n/m*	XP_002925784.1	Class E bHLH protein 23
Oligo	GpOligo1	Oligo1	91	85	98	XP_002919636.1	Oligo1
	GpOligo2	Oligo2	88	59	56	XP_002919637.1	Oligo2
	GpOligo3	Oligo3	90	73	98	XP_002915132.1	Oligo3
Net	GpAth6	Math6	100	100	100	XP_002928677.1	Ath8
Mesp	GpMesp1 a	Mesp1	99	78	n/m*	XP_002919616.1	Mesp1
	GpPMeso1	pMeso1	100	100	97	XP_002915045.1	Mesogenin-1
Twist	GpTwist	Twist	91	64	n/m*	XP_002915415.1	Hypothetical protein
	GpDermo1	Dermo1	90	55	90	XP_002922521.1	Twist-2-like
Paraxis	GpParaxis	Paraxis	78	70	86	XP_002925450.1	Transcription factor 15
MyoRa	GpMyoR	MyoR	75	64	84	XP_002922844.1	Musculin
	GpPod1	Pod1	78	25	n/m*	XP_002922333.1	Transcription factor 21
MyoRb	GpMyoRb1	MyoRb1	100	100	100	XP_002916432.1	Hypothetical protein
	GpMyoRb2	MyoRb2	100	99	93	XP_002913861.1	Transcription factor 23
Hand	GpDHand	dHand	100	64	n/m*	XP_002912726.1	Hand 2
	GpEHand	eHand	99	99	99	XP_002917201.1	Hand 1
PTFa	GpPTFa	PTFa	100	100	97	XP_002913208.1	Hypothetical protein
PTFb	GpPTFb	PTFb	100	100	99	XP_002915418.1	Fer3
SCL	GpTal1	Tal1	100	71	85	hmm534354.p	Hypothetical protein
	GpTal2	Tal2	99	94	88	XP_002927719.1	Tal2
	GpLyl1	Lyl1	99	99	100	XP_002921032.1	Lyl1
NSCL	GpHen1 a	Hen1	100	100	89	XP_002928490.1	HLH protein-1-like
	GpHen2	Hen2	60	40	n/m*	XP_002925359.1	HLH protein-2-like
SRC	GpSRC1	SRC1	100	99	98	XP_002913840.1	NcoA 1
	GpSRC2	SRC2	100	100	99	XP_002918551.1	NcoA 2
	GpSRC3	SRC3	100	100	99	XP_002927046.1	NcoA 3
FIGα	GpFiga	Figa	100	100	100	XP_002914962.1	Figa
Myc	GpN-Myc	N-Myc	99	70	85	XP_002923116.1	N-Myc
	GpC-Myc	C-Myc	100	100	100	XP_002915028.1	C-Myc
	GpL-Myc	L-Myc	100	100	99	XP_002927604.1	L-Myc
Mad	GpMxi1	Mxi1	100	99	96	XP_002930845.1	Mxi1
	GpMad1	Mad1	100	100	92	XP_002914951.1	Mad1
	GpMad3	Mad3	100	98	91	XP_002928184.1	Mad3
	GpMad4	Mad4	98	87	91	XP_002916603.1	Mad4
Mnt	GpMnt	Mnt	100	100	99	XP_002918069.1	Mnt
Max	GpMax	Max	100	100	100	XP_002914193.1	Max
USF	GpUSF1	USF1	100	98	100	XP_002928795.1	USF1
	GpUSF2	USF2	92	38	99	XP_002920933.1	USF2
MITF	GpMITF	MITF	75	n/m*	n/m*	XP_002927657.1	MITF
	GpTFEb	TFEb	100	100	98	XP_002914561.1	TFEb
	GpTFEc	TFEc	98	97	96	XP_002923929.1	TFEc
	GpTFE3	TFE3	63	52	n/m	XP_002917800.1	TFE3
SREBP	GpSREBP1	SREBP1	100	99	n/m*	XP_002923179.1	SREBP1
	GpSREBP2	SREBP2	100	100	100	XP_002929331.1	SREBP2
AP4	GpAP4	AP4	100	100	99	XP_002924645.1	AP4
MLX	GpMlx	Mlx	100	99	92	XP_002923532.1	WBSCR14
	GpMondoA	MondoA	100	100	97	XP_002913172.1	Mlx-interacting protein
TF4	GpTF4	TF4	100	100	100	XP_002922185.1	Max-like protein X
Clock	GpClk	Clk	100	95	100	XP_002919413.1	Clk
	GpNPAS2	NPAS2	100	99	100	XP_002919235.1	NPAS2
ARNT	GpARNT1	ARNT1	97	61	n/m*	XP_002919403.1	ARNT1
	GpARNT2	ARNT2	96	87	97	XP_002919129.1	ARNT2
Bmal	GpBmal1	Bmal1	100	99	97	XP_002926157.1	ARNT-like protein 1
	GpBmal2 a	Bmal2	100	100	100	XP_002917162.1	ARNT-like protein 2
Sim	GpSim1	Sim1	97	74	96	XP_002922016.1	Sim1
	GpSim2	Sim2	97	83	90	hmm348774.p	Hypothetical protein
AHR	GpAHR1	AHR1	100	99	100	XP_002917450.1	AHR1
	GpAHR2	AHR2	81	64	n/m	XP_002926684.1	AHRR
Trh	GpNPAS3	NPAS3	100	91	82	hmm740504.p	Hypothetical protein
HIF	GpHif1a	Hif1a	100	100	100	XP_002913080.1	Hif1a
	GpHif3a	Hif3a	100	100	100	XP_002923099.1	Hif3a
	GpNPAS1	NPAS1	100	100	95	XP_002923107.1	NPAS1
	GpEPAS1	EPAS1	100	99	100	XP_002912483.1	EPAS1
Emc	GpId1	Id1	93	57	n/m*	hmm387023.p	Hypothetical protein
	GpId2	Id2	87	82	56	XP_002923275.1	Id2
	GpId3	Id3	99	92	100	XP_002913316.1	Id3
	GpId4	Id4	100	90	76	hmm7844.p	Hypothetical protein
Hey	GpHerp1	Herp1	96	86	96	XP_002927896.1	Herp1
	GpHerp2	Herp2	96	50	n/m*	XP_002915182.1	Herp2
	GpHEYL	HEYL	98	94	98	XP_002930399.1	HEYL
	GpHey4	Hey4	100	100	92	XP_002914075.1	HELT-like protein
H/E(spl)	GpDec1	Dec1	76	67	99	XP_002920034.1	Class E bHLH protein 40
	GpDec2	Dec2	73	n/m*	n/m*	hmm164814.p	Hypothetical protein
	GpHes1a	Hes1	99	66	84	XP_002930213.1	Hes4-like protein
	GpHes1b	Hes1	100	100	100	XP_002923794.1	Hes1
	GpHes2	Hes2	97	67	88	XP_002923913.1	Hes2
	GpHes3	Hes3	100	97	98	XP_002923915.1	Hes3
	GpHes7	Hes7	100	100	97	hmm475304.p	Hypothetical protein
COE	GpEBF1	EBF1	95	45	58	XP_002912553.1	COE1
	GpEBF2	EBF2	94	89	56	XP_002914472.1	COE2
	GpEBF3	EBF3	72	n/m*	n/m*	XP_002922830.1	COE3
Orphan	GpOrphan2	Orphan2	100	100	59	XP_002913251.1	MAX gene-associated
	GpOrphan3	Orphan3	100	100	n/m*	XP_002923506.1	Sohlh2-like protein
	GpOrphan4	Orphan4	100	100	100	Not available	/

Note: Giant panda bHLH genes were named according to their mouse homologues. Bootstrap values were from in-group phylogenetic analyses with mouse bHLH motif sequences using NJ, MP, and ML algorithms, respectively. OsRa (the rice bHLH motif sequence of R family) was used as the outgroup in every constructed tree except those for GpHen1 and GpBmal2 which used separate outgroup sequence. n/m means that a giant panda bHLH does not form a monophyletic group with any other single bHLH motif sequence. n/m* means that a giant panda bHLH does not form a monophyletic clade with any specific bHLH motif sequence but forms a monophyletic clade with other bHLH proteins of the same family.

means that the gene's orthology was defined by in-group phylogenetic analyses with corresponding whole bHLH protein sequences from mouse. The accession numbers are from different protein resources. Those labeled as “XP” are from ‘RefSeq protein’ and those labeled as “hmm” were from ‘Ab initio protein’ databases of giant panda.

Identification of Orthologous Families

Ortholog identification has had much uncertainty since there is no absolute criterion that can be used to decide whether two genes are orthologous [17]. In our previous studies [9], [10], in-group phylogenetic analysis was adopted to identify homologues for the unknown sequences that would form a monophyletic clade among themselves by using a more certain criterion based on the criterion used by Ledent et al. [17], [24]: If an unknown single giant panda bHLH forms a monophyletic clade with another bHLH of known family in phylogenetic trees constructed with different methods and all the bootstrap values exceed 50, the known member will be regarded as a homologue of the unknown sequence. Figure S3, as an example here, shows NJ, MP and ML phylogenetic trees constructed with one GpbHLH member (GpAsh1) and eight group A bHLH members from mouse. In all three trees, GpAsh1 formed monophyletic clade with Mash1 of mouse with bootstrap values ranging from 92 to 100. Therefore, GpAsh1 was considered as an ortholog of Mash1 of mouse. The similar in-group phylogenetic analyses were conducted to each of the identified GpbHLH members by referencing Figure S2 to select appropriate related mouse bHLH members for the analysis. All the bootstrap values of constructed NJ, MP and ML trees were listed in Table 1 without showing the correspondent constructed trees. Table 1 showed that the orthology of GpbHLH members with mouse can be divided into the following categories.

Firstly, among the 107 GpbHLH members, 83 GpbHLH members have all the bootstrap values over 50 (55≦bootstrap values≦100) in constructed NJ, MP and ML trees. We have sufficient confidence to define orthology of these GpbHLH motifs to their corresponding mouse bHLH orthologs.

Secondly, 4 GpbHLH members, namely GpTCF4, GpNDF1, GpUSF2 and GpEBF1, formed monophyletic clade with bootstrap values over 50 in NJ and ML trees. Although they also formed monophyletic clade in MP trees, their bootstrap values ranged from 21 to 45. Therefore, the orthology of these 4 GpbHLH members have been defined according to the statistical support from NJ and ML trees. And 10 GpbHLH members, namely GpMist1, GpAHR2, GpTwist, GpDHand, GpARNT1, GpSREBP1, GpId1, GpHerp2 and GpOrphan3, formed monophyletic clade with bootstrap values ranging from 50 to 100 in NJ and MP trees, but did not form monophyletic group with any single bHLH sequence in ML trees (marked with n/m* or n/m in Table 1). For these 9 GpbHLH members, we have defined their orthology according to the statistical support from NJ and MP trees.

Thirdly, 2 GpbHLH members, namely GpPod1 and GpHen2 formed monophyletic clade in NJ and MP trees with bootstrap values ranging from 20 to 79 but did not form monophyletic group in ML tree. And 4 other GpbHLH members, namely GpTF12, GpMITF, GpDec2 and GpEBF3, formed monophyletic clade with bootstrap values ranging from 72 to 82 in NJ tree, but did not form monophyletic clade in MP and ML trees. Although these 6 GpbHLH members did not have sufficient bootstrap support, we defined orthologs for them because they all have one or two bootstrap support to testify their orthology to the correspondent mouse ortholog. This phylogenetic divergence of bHLH motif sequences between giant panda and mouse probably means that these two mammals have evolved in quite different circumstances.

Finally, there are 4 GpbHLH sequences which did not form monophyletic clade with most of the mouse bHLH motif sequences in all constructed phylogenetic trees. They are GpBeta3b, GpMesp1, GpHen1 and GpBmal2 of which whole protein sequences were used to conduct in-group phylogenetic analyses with whole sequences of corresponding mouse bHLH proteins for defining their orthology (marked with a in Table 1).

Protein Sequences and Genomic Coding Regions of Giant Panda bHLH Genes

Protein sequence accession numbers of all the identified GpbHLH motifs were listed in Table 1. It was found that there are 95 GpbHLH motifs of which protein sequence accession numbers were found in ‘Non-RefSeq protein’ database (shown as ‘XP’ plus number). Protein sequence accession numbers of 9 GpbHLH motifs were only found in ‘Ab initio protein’ database in which all protein sequences were predicted from their corresponding genomic sequences (shown as ‘hmm’ plus number). They are GpAsh3b, GpAsh3c, GpTal1, GpSim2, GpNPAS3, GpId1, GpId4, GpDec2 and Hes7, respectively. There are also 3 GpbHLH protein sequences of which accession numbers were not found in any protein databases. They are GpKA1, GpMist1 and GpOrphan4, respectively.

Table 1 showed that, among the 104 bHLH protein sequences deposited in giant panda databases, 58 were annotated in full agreement with our analytical result (shown as the same name in the column of “annotation in GenBank” with that in the column of “gene name”), 33 were annotated differently with our result (shown as a different name in the column of “annotation in GenBank” with that in the column of “gene name”), and 13 were merely predicted proteins (indicated as “hypothetical protein”). Therefore, our work not only newly identified the 13 protein sequences as bHLH family members but also provided additional information for further investigations on the 33 differently annotated bHLH protein sequences.

The coding regions and intron analysis for 107 giant panda bHLH motifs are listed in Table 2. The data of intron analyses showed that there are 47 GpbHLH members with introns in their bHLH motifs. It was found that: (i) 26 GpbHLH members have one intron, among which 13 GpbHLH members have introns in the basic region, 12 have introns in the loop region, and 1 has introns in the helix 2 region. (ii) 19 GpbHLH members have two introns, among which 15 have introns in the basic and loop regions respectively, 3 have introns in the basic and helix 2 regions respectively, and 1 has introns in the helix1 and helix 2 regions respectively. (iii) 2 GpbHLH members have three introns among which two were located in basic region and one was located in helix 2 region. There are altogether 70 introns being identified in the 47 GpbHLH motifs. The longest intron in GpbHLH motif is 45,217 bp (base pairs), and the average length of intron is 4,393 bp. These data are comparable with those of mouse. In mouse, there are also 47 bHLH members having introns in their bHLH motifs. The total number of introns identified is 73, with the longest one of 48,288 bp and the average length of 4,286 bp (data not shown).

Table 2

Coding regions, intron location and length of 107 giant panda bHLH motifs.

Family	Gene name	Genomic coding sequence(s)			Intron (location: length)
		Contig No.	Frame	Coding region(s)
ASCa	GpAsh1	NW_003217384.1	+1	937516-937674
	GpAsh2	NW_003217681.1	−2	1024199-1024041
ASCb	GpAsh3a	NW_003217414.1	+1	1397734-1397892
	GpAsh3b	NW_003217343.1	+3	1118238-1118387
	GpAsh3c	NW_003217785.1	−3	1162741-1162583
MyoD	GpMyoD	NW_003218874.1	+3	47166-47321
	GpMyoG	NW_003218226.1	−1	585332-585177
	GpMyf5	NW_003217445.1	−1	705066-704911
	GpMyf6	NW_003217445.1	−3	714220-714065
E12/E47	GpTF12	NW_003217723.1	−3	878591-878430
	GpE2A	NW_003217991.1	−2	930301-930140
	GpKA1	NW_003217991.1	−1	932483-932322
	GpTCF4	NW_003217346.1	+1	1744351-1744512
Ngn	GpAth4a	NW_003218321.1	−3	24207-24049
	GpAth4b	NW_003217318.1	+1	2044831-2044989
	GpAth4c	NW_003217300.1	−3	4043793-4043635
NeuroD	GpNDF1	NW_003217851.1	+3	616848-617006
	GpNDF2	NW_003217447.1	+2	18473-18631
	GpAth2	NW_003217612.1	−1	1092508-1092350
	GpAth3	NW_003219813.1	−3	40317-40159
Atonal	GpAth1	NW_003217374.1	+2	262304-262462
	GpAth5	NW_003217318.1	+3	3168366-3168524
Mist	GpMist1	NW_003218585.1	−1	220417-220259
Beta3	GpBeta3b	NW_003218276.1	+1	208657-208821
Oligo	GpOligo1	NW_003217632.1	−2	1158013-1157843
	GpOligo2	NW_003217632.1	−3	1198932-1198768
	GpOligo3	NW_003217365.1	−3	2603795-2603631
Net	GpAth6	NW_003218843.1	+3	61194-61271	Loop: 8,330 bp
			+2	69602-69682
Mesp	GpMesp1	NW_003217631.1	−3	1622851-1622687
	GpPMeso1	NW_003217360.1	+3	779709-779873
Twist	GpTwist	NW_003217378.1	−2	1057777-1057622
	GpDermo1	NW_003217871.1	−3	1180296-1180141
Paraxis	GpParaxis	NW_003218220.1	−1	310143-310117	Basic: 29,249 bp
			−3	280867-280736
MyoRa	GpMyoR	NW_003217902.1	−2	213023-212865
	GpPod1	NW_003217853.1	+1	879529-879687
MyoRb	GpMyoRb1	NW_003217428.1	−3	2043881-2043723
	GpMyoRb2	NW_003217319.1	−1	759695-759537
Hand	GpDHand	NW_003217296.1	+2	4789691-4789849
	GpEHand	NW_003217471.1	+1	889240-889398
PTFa	GpPTFa	NW_003217305.1	+2	3160973-3161131
PTFb	GpPTFb	NW_003217378.1	−2	1090543-1090385
SCL	GpTal1	NW_003218755.1	+2	402998-403156
	GpTal2	NW_003218606.1	+3	375012-375170
	GpLyl1	NW_003217749.1	−1	440560-440402
NSCL	GpHen1	NW_003218810.1	−1	310980-310822
	GpHen2	NW_003218212.1	+3	118701-118850	No intron (two separate contigs)
		NW_003222115.1	−1	2942-2934
SRC	GpSRC1	NW_003217319.1	−3	2757930-2757913	Basic: 6,972 bp
			−3	2750940-2750785
	GpSRC2	NW_003217553.1	−1	1113592-1113587	Basic: 2,669 bp
			−3	1110917-1110750
	GpSRC3	NW_003218488.1	−3	105991-105983	Basic: 876 bp
			−3	105106-104942
FIGα	GpFiga	NW_003217356.1	+3	2013393-2013428	Basic: 5,738 bp
			+2	2019167-2019289
Myc	GpN-Myc	NW_003217942.1	−3	698202-698044
	GpC-Myc	NW_003217359.1	−3	2278151-2277993
	GpL-Myc	NW_003218583.1	−2	137657-137499
Mad	GpMxi1	NW_003219956.1	−1	80826-80792	Basic: 29,318 bp
			−3	50884-50770	Helix 2: 613 bp
			−1	50156-50148
	GpMad1	NW_003217356.1	−1	2726736-2726732	Basic: 4,015 bp
			−3	2721716-2721687	Basic: 14,277 bp
			−3	2707409-2707295	Helix 2: 1,545 bp
			−3	2705749-2705741
	GpMad3	NW_003218733.1	+2	210165-210168	Basic: 647 bp
			+2	210816-210841	Basic: 128 bp
			+1	210970-211089	Helix 2: 2,020 bp
			+2	213110-213118
	GpMad4	NW_003217437.1	−1	395863-395833	Basic: 563 bp
			−2	390270-390154	Helix 2: 948 bp
			−2	389205-389197
Mnt	GpMnt	NW_003217523.1	+3	590949-590983	Basic: 142 bp
			+1	591126-591237	Helix 2: 5,001 bp
			+1	596239-596247
Max	GpMax	NW_003217331.1	+3	1173984-1174085	Loop: 13,160 bp
			+2	1187246-1187299
USF	GpUSF1	NW_003218870.1	+3	285504-285525	Basic: 121 bp
			+1	285647-285741	Loop: 250 bp
			+3	285990-286040
	GpUSF2	NW_003217744.1	+3	363972-363992	Basic: 6,252 bp
			+1	370243-370338	Loop: 103 bp
			+2	370442-370492
MITF	GpMITF	NW_003218591.1	+3	401976-401997	Basic: 5,362 bp
			+1	407360-407436	Loop: 3,549 bp
			+1	410986-411048
	GpTFEb	NW_003217341.1	+3	3067443-3067464	Basic: 617 bp
			+2	3068082-3068158	Loop: 644 bp
			+1	3068803-3068865
	GpTFEc	NW_003218029.1	+2	296780-296801	Basic: 3,864 bp
			+2	300666-300741	Loop: 4,628 bp
			+1	305370-305433
	GpTFE3	NW_003217513.1	+2	815657-815678	Basic: 172 bp
			+3	815851-815927	Loop: 1,124 bp
			+2	817052-817114
SREBP	GpSREBP1	NW_003217951.1	+1	876259-876357	Loop: 642 bp
			+1	877000-877053
	GpSREBP2	NW_003219081.1	−2	42239-42141	Loop: 1,224 bp
			−2	40916-40863
AP4	GpAP4	NW_003218113.1	+3	891114-891224	Loop: 117 bp
			+3	891342-891386
MLX	GpMlx	NW_003217989.1	−2	869754-869644	Loop: 186 bp
			−2	869457-869404
	GpMondoA	NW_003217304.1	+1	2129887-2129982	Loop: 108 bp
			+1	2130091-2130147
TF4	GpTF4	NW_003217842.1	+2	991667-991717	Helix 1: 260 bp
			+1	991978-992077	Helix 2: 194 bp
			+3	992272-992297
Clock	GpClk	NW_003217618.1	−3	1508321-1508314	Basic: 3,666 bp
			−3	1504647-1504503
	GpNPAS2	NW_003217607.1	−1	787626-787622	Basic: 236 bp
			−3	767385-767238
ARNT	GpARNT1	NW_003217617.1	+1	1009551-1009555	Basic: 2,103 bp
			+3	1011659-1011815
	GpARNT2	NW_003217598.1	−1	1385223-1385219	Basic: 7,467 bp
			−2	1377751-1377595
Bmal	GpBmal1	NW_003218335.1	+2	511835-511839	Basic: 1,225 bp
			+3	513065-513221
	GpBmal2	NW_003217468.1	−2	955835-955831	Basic: 3,125 bp
			−3	952705-952549
Sim	GpSim1	NW_003217828.1	+1	640339-640500
	GpSim2	NW_003217464.1	+2	2035250-2035411
AHR	GpAHR1	NW_003217483.1	+2	1606550-606711
	GpAHR2	NW_003218420.1	−1	471842-471681
Trh	GpNPAS3	NW_003219637.1	−2	62695-2534
HIF	GpHif1a	NW_003217302.1	+2	4505891-4506052
	GpHif3a	NW_003217939.1	−2	1043816-1043655
	GpNPAS1	NW_003217939.1	−2	538490-538344
	GpEPAS1	NW_003217290.1	+2	3566561-3566722
Emc	GpId1	NW_003217538.1	−3	1629899-1629801
	GpId2	NW_003217962.1	+1	246616-246714
	GpId3	NW_003217307.1	−2	256780-256682
	GpId4	NW_003218297.1	+3	402159-402257
Hey	GpHerp1	NW_003218647.1	+3	207195-207212	Basic: 125 bp
			+2	207338-207421	Loop: 276 bp
			+2	207698-207763
	GpHerp2	NW_003217369.1	−1	1395712-1395695	Basic: 132 bp
			−1	1395562-1395479	Loop: 2,267 bp
			−3	1393211-1393446
	GpHEYL	NW_003219577.1	−1	109083-109066	Basic: 1,029 bp
			−1	108036-107953	Loop: 1,266 bp
			−3	106684-106619
	GpHey4	NW_003217325.1	+3	3706089-3706190	Loop: 271 bp
			+1	3706462-3706527
H/E(spl)	GpDec1	NW_003217667.1	+1	608623-608724	Loop: 941 bp
			+3	609666-609731
	GpDec2	NW_003217468.1	+3	2062578-2062679	Loop: 321 bp
			+1	2063002-2063067
	GpHes1a	NW_003219474.1	−2	115790-115785	Basic: 82 bp
			−3	115702-115607	Loop: 96 bp
			−3	115510-115439
	GpHes1b	NW_003218013.1	−1	184110-184105	Basic: 135 bp
			−1	183969-183874	Loop: 194 bp
			−3	183679-183608
	GpHes2	NW_003218027.1	+3	663288-663293	Basic: 86 bp
			+2	663380-663475	Loop: 208 bp
			+3	663684-663755
	GpHes3	NW_003266724.1	−3	806524-806423	Loop: 101 bp
			−2	806321-806253
	GpHes7	NW_003217758.1	+2	1201700-1201801	Loop: 589 bp
			+3	1202391-1202462
COE	GpEBF1	NW_003217293.1	−1	2621749-2621723	Basic: 45,217 bp
			−2	2576505-2576416	Loop: 16,756 bp
			−3	2559659-2559615
	GpEBF2	NW_003217339.1	+3	2341320-2341347	Basic: 19,688 bp
			+2	2361036-2361124	Loop: 1,270 bp
			+3	2362395-2362439
	GpEBF3	NW_003217894.1	+2	583319-583346	Basic: 17,574 bp
			+2	600921-601009	Loop: 5,187 bp
			+2	606197-606241
Orphan	GpOrphan2	NW_003217306.1	−2	2667874-2667746	Helix 2: 1,301 bp
			−1	2666444-2666418
	GpOrphan3	NW_003217988.1	+1	82414-82451	Basic: 16,940 bp
			+3	99392-99509
	GpOrphan4	NW_003218276.1	+2	325310-325347	Basic: 1,985 bp
			+1	327333-327441

Note: Basic, helix 1, loop, and helix 2 regions are delineated as shown in Figure S1.

The Giant Panda bHLH Repertoire

Compared to the 114 bHLH family members of mouse, it was found that the giant panda has one less member in each of the 7 bHLH families namely Beta3, Mesp, Paraxis, Myc, Hes, COE and Orphan. The missing bHLH family members are Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6), EBF4 and Orphan 1, respectively. Based on the available data, it is difficult to say whether giant panda does lack these bHLH genes. At present, there are three mammalian species (human, mouse and rat) of which bHLH family members have been identified and classified [4], [7]. While human has different members with mouse and rat in only 2 bHLH families, i.e. Myc and H/E(spl), it is hard to believe that giant panda could have different members in 7 bHLH families. Moreover, among the 7 family members missing in giant panda, zebrafish and chicken are found to lack only one (S-Myc) and two (S-Myc and EBF4) members, respectively [11], [12]. Therefore, it is thought that additional bHLH members may be found after a new and higher quality version of giant panda genome sequence is released. Nevertheless, given that very little information is available on bHLH genes and their functions among bear speices, our data provide a good background information for further studies on regulatory functions of bHLH proteins in giant panda and other bear species.

Alignment of 107 giant panda bHLH family members. Designation of basic, helix 1, loop and helix 2 follows Ferre-D'Amare et al. [25]. The family names and high-order groups have been organized according to Table 1 of Ledent et al. [24]. Highly conserved sites are indicated with asterisks on the top. The first five amino acids of NPAS1 were not available due to incompleteness of the correspondent genomic contig sequences.

(TIF)

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Phylogenetic relationship of 107 giant panda and 114 mouse bHLH members. The tree was constructed with neighbor-joining algorithm with OsRa (the rice bHLH motif sequence of R family) as outgroup. For simplicity, branch lengths of the tree are not proportional to distances between sequences, and bootstrap values less than 50 are not shown. The higher-order group labels are in accordance with Ledent et al. [24].

(TIF)

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In-group phylogenetic analyses of GpAsh1. (a), (b) and (c) are NJ, MP and ML trees constructed with one giant panda bHLH member (GpAsh1) and nine group A bHLH members from mouse, respectively. In all trees, OsRa was used as the outgroup.

(TIF)

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Amino acid sequences of 107 giant panda bHLH motifs. The giant panda bHLH family members are arranged as those in Tables 1 and 2, in which their family assignment, protein and coding region information can be found accordingly.

(DOC)

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