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Open Access

Phylogenetic analysis of the human basic helix-loop-helix proteins

Genome Biology20023:research0030.1

https://doi.org/10.1186/gb-2002-3-6-research0030

Received: 29 November 2001

Accepted: 15 April 2002

Published: 30 May 2002

Abstract

Background

The basic helix-loop-helix (bHLH) proteins are a large and complex multigene family of transcription factors with important roles in animal development, including that of fruitflies, nematodes and vertebrates. The identification of orthologous relationships among the bHLH genes from these widely divergent taxa allows reconstruction of the putative complement of bHLH genes present in the genome of their last common ancestor.

Results

We identified 39 different bHLH genes in the worm Caenorhabditis elegans, 58 in the fly Drosophila melanogaster and 125 in human (Homo sapiens). We defined 44 orthologous families that include most of these bHLH genes. Of these, 43 include both human and fly and/or worm genes, indicating that genes from these families were already present in the last common ancestor of worm, fly and human. Only two families contain both yeast and animal genes, and no family contains both plant and animal bHLH genes. We suggest that the diversification of bHLH genes is directly linked to the acquisition of multicellularity, and that important diversification of the bHLH repertoire occurred independently in animals and plants.

Conclusions

As the last common ancestor of worm, fly and human is also that of all bilaterian animals, our analysis indicates that this ancient ancestor must have possessed at least 43 different types of bHLH, highlighting its genomic complexity.

Background

The basic helix-loop-helix (bHLH) family of transcriptional regulators are key players in a wide array of developmental processes in metazoans, including neurogenesis, myogenesis, hematopoiesis, sex determination and gut development (reviewed in [1,2,3,4,5]). The bHLH domain is approximately 60 amino acids long and comprises a DNA-binding basic region (b) followed by two α helices separated by a variable loop region (HLH) (reviewed in [4]). The HLH domain promotes dimerization, allowing the formation of homodimeric or heterodimeric complexes between different family members. The two basic domains brought together through dimerization bind specific hexanucleotide sequences.

Over 400 bHLH proteins have been identified to date in organisms ranging from the yeast Saccharomyces cerevisiae to humans (see, for example [6,7,8]). In previous work, we took advantage of the complete sequencing of the nematode [9] and fly [10] genomes to extract a large, and possibly complete, set of bHLH genes from these two organisms [8]. A phylogenetic analysis of the amino acid sequences of these bHLHs, together with a large number (> 350) of bHLH from other sources, in particular from mouse, led us to define 44 orthologous families (that is, groups of orthologous sequences that derive from the duplication of a common ancestor), among which 36 include bHLH from metazoans only, and 2 have representatives in both yeasts and metazoans [8] (Table 1). We also identified two bHLH motifs present only in yeast, and four that are present only in plants [8].
Table 1

The 44 families of animal bHLH defined by our phylogenetic analyses

Family name

Number of worm genes

Number of fly genes

Number of mouse genes

Number of human genes

Number of sea squirt genes

Number of pufferfish genes

Group

Achaete-Scute a

4

4

2

2

0

3

A

Achaete-Scute b

1

0

1

3

1

3

A

MyoD

1

1

4

4

1

1

A

E12/E47

1

1

4

6

1

1

A

Neurogenin

1

1

3

3

1

2

A

NeuroD

1

0

4

4

0

5

A

Atonal

1

3

2

2

1

4

A

Mist

0

1

1

1

1

0

A

Beta3

1 or 2*

1

2

2

0

2

A

Oligo

0 or 1*

0

3

3

0

3

A

Net

1

1

1

1

1

0

A

Mesp

0

1

3

4

1

2

A

Twist

1

1

2

1

0

3

A

Paraxis

0

1

2

1

0

2

A

MyoR

1

1

2

4

0

3

A

Hand

1

1

2

2

0

1

A

PTFa

0

1

1

1

0

1

A

PTFb

1

2

0

1

0

2

A

SCL

0

1

4

3

0

1

A

NSCL

1

1

2

2

0

1

A

SRC

0

1

3

3

0

2

B

Figa

0

0

1

1

1

0

B

Myc

0

1

4

5

0

3

B

Mad

1

0

4

5

1 or 2

0

B

Mnt

0

1

1

1

0 or 1†

0

B

Max

2

1

1

1

0

1

B

USF

1

1

2

3

0

1

B

MITF

1

0

4

5

0

2

B

SREBP

1

1

2

2

0

2

B

AP4

1

1

1

1

0

0

B

MLX

0 or 1§

1

1

2

0

3

B

TF4

0 or 1§

1

1

2

1

1

B

Clock

1

3

2

2

0

1

C

ARNT

1

1

2

2

1

2

C

Bmal

0

1

1

2

0

2

C

AHR

1

2

1

4

0

1

C

Sim

0 to 1

1

2 or 3

2 or 3

1 or 2

4 or 5

C

Trh

0 to 1

1

1 or 2

1 or 2

0 or 1

0 or 1

C

HIF

1 to 2

1

3 or 4

3 or 4

0 or 1

2 or 3

C

Emc

0

1

4

5

0

4

D

Hey#

0

2

2

5

1

2

E

Hairy#

0

3

1

2

1

4

E

E (spl) #

1

8

8

8

1

8

E

COE

1

1

4

4

0

0

F

Orphans

6

1

0

3

0

0

No

Families have been named according to the name (or its common abbreviation) of the first discovered or best-known member of the family. The number of members per family in worm, fly and human (complete genomes) as well as in mouse, sea squirt, and pufferfish (uncompleted genomes) is reported. Each family has been tentatively assigned to a high-order group using the classification of Atchley and Fitch [6] and Ledent and Vervoort [8]. Genes that cannot be assigned to any families are categorized as 'orphan' genes. *Beta3 and Oligo are closely related families, one C. elegans gene (F38C2.2) belongs to the Beta3 family while another (DY3.3) is equally related to both Beta3 and Oligo families. Mad and Mnt are closely related families, one Ciona gene (Not7) belongs to the Mad family while another (LQW20007) is equally related to both Mad and Mnt families. These two families also include yeast genes. §TF4 and Mlx are closely related families, one C. elegans gene (T20B12.6) is equally related to both families. The Hif, Sim, and Trh families form a strongly supported monophyletic group (bootstrap value, 95%). A few genes that are included in this group cannot be clearly related to one of the three families (see Additional data for details). #The Hey, Hairy and Enhancer of split families genes form a well-supported monophyletic group (group E; see Figure 1). Two clear families (Hairy and Hey families) with high bootstrap support emerge from this group. All the remaining sequences have been grouped in a single family (named Enhancer of split), which has no real phylogenetic support. A phylogenetic tree of the group can be found in the Additional data.

In addition, we defined higher-order groups which include several evolutionarily related families that share structural and biochemical properties [8]. The different groups were named A, B, C, D, E and F, in agreement with the nomenclature of Atchley and Fitch [6]. Figure 1 shows the phylogenetic relationships between animal families and their tentative inclusion into the different higher-order groups. The properties of these groups have been described elsewhere [4,6,8].
Figure 1

Phylogenetic relationships and high-order grouping of the bHLH families. A neighbor-joining (NJ) tree showing the evolutionary relationships of the 44 animal bHLH families listed in Table 1 is shown. We used one gene (usually from mouse) per family to construct this tree. Although there are strong theoretical reasons for preferring the unrooted tree, we show a rooted tree because it is easier to display compactly and more clearly represents the relationships at the tip of the branches. This tree is just a representation of an unrooted tree with rooting that should be considered arbitrary. We used the plant bHLH family (R family) as outgroup. For simplicity, we show a tree in which branch lengths are not proportional to distances between sequences. High-order groups [6,8] are shown. Some of these groups (A and E) are monophyletic groups, others (D and F) correspond to only one family, and yet others (B and C) are paraphyletic (the last common ancestor of the different families that constitute the group is also that of bHLHs that do not belong to that group). A subgroup of group A families (the Atonal 'superfamily' [8]) is also highlighted and is displayed in more detail in Figure 2.

Figure 2

Some examples of phylogenetic relationships among human and mouse bHLH. Rooted NJ trees are shown. Numbers above branches indicate per cent support in bootstrap analyses (1,000 replicates). As in Figure 1, the rooting should be considered arbitrary. Branch lengths are proportional to distance between sequences. Mouse genes are shown in red, human genes in blue, and other species in black. Species abreviations are as followed: Br, Brachydanio rerio; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Gg, Gallus gallus; Tr, Takifugu rubripes; Xl, Xenopus laevis. (a) Evolutionary relationships among Atonal 'superfamily' members (see Figure 1). The different constituting families are pointed out. For sake of simplicity, only mouse, human and fly genes are shown. This tree is rooted using the closely related twist gene from mouse (see Figure 1) as outgroup. In all cases, a human and a mouse sequence cluster together with high bootstrap values, indicating orthology relationships. (b) Evolutionary relationships among Mesp family members. This tree is rooted using the closely related MATH1 gene from mouse (see Figure 1) as outgroup. Whereas one human and one mouse bHLH (N015926 and pMeso1, respectively) are clearly orthologs, there is no one-to-one relationship between two mouse bHLH (Mesp1 and Mesp2) and three human bHLH (N010356a, b, c), although these bHLH cluster together with a high bootstrap value. (c) Evolutionary relationships among TF4 and MLX family members. This tree is rooted using the closely related MITF gene from mouse (see Figure 1) as outgroup. Two human genes have clear mouse orthologs but two others (Q9HAP2 and N005106) have no such orthologs. (d) Evolutionary relationships among SCL family members. This tree is rooted using the closely related Hen1 gene (NSCL family) from mouse (see Figure 1) as outgroup. The Lyl1 and Lyl2 mouse genes are collectively orthologs to one human gene (P12980), indicating a probable gene duplication specific to mouse.

In brief, groups A and B include bHLH proteins that bind core DNA sequences referred to as E boxes (CANNTG), respectively CACCTG or CAGCTG (group A) and CACGTG or CATGTTG (group B). Group C corresponds to the family of bHLH proteins known as bHLH-PAS, as they contain a PAS domain in addition to the bHLH. They bind to ACGTG or GCGTG core sequences. Group D corresponds to HLH proteins that lack a basic domain and are hence unable to bind DNA. These proteins act as antagonists of group A bHLH proteins. Group E includes proteins related to the Drosophila Hairy and Enhancer of split bHLH (HER proteins). These proteins bind preferentially to sequences referred to as N boxes (CACGCG or CACGAG). They also contain two characteristic domains in addition to the bHLH, the 'Orange' domain and a WRPW peptide in their carboxy-terminal part. Group F corresponds to the COE family, which is characterized by the presence of an additional domain involved both in dimerization and in DNA binding, the COE domain. Yeast and plant bHLHs are all included in group B [6,8].

The completion of the human genome sequencing project [11,12] now allows us to derive the complete set of bHLH present in a vertebrate genome. TBLASTN searches [13] on the human genome draft sequence enabled us to identify 125 different human bHLHs. After exhaustive searches with BLASTP in protein databases and the use of the SMART database (Simple Modular Architecture Research Tool [14,15]) we also identified additional fly, worm and mouse bHLH sequences (total number: 58 in fly, 39 in worm, and 102 in mouse). In addition, we made TBLASTX searches on the incompletely sequenced genomes of the pufferfish Takifugu rubripes and the sea squirt Ciona intestinalis and retrieved 84 and 18 different bHLHs, respectively. We also retrieved, through BLASTP searches, eight different bHLH genes from the completely sequenced yeast genome.

Phylogenetic analysis of all these sequences allowed us to define 44 orthologous families of bHLH proteins in metazoans (the 38 families defined in our previous report plus 6 additional ones, arising out of the additional sequences used in this analysis). Our work now enables comparison of the putative complete repertoires of bHLHs in metazoans belonging to the two main subdivisions of bilaterian animals (the Bilateria; see [16] for a recent overview of the classification of metazoans) - the deuterostomes (human) and the protostomes (fly and worm). This comparison gives us the opportunity to analyze evolution of the diversity of the bHLHs on a metazoan-wide scale, thus giving useful insights into the evolution of multigenic families. In addition, our results allow us to reconstruct the minimum complement of bHLH genes that were present in the bilaterian common ancestor. We also discuss the evolution of the bHLH gene family.

Results and discussion

Isolation of bHLH sequences from protein and genome databases

To isolate human bHLH genes, we made TBLASTN searches [13] on the human genome draft sequence [11], as described in Materials and methods. We completed the list of the retrieved bHLH using the SMART database [14,15]. We eventually got 125 different human bHLH sequences, which are listed in Table 2. All retrieved sequences were used to make BLASTP searches against protein databases in order to detect those sequences that were already identified. We found that 80 sequences were already present in protein databases; 45 of the retrieved sequences from the human genome correspond to previously uncharacterized genes. We similarly retrieved, by TBLASTN, 84 and 18 different bHLH sequences from the incompletely sequenced genomes of the pufferfish T. rubripes and the sea squirt C. intestinalis, respectively (see Additional data files). In addition, we retrieved the complete set of bHLH genes present in the fly (total 58), worm (39), and yeast (8) genomes, as well as all the cloned mouse bHLH genes to date (102), as described in Materials and methods. These sequences with their accession numbers and some information (genomic localization and orthology relationships) are listed in Tables 3,4,5,6.
Table 2

The complete list of bHLH genes from Homo sapiens

Sequence identification

Gene name

Family

Mouse ortholog(s)

Contigs

Chromosome localization

P50553

Hash1

Achaete-Scute a

Mash1

NT_009439.3

12q22-q23

Q99929

Hash2

Achaete-Scute a

Mash2

NT_009368.3

11p15.5

N024228

Hash3a *

Achaete-Scute b

Mash3

NT_024228.3

11p15.3

N004680

Hash3b *

Achaete-Scute b

?

NT_004680.3

1q31-q32

N009720

Hash3c *

Achaete-Scute b

?

NT_009720.3

12q23-q24

P15173

Myf4

MyoD

Myogenin

NT_004662.3

1q31-41

P23409

Myf6

MyoD

Myf6

NT_024473.2

12q21

P15172

Myf3

MyoD

MyoD

NT_009307.3

11p15.4

P13349

Myf5

MyoD

Myf5

NT_024473.2

12q21

N011269

E2A *

E12/E47

E2A

NT_011269.3

19p13.3

Q99081

TF12

E12/E47

TF12

NT_010289.3

15q21

P15884

TCF4

E12/E47

TCF4

NT_011059.5

18q21.1

P15884 D

TCF4b *

E12/E47

TCF4

NT_029427.1

12

P15923

TCF3

E12/E47

?

NT_011269.3

19p13.3

N008413

 

E12/E47

?

NT_008413.3

9p22-q22

Q92858

Hath1

Atonal

Math1

?

4q22

N029388

Hath5 *

Atonal

Math5

NT_029388.3

10q21-q26

N007816

Mist1 *

Mist

Mist1

NT_007816.3

7q21-q31

N011512

Oligo1 *

Oligo

Oligo 1

NT_011512.3

21q21-q22

Q9NZ14

Oligo2 *

Oligo

Oligo 2

?

?

N025741

Oligo3 *

Oligo

Oligo 3

NT_025741.3

6q22-q24

N030199

Beta3a *

Beta3

Beta3

NT_030199.1

8q21

N011333

Beta3b *

Beta3

Q9H494

NT_011333.4

20p11-q13

Q9H2A3

Neurogenin2

Neurogenin

Math4a

NT_022859.3

4

N024089

Hath4b

Neurogenin

Math4b

NT_024089.3

10q21.3

Q92886

NDF3

Neurogenin

NDF3

NT_007091.3

5q23-Q31

N009563

Hath3 *

NeuroD

Math3

NT_009563.3

12q13-q14

N007819

Hath2 *

NeuroD

Math2

NT_007819.6

7p14-p15

Q15784

NDF2

NeuroD

NDF2

NT_010685.3

17q12

Q13562

NDF1

NeuroD

NDF1

NT_005272.3

2q32

N010356a

Mesp1 *

Mesp

?

NT_010356.6

15q25-q26

N010356b

Mesp2 *

Mesp

?

NT_010356.6

15q25-q26

N010356c

Mesp3 *

Mesp

?

NT_010356.6

15q25-q26

N015926

Mesp4 *

Mesp

pMeso1

NT_015926.3

2p24

N015805

Hath6 *

Net

Math6

NT_015805.6

2p11-q24

N005204a

MyoR1 *

MyoR

Pod1

NT_005204.6

2p21-p25

N008253

MyoR2 *

MyoR

MyoR

NT_008253.3

8q13

N005204b

MyoR3 *

MyoR

?

NT_005204.6

2p21-p25

N008166

MyoR4 *

MyoR

?

NT_008166.3

8q13-q22

Q9HC25

P48

PTFa

PTF1

NT_008895.6

10p12-q22

N007918

PTFb *

PTFb

?

NT_007918.6

7p15-p21

O96004

ehand

Hand

eHand

NT_026280.4

5q33

O95300

dHand

Hand

dHand

NT_006257.3

4q31-q33

Q15672

twist

Twist

Twist

NT_007918.3

7p21

N011493

paraxis *

Paraxis

paraxis

NT_011493.3

20

Q02577

NSCL-2

NSCL

Hen2

NT_021883.3

1p11-p12

Q02575

NSCL-1

NSCL

Hen1

NT_004406.3

1q22

Q16559

Tal2

SCL

Tal2

NT_008470.3

9q31

P17542

Tal1

SCL

Tal1

NT_004701.3

1p32

P12980

Lyl

SCL

Lyl1+Lyl2

NT_011247.3

19p13.2

Q01664

AP4

AP4

AP4

NT_015360.3

19p13

Q99583

Mnt

Mnt

Mnt

NT_010692.3

17p13.3

Q14582

Mad4

Mad

Mad4

NT_022865.3

4p16.3

Q9H7H9

Mad4b *

Mad

?

NT_022865.3

4p16.3

P50539

Mxi1

Mad

Mxi1

NT_024048.3

10q25

Q05195

Mad1

Mad

Mad1

NT_005420.3

2p12-p13

AAH00745

Mad3

Mad

Mad3

?

?

P25912

Max

Max

Max

NT_025892.3

14q23

P04198

N-Myc

Myc

N-Myc

NT_026240.1

2p24.1

P01106

C-Myc

Myc

C-Myc

NT_008012.3

8q24

P12524

L-Myc1

Myc

L-Myc

NT_004893.3

1p34

P12525

L-Myc2

Myc

?

NT_011762.3

Xq22-q23

N011572

L-Myc3 *

Myc

?

NT_011572.3

Xq27

O43792

SRC1

SRC

SRC1

NT_005204.3

2p22-p25

Q15596

SRC2

SRC

SRC2

NT_023676.3

8p22-q21

Q9Y6Q9

SRC3

SRC

SRC3

NT_011371.3

20q12

O75030

MITF

MITF

MITF

NT_005510.3

3p12-p14

P19532

TFE3

MITF

TFE3

NT_011611.3

Xp11

P19484

TFEB

MITF

TFEB

NT_023409.3

6p21

O14948

TFEC1

MITF

TFEC

NT_026338.1

7

N009714

TFEC2 *

MITF

TFEC

NT_009714.3

12p11-q14

P36956

SREBP1

SREBP

SREBP1

NT_010657.3

17p11.2

Q12772

SREBP2

SREBP

SREBP2

NT_011520.3

22q13

P22415

USF1

USF

USF1

NT_026219.3

1q22-q23

Q15853

USF2

USF

USF2

NT_011294.3

19q13

N009711

USF2b *

USF

USF2

NT_009711.6

12

Q9NP71

MLXa

MLX

MLX

NT_023557.3

7q11

Q9HAP2

Mondoa

MLX

?

?

12q21

Q9UH92

TF4a *

TF4

TF4

NT_010771.3

17q21.1

N005106

TF4b *

TF4

?

NT_005106.6

2p24-q36

N005420

Figa

Figa

Figa

NT_005420.3

2p13-p24

O00327

Bmal1

Bmal

Bmal1

NT_017854.3

11p15

Q9NYQ5

Bmal2

Bmal

?

NT_009622.3

12p11-p12

P27540

ARNT1

ARNT

ARNT1

NT_004811.3

1q21

Q9HBZ2

ARNT2

ARNT

ARNT2

NT_004811.6

1q21

O15516

clock1

Clock

clock

NT_029271.2

4q12

Q99743

NPAS2

Clock

NPAS2

NT_022171.6

2q13

Q16665

Hif1a

HIF

Hif1a

?

14q21-q24

Q99814

EPAS1

HIF

EPAS1

NT_029237.2

2p21-p16

O95262

Hif3a

HIF

Hif3a

NT_011166.6

19q13

Q99742

NPAS1

HIF/Sim/Trh

NPAS1

NT_011166.3

19q13

P81133

Sim1

Sim

Sim1

NT_019424.3

6q16-q21

Q14190

Sim2

Sim

Sim2

NT_011512.3

21q22.13

Q9H323

NPAS3

Trh

NPAS3

NT_010164.3

14q12-13

Q13804

AHR1

AHR

AHR

NT_007755.4

7p15

N016866

AHR2 *

AHR

?

NT_016866.3

5p15

Q9HAZ3

AHR3 *

AHR

?

NT_016866.3

5p15

N030106

AHR4 *

AHR

?

NT_030106.1

11q12-q13

Q9Y5J3

Herp2

Hey

Hey1

NT_023700.5

8q21

Q9UBP5

Herp1

Hey

Herp1

?

?

Q9NQ87

HEYL

Hey

?

NT_004893.5

1p34.3

Q9NQ87D

HEYLb *

Hey

?

NT_004893.5

1p34.3

N029966

Hey4 *

Hey

?

NT_029966.1

4

O14503

Dec1

E(spl)

Dec1

NT_005927.3

3p24-p26

BAB21502

Dec2

E(spl)

Dec2

NT_009471.3

12p11-p12

N029854

Hes5 *

E(spl)

Hes5

NT_029854.1

1p36

Q9BYEO

Hes7

E(spl)

Hes7

NT_010841.2

17p12-p13

N019265

Hes3 *

E(spl)

Hes3+BAA9469

NT_019265.6

1p36

Q9P2S3

Hes6 *

E(spl)

Hes6

NT_005139.6

2q36-q37

Q9Y543

Hes2

E(spl)

Hes2

NT_019265.6

1p36

Q9BYW0

Cha

E(spl)

?

NT_011333.4

20q11-q13

Q14469

Hes1

Hairy

Hes1

NT_005571.3

3q28-q29

Q9HCC6

Hes4

Hairy

?

NT_025635.5

1p36

P41134

Id1

Emc

Id1

NT_028392.4

20q11

N00599

Id2

Emc

?

NT_005999.3

3p21-q13

Q02363

Id2

Emc

Id2

NT_022194.3

2p25

Q02535

Id3

Emc

Id3

NT_004359.6

1p36

P47928

Id4

Emc

Id4

NT_027049.3

6p21-p22

Q9UH73

EBF1

Coe

Coe1

NT_007006.4

5q34

Q9BQW3

Coe2 *

Coe

?

NT_023666.3

8p21-p22

Q9H4W6

EBF3

Coe

?

NT_008818.6

10q25-q26

Q9NUB6

Coe4 *

Coe

?

?

20p11-p13

N010809

?

Orphan

?

NT_010809.6

17p13.3

Q9NX45

?

Orphan

?

NT_030131.1

13q12-q14

Q9H8R3

?

Orphan

?

NT_010194.6

15q14-q22

Human sequences are identified using their accession number from Swissprot, Trembl, Smart or NCBI genome project sequences. In the latest case, the accession number is that of the contig which includes the bHLH gene. This accession number NT_XXXXXX.Y (XXXXXX identifies the contig and Y the version of the draft) has been abbreviated as NXXXXXXX. Gene names are those reported in protein databases or have been assigned by us on the basis of the orthology relationships with mouse genes (these names are marked by an asterisk). The identification of the contig in which each of the bHLH gene is included is also given. In a few cases (marked with a question mark), we were unable to retrieve, in the genome sequence, previously cloned genes. This may be due to the fact that these genes lie in still unsequenced regions of the genome, or to some limitations of the current version of BLAST (see text for details). Chromosomal localizations are given as reported in the NCBI human genome sequence database (LocusLink and/or OMIM [77,78]).

Table 3

The complete list of bHLH genes from Drosophila melanogaster

Full gene name

Symbol

ID

Family

Localization

Accession number

daughterless

da

CG5102

E12/E47

31D11-E1

pir||A31641

nautilus

nau

CG10250

MyoD

95B3-5

SW:P22816

achaete

ac

CG3796

Achaete-Scute a

1B1

gb|AAF45498.1

scute

sc

CG3827

Achaete-Scute a

1B1

gb|AAA28313.1

lethal of scute

l'sc

CG3839

Achaete-Scute a

1B1

gb|AAF45500.1

asense

ase

CG3258

Achaete-Scute a

1B1

gb|AAF45502.1

target of poxn (biparous)

tap (bp)

CG7659

Neurogenin

74B1-2

emb|CAA65103.1

Mist1-related

Mistr

CG8667

Mist

39D3

gb|AAF53991.1

Olig family

Oli

CG5545

Beta3

36C6-7

gb|AAF53631.1

cousin of atonal

cato

CG7760

Atonal

53A1-2

gb|AAF58026.1

atonal

ato

CG7508

Atonal

84F6

gb|AAF54209.1

absent MD and olfactory sensilla

amos

CG10393

Atonal

37A1-2

gb|AAF53678.1

net

net

CG11450

Net

21A5-B1

gb|AAF51562.1

HLH54F (MyoR * )

MyoR *

CG5005

MyoR

54E7-9

gb|AAF57795.1

salivary gland-expressed bHLH

sage

CG12952

Mesp

85D7-10

gb|AAF54351.1

paraxis *

Pxs *

CG12648

Paraxis

9A4

sp|Q9W2Z5

twist

twi

CG2956

Twist

59C2-3

emb|CAA32707.1

48 related 1

Fer1

CG10066

PTFa

84C3-4

gb|AAF54058.1

48 related 2

Fer2

CG5952

PTFb

89B9-12

gb|AAF55280.1

48 related 3

Fer3

CG6913

PTFb

86F1-2

gb|AAF54684.1

hand

Hand

CG18144

Hand

31D1-6

gb| AAF52900.1

HLH3b (SCL * )

SCL *

CG2655

SCL

3B3-4

gb|AAF45802.1

HLH4C (NSCL * )

NSCL *

CG3052

NSCL

4C6-7

gb|AAF45967.1

EG:114E2.2 (Mnt * )

Mnt *

CG2856

Mnt

3F2-3

sp|O46042

max

max

CG9648

Max

76A3

gb|AAF49179.1

diminutive

dm

CG10798

Myc

3D3-4

gb|AAB39842.1

USF

USF

CG17592

USF

4C4

gb|AAF45953.1

cropped

crp

CG7664

AP4

35F6-7

gb|AAF53510.1

bigmax

bmx

CG3350

TF4

97F5-6

gb|AAF56696.1

MLX *

MLX *

CG18362

MLX

39D1-2

gb|AAF53989.1

HLH106 (SREBP * )

SREBP *

CG8522

SREBP

76D1-3

gb|AAF49115.1

taiman

tai

CG13109

SRC

30A7-8

sp|Q9VLD9

clock

clk

CG7391

Clock

66A11-B1

gb|AAD10630.1

Resistance to Juvenile Hormone

Rst(1)JH

CG1705

Clock

10C6-8

gb|AAC14350.1

germ cell-expressed bHLH-PAS

gce

CG6211

Clock

13C1

gb|AAF48439.1

AHR2 *

AHR2 *

CG12561

AHR

96F14-97A1

gb|AAF56569.1

spineless

ss

CG6993

AHR

89C1-2

gb|AAD09205.1

single-minded

sim

CG7771

Sim

87D12-13

gb|AAF54902.1

trachealess

trh

CG6883

Trh

61C1

gb|AAA96754.1

similar (Hif-1)

sima

CG7951

HIF

99D5-F1

gb|AAC47303.1

tango

tgo

CG11987

ARNT

85C5-7

gb|AAF54329.1

cycle

cyc

CG8727

BMAL

76D2-3

gb|AAF49107.1

extramacrochaete

emc

CG1007

Emc

61D1-2

gb|AAF47413.1

Hey

Hey

CG11194

Hey

43F9-44A1

gb|AAF59152.1

Sticky ch1

Stich1

CG17100

Hey

86A5-6

gb|AAF24476.1

hairy

h

CG6494

Hairy

66D11-12

emb|CAA34018.1

deadpan

dpn

CG8704

Hairy

44B3-4

gb|AAB24149.1

similar to deadpan

side

CG10446

Hairy

37B9-11

gb|AAF53741.1

Enhancer of split m3

E(spl) m3

CG8346

E (spl)

96F10-12

gb|AAF56550.1

Enhancer of split m5

E(spl) m5

CG6096

E (spl)

96F10-12

emb|CAA34552.1

Enhancer of split m8

E(spl) m8

CG8365

E (spl)

96F10-12

sp|P13098

Enhancer of split m7

E(spl) m7

CG8361

E (spl)

96F10-12

emb|CAA34553.1

Enhancer of split mB (g)

E(spl) mB (g)

CG8333

E (spl)

96F10-12

gb|AAA28910.1

Enhancer of split mC (d)

E(spl) mC (d)

CG8328

E (spl)

96F10-12

gb|AAA28911.1

Enhancer of split mA (b)

E(spl) mA (b)

CG14548

E (spl)

96F10-12

gb|AAA28909.1

HES-related

Her

CG5927

E (spl)

17A3

gb|AAF48810.1

knot (collier)

kn (col)

CG10197

COE

51C2-5

gb|AAF58204.1

Delilah

del

CG5441

Orphan

97B1-2

gb|AAF56590.1

Gene names (with commonly used synonyms in some cases) and their usual abbreviation are as reported in FlyBase [79,80], except those marked by an asterisk. In these cases, we propose names based on the orthology relationships with well-characterized vertebrate genes. Identification numbers are those from the Berkeley Drosophila Genome Project [81]. Sequences are listed with the family in which there are included (or stated as orphan genes), their chromosomal localization (position on the polytene chromosomes map as found in FlyBase), and their accession number. The 'orphan' gene Delilah clearly belongs to the high-order group A and is most probably a highly divergent NeuroD family gene (see [8] for discussion).

Table 4

The complete list of bHLH genes from Caenorhabditis elegans

Sequence name

Family

Localization

Accession number

HLH-2 (MO5B5.5)

E12/E47

I: 1,82

TR:Q17588

HLH-1 (BO3O4.1)

MyoD

II: -4,51

SW:P22980

HLH-3 (T29B8.6)

Achaete-Scute a

II: 1

gb|AAB38323.1

C18A3.8

Achaete-Scute a

II:1,11

TR:Q09961

F57C12.3

Achaete-Scute a

X: -19,47

TR:Q20941

C28C12.8

Achaete-Scute a

IV: 3,86

TR:Q18277

T15H9.3

Achaete-Scute b

II: 1,51

emb|CAA87416.1

C34E10.7 (cnd-1)

NeuroD

III: -2,01

sp|P46581

Y69A2AR

Neurogenin

****

****

F38C2.2

Beta3

IV: 24,06

TR:O45489

DY3.3

Beta3/Oligo

I: 3,04

TR:O45320

T14F9.5 (lin-32)

Atonal

X: -15,13

TR:10574

T05G5.2

Net

III:0,92

sp|P34555|YNP2

ZK682.4

MyoR

V: 1,87

TR:Q23579

HLH-8 (CO2B8.4)

Twist

X: -0,63

gb|AAC26105.1

C44C10.8

Hand

X: 5,8

TR:Q18612

F48D6.3

PTFb

X: -8,42

TR:Q20561

C43H6.8

NSCL

X: -14

TR:Q18590

F46G10.6

Max

X: 12,32

TR/Q18711

T19B10.11

Max

V: 3,05

TR:P90982

RO3E9.1 (MDL-1)

Mad

X:-2,63

sp|Q21663

F40G9.11

USF

III: -28,29

gb|AAC68792

W02C12.3

MITF

IV: -1, 14

TR:P91527

F58A4.7

AP4

III: 0,63

SW:P34474

Y47D3B.7

SREBP

III: 8,9

TR:Q9XX00

T20B12.6

TF4/Mlx

III:-0,71

gb|AAA19059.1

C15C8.2

Clock

V: 4,63

emb|CAA99775.1

C41G7.5

AHR

I: 3,75

emb|CAB51463.1

F38A6.3

HIF

V: 27,08

pir||T21944

T01D3.2

HIF/Sim/Trh

V: 5,39

TR:P90953

C25A11.1 (AHA-1)

ARNT

X: 0,43

TR:O02219

lin-22

E (spl)

IV: 6,9

gb|AAB68848.1

Y16B4A.1 (Unc-3)

COE

X: 19,39

gb|AAC06226.1

Y39A3CR.6

Orphan

III: -19,16

gb|AAF605231

T01E8.2 (REF-1) *

Orphan

II: 2,22

emb|CAA88744.1

C17C3.10 *

Orphan

II: -1,28

gb|AAB52693.1

F31A3.4 (F31A3.2) *

Orphan

X: 24,06

TR:Q19917

C17C3.7 *

Orphan

II: -1,28

gb|AAK31421

C17C3.8 *

Orphan

II:1,28

TR:Q18053

Gene identifications are those of the C. elegans genome project. The localization of the genes referred to the worm recombination genetic map as found in Wormbase [82]. Sequences marked with an asterisk form a well supported monophyletic group and encode proteins with two bHLH (see text for details). The Y69A2AR gene was not found in the databases. Its sequence comes from [45].

Table 5

The complete list of bHLH genes from Mus musculus

Gene name

Family

Human ortholog(s)

Accession number

Mash1

Achaete-Scute a

P50553

gb|AAB28830.1

Mash2

Achaete-Scute a

Q99929

gb|AAD33794.1

Mash3

Achaete-Scute b

N024228

sp|CAC37689

Myogenin

MyoD

P15173

sp|P12979

Myf6

MyoD

P23409

ref|NP_032683.1

MyoD

MyoD

P15172

sp|P10085

Myf5

MyoD

P13349

ref|NP_032682.1

E2A

E12/E47

N011269

sp|15806

TF12

E12/E47

Q99081

ref|NP_035674.1

TCF4

E12/E47

P15884/P15884 D

ref|NP_038713.1

KA1

E12/E47

?

dbj|BAA06218.1

Math1

Atonal

Q92858

dbj|BAA07791.1

Math5

Atonal

N024033

gb|AAC68868.1

Mist1

Mist

N007757

gb|AAF17706.1

Oligo1

Oligo

N011512

ref|NP_058664.1

Oligo2

Oligo

Q9NZ14

sptrembl|Q9EQW6

Oligo3

Oligo

N025741

sptrembl|Q9EQW5

Beta3

Beta3

N023718

gb|AAF32324.1

Q9H494

Beta3

N011476

sptrembl|Q9H494

Math4a

Neurogenin

Q9H2A3

gb|AAC53028.1

Math4b

Neurogenin

N024089

emb|CAA70366.1

Math4C

Neurogenin

Q92886

sp|P70660

Math2

NeuroD

N007825

dbj|BAA07923.1

Math3

NeuroD

N009563

gb|AAC15969.1

NDF1

NeuroD

Q13562

sp|Q62414

NDF2

NeuroD

Q15784

gb|AAC52203.1

Math6

Net

N005263

spnew|BAB39468

Mesp1

Mesp

?

gb|AAF70375.1

Mesp2

Mesp

?

gb|AAB51199.1

pMeso1

Mesp

N015926

ref|NP_062417.1

Pod1

MyoR

N007203

gb|AAC62513.1

MyoR

MyoR

N008253

gb|AAD10053.1

PTF1

PTFa

Q9HC25

emb|CAB65273.1

eHand

Hand

O96004

gb|AAB35104.1

dHand

Hand

O95300

gb|AAC52338.1

Twist

Twist

Q15672

gb|AAA40514.1

Dermo1

Twist

?

emb|CAA69333.1

Paraxis

Paraxis

N011493

gb|AAA86825.1

Scleraxis

Paraxis

?

gb|AAB34266.1

Hen1

NSCL

Q02575

gb|AAA39840.1

Hen2

NSCL

Q02577

gb|AAB22580.1

Tal1

SCL

P17542

emb|CAB72256.1

Tal2

SCL

Q16559/N024631

gb|AAA40162.1

Lyl1

SCL

P12980

emb|CAA40870.1

Lyl2

SCL

P12980

pir||B43814

Figa

Figa

N005420

sptrembl|O55208

AP4

AP4

Q01664

gb|AAF80448.1

Mnt

Mnt

Q99583

swissprot|O08789

Mxi1

Mad

P50539

swissprot|P50540

Mad1

Mad

Q05195

swissprot|P50538

Mad3

Mad

AAH00745

sptrembl|Q60947

Mad4

Mad

Q14582

pir||S60006

Max

Max

P25912

sp|P28574

N-Myc

Myc

P04198

gb|AAA39833.1

C-Myc

Myc

P01106

emb|CAA25508.1

L-Myc

Myc

P12524

emb|CAA32128.1

S-Myc

Myc

?

ref|NP_034980.1

SRC1

SRC

O43792

gb|AAB01228.1

SRC2

SRC

Q15596

gb|AAB06177.1

SRC3

SRC

Q9Y6Q9

sp|O09000

MITF

MITF

O75030

gb|AAF81266.1

TFE3

MITF

P19532

gb|AAB21130.1

TFEB

MITF

P19484

gb|AAD20979.1

TFEC

MITF

N009714/O14948

gb|AAD24426.1

SREBP1

SREBP

P36956

dbj|BAA74795.1

SREBP2

SREBP

Q12772

gb|AAG01859.1

USF1

USF

P22415

emb|CAA64627.1

USF2

USF

Q15853/N026304

pir||A56522

Mlx

MLX

Q9NP71

gb|AAK20940.1

TF4

TF4

Q9UH92

gb|AAB51368.1

Bmal1

BMAL

O00327

dbj|BAA76414.1

ARNT1

ARNT

P27540

gb|AAA56717.1

ARNT2

ARNT

Q9HBZ2

dbj|BAA09799.1

Clock

Clock

O15516

swissnew|O08785

NPAS2

Clock

Q99743/N023384

gb|AAB47249.1

Hif1a

HIF

Q16665

emb|CAA70306.1

EPAS1

HIF

Q99814

gb|AAC12871.1

Hif3a

HIF

O95262

gb|AAC72734.1

NPAS1

HIF/Sim/Trh

Q99742

gb|AAB47247.1

Sim1

Sim

P81133

gb|AAC05481.1

Sim2

Sim

Q14190

gb|AAB84099.1

NPAS3

Trh

Q9H323

gb|AAF14283.1

AHR

AHR

Q13804

dbj|BAA07469.1

Id1

Emc

P41134

sp|P20067

Id2

Emc

Q02363

gb|AAA79771.1

Id3

Emc

Q02535

sp|P41133

Id4

Emc

P47928

emb|CAA05120.1

Hey1

Hey

Q9Y5J3

emb|CAB51321.1

Herp1

Hey

Q9UBP5

gb|AAF37298.1

Hes1

Hairy

Q14469

dbj|BAA03931.1

Dec1

E(spl)

O14503

sptrembl|O14503

Dec2

E(spl)

BAB21502

spnew|BAB21503

Hes2

E(spl)

Q9Y543

dbj|BAA24091.1

Hes3

E(spl)

N019265

dbj|BAA19799.1

Hes5

E(spl)

N004350

dbj|BAA06858.1

Hes6

E(spl)

Q9P2S3

gb|AAF63757.1

Hes7

E(spl)

Q9BYEQ

spnew|BAB39526

BAA9469

E(spl)

N019265

spnew|BAA9469

Coe1

Coe

Q9UH73

swissprot|Q07802

MOTF1

Coe

?

gb|AAB58423.1

Coe2

Coe

?

swissprot|O08792

Coe3

Coe

?

swissprot|O08791

Mouse genes are listed with the family in which they are included, the identification of their human ortholog(s) (? indicates that no clear ortholog was found, see text for details), and their accession number. In most cases, several names exist for each gene. We report here only one name; synonyms can be found in the protein databases using the reported accession numbers.

Table 6

The complete list of bHLH genes from Saccharomyces cerevisiae

Gene name

Accession number

Family

RTG3P

gb|AAA86842.1

RTG3P

RTG1

sp| P32607

MITF

TYE7

sp|P33122

SREBP

HMS1

sp|Q12398

SREBP

Pho4

ref|NP_011227.1

Pho4

CBP

gb|AAA34490.1

CBP

Ino2

sp| P26798

Orphan

Ino4

sp|P13902

Orphan

Yeast genes are listed with the family in which they are included and their accession number.

Determination of orthology relationships

To carry out evolutionary analyses of multigene families requires one to distinguish orthologs, which have evolved by vertical descent from a common ancestor, from paralogs, which arise by duplication and domain shuffling within a genome [17]. Failure to do so can result in functional misclassification and inaccurate molecular evolutionary reconstructions [18,19]. The overall similarity (as determined by the BLAST E-value) is often used as a criterion to determine orthology relationships within large data sets such as complete genomes [20,21,22,23], but there is evidence that more rigorous phylogenetic reconstructions are required to confidently determine orthologies [22,24]. We therefore constructed phylogenetic trees to define groups of orthologous sequences, as we did previously [8] (see Materials and methods).

We determined 44 orthologous families that contain most of the metazoan bHLH families (Table 1 and Additional data). Two of these families also contain yeast genes. The criterion we used to define orthologous families was as in [8,25]; that is, orthologous families are monophyletic groups found in the gene trees constructed by different phylogenetic methods and whose monophyly is supported by bootstrap values larger than 50%. We named each family according to its first discovered member or, in a few cases, its best-characterized member. This analysis gave similar results to that described in [8], except that the additional sequences included in the present phylogenetic analyses led us to define six additional families of bHLHs from metazoans, compared with our previous report. We have also to mention the existence of three yeast-specific families.

Comparison of the human and mouse bHLH repertoires

We found a total of 125 and 102 different bHLH sequences in human and mouse, respectively (Tables 2 and 5). These sequences were used to make phylogenetic reconstructions as described above and in Materials and methods. This allows us to infer orthology relationships between mouse and human sequences. Two sequences were considered as orthologs if they are more closely related to each other than to any other mouse or human sequences. This can be easily detected in the phylogenetic trees, as the two sequences will form an exclusive monophyletic group (Figure 2a). Among the 125 human sequences, 94 can be accurately related to 1 (or in a few cases 2, see below) mouse genes (Table 2) and, conversely, human orthologs can be confidently assigned to 93 of the 102 mouse genes (Table 5). Among the 31 human genes and 9 mouse genes that do not show clear orthology relationships to any mouse or human genes, respectively, 8 human genes and 6 mouse genes are members of families in which phylogenetic relationships are uncertain - the Mesp, E12 and Coe families (Figure 2b and Additional data). The Mesp family contains four human genes and three mouse genes, the E12 family seven human and four mouse genes, and the Coe family four human and four mouse genes. Some of these genes cannot be clearly linked to each other (see Figure 2b for an example). It is, however, conceivable that such relationships do exist but that phylogenetic reconstruction methods fail to detect them. We therefore consider that, in the Mesp family for example (Figure 2b), three of the four human genes correspond to the three mouse genes, and so, to date, one human gene lacks an ortholog among the cloned mouse genes.

Applying the same reasoning to the E12 and Coe families leads us to conclude that at least 26 human genes (20% of the total) do not have orthologs among the mouse bHLH genes cloned to date and only 3 mouse bHLHs (3%) have no orthologs in the bHLH set we derived from the human genome sequence draft. Figure 2c shows a typical phylogenetic tree of a family containing human genes that lack mouse orthologs. The fact that only three mouse genes lack human orthologs strongly argues that, although our analysis was made on a draft version of the human genome sequence, the set of bHLH we retrieved is likely to be almost complete, and hence gives a highly accurate view of the bHLH repertoire of a human being. Additional BLAST searches for human orthologs of the three mouse bHLHs that lacked orthologs (Scleraxis, Dermo-1 and S-Myc) were unsuccessful, suggesting that these orthologs either do not exist in humans or are not in the draft sequence. We were recently made aware that there is some incompatibility between the current version of BLAST and the human genome sequence (probably due to the large number of Ns (unassigned nucleotides) in the sequence), which makes BLAST unable to locate some of the best or even exact matches of small query sequences (J.A.M. Leunissen, personal communication). This may explain why we missed the four genes cited above, and also why, in a few cases, we were unable to find known cloned human genes in the genome sequence (see Table 2).

We also found eight cases in which two human genes group together (with high statistical support) to the exclusion of any other genes and are often orthologs of a single mouse gene (Figure 2b and Additional data). Conversely, we found two cases in which two mouse genes are, collectively, orthologs of a single human gene (Figure 2d). This may reveal relatively recent duplications specific to the human or mouse lineage. In agreement with this, in all cases amino-acid identity between the two duplicates is high and is not confined to the bHLH. In addition, we found that in two cases (human sequences Q9UH92/N005106 and Q02363/N005999), one of the two duplicates lacks introns. The two copies are, furthermore, on different chromosomes. This strongly suggests that the duplications have occurred by retrotransposition, a type of event that appears to be rather frequent in humans [26]. In both cases, the copy lacking introns has stop codons in the bHLH, suggesting that it is a pseudogene.

Proteins with two bHLHs

Among the 39 bHLH from the worm, 6 cannot be assigned to any family (orphan genes; see Tables 1 and 4). Five of these have an unusual architecture in they contain two bHLH domains (see also [27,28]). Phylogenetic analysis of these proteins indicates that they result from the duplication of an ancestral gene that already contained two bHLHs (Figure 3). Both bHLH domains are loosely related (on the basis of overall similarity) to HER proteins (group E; Figure 1), but their inclusion in group E is not supported by phylogenetic reconstruction (Figure 3). In addition, they lack the Orange domain, which is characteristic of most HER proteins and provides them with functional specificity [29]. They also lack the WRPW motif found in the carboxy-terminal region of almost all HER proteins and which allows interaction with the Groucho represser protein [30,31,32]. Moreover, they lack a conserved proline in the basic domain that confers DNA-binding specificity on the HER proteins [30].
Figure 3

Worm proteins with two bHLH domains. A rooted NJ tree is shown that depicts the phylogenetic relationships of the five worm proteins with two bHLH domains. Mouse genes representative of some of the animal families have been included in this analysis. Rooting is as in Figure 1. Numbers above branches indicate per cent support in bootstrap analyses (1,000 replicates). As in Figure 1, the rooting should be considered arbitrary. Branch lengths are proportional to distance between sequences. Mm, Mus musculus; Ce, Caenorhabditis elegans. The sequences of the first bHLH of each worm proteins are shown in blue, the second in red. Both form monophyletic groups with high bootstrap values, indicating that these proteins originate from an ancestral protein that already had two bHLH domains. There is, furthermore, a weaker support (40% bootstraps) for an association of the two bHLH domains into a monophyletic group (not shown in the figure, as only nodes with 50% or more support are shown), suggesting that the ancestral protein may have acquired its two bHLH domains through tandem duplication rather than by association of unrelated bHLH domains.

No other protein with two bHLHs has been reported in other metazoans and we were unable to find such proteins in the fly and human genomes. A protein with two bHLH domains is found in rice (Oryza sativa; protein P0498B01.20; accession number BAB61947) but its sequence is completely unrelated to that of the worm protein. Several bHLH proteins do contain other DNA-binding and/or dimerization domains in addition to their bHLH, such as the PAS domain, leucine zippers or the Coe domain [6,33,34]. It is conceivable that these domains may cooperate and thereby confer particular functions on the proteins containing them. Similarly, the presence of two bHLHs might modify the specificity of the proteins containing them.

The establishment of the bHLH gene family

bHLH genes are found in all major subdivisions of the eukaryotes: metazoans, fungi and plants. In contrast, no bHLH sequences can be found in prokaryotes. It seems, therefore, that the bHLH motif was established in early eukaryote evolution. We have found eight different bHLH genes in the unicellular eukaryote, the yeast S. cerevisiae. Most of these genes were already cloned and have been functionally characterized (reviewed in [7]). These genes often regulate biochemical pathways (such as phosphate utilization, phospholipid and amino-acid biosynthesis, glycolysis) through the transcriptional activation of more-or-less large sets of genes involved in these pathways [7]. Orthologs of these genes are found in other distantly related yeasts such as Schizosaccharomyces pombe and Kluyveromyces lactis (our unpublished observations), indicating an ancient origin for the different bHLH genes among yeasts.

The relatively small number of bHLH genes found in the unicellular yeast contrasts with the large number found in multicellular eukaryotes such as animals and plants. We report here the existence of 39 different bHLH genes in C. elegans, 58 in D. melanogaster, and 125 in humans. Preliminary analysis of plant genomes, in particular of Arabidopsis thaliana and O. sativa, similarly indicates a large number of bHLH genes (more than 100 in the completely sequenced genome of A. thaliana, our unpublished observations). This important diversification of the bHLH repertoire in animals and plants has occurred independently, as plant and animal bHLH genes are never found in a same family. The current view of eukaryote phylogeny suggests that fungi and animals are more closely related to each other than to the plants [35]. Nevertheless, we found that only two families contain both yeast and animal genes (see Table 1), suggesting that the common ancestor of fungi and animals may have possessed even fewer bHLH genes than the present-day yeasts. In the near future, the genome projects currently underway on various 'basal' eukaryotes (see [36,37]) may give important insights into the very early evolutionary history of the bHLH family.

We suggest that the diversification of bHLH genes is directly linked to the acquisition of multicellularity and hence to the recruitment of genes involved in cell functions such as metabolism into the developmental processes required to build multicellularity. Indeed, in animals, bHLH genes are generally involved in development and in tissue-specific gene regulation (reviewed in [1,2,3,4,5]). A similar situation may exist in plants, although very few bHLH genes have been functionally characterized. In addition, in both animals and plants, the diversification of bHLH genes seems to have occurred early in the evolution of these lineages.

Indeed, our phylogenetic analysis of animal bHLH genes shows that most belong to 44 different orthologous families. Of these families, 43 contain representatives from both protostomes and deuterostomes, and must therefore be represented in their common ancestor (often called Urbilateria) [38], which lived in pre-Cambrian times (600 million years ago). In addition, the few bHLH genes that have been cloned from cnidarians, which are not bilaterians, are clearly included in families (see the Twist, MyoD and ASC families in Additional data), suggesting that the establishment of at least some families predates the divergence of bilaterians and non-bilaterians. Further analyses of bHLH genes in cnidarians, sponges and slime molds will help to resolve the issue of the early evolution of bHLH genes in animals.

Our preliminary analyses of plant bHLH genes are consistent with an early diversification in plants, as in animals. Indeed, many A. thaliana bHLH genes have clear orthologs in a distantly related plant, O. sativa, whose genome has been partially sequenced (our unpublished observations). Arabidopsis is a eudicotyledon and Oryza a member of the Liliopsida (a monocotyledon), and given the phylogenetic relationships of these clades [39] this suggests that the possession of numerous bHLH genes might be ancestral to angiosperms. Further analysis of the evolution of bHLH in plants will require the completion of the genome projects currently underway on rice and tomato (a eudicotyledon of a different lineage from Arabidopsis), as well as the isolation of bHLH in a broader spectrum of plant species, in particular in basal angiosperms and non-angiosperms.

Evolution of bHLH genes in metazoans

Comparison of the bHLH repertoires found in the protostomes and the deuterostomes gives important insights in to the evolution of the bHLH family in metazoans. The conclusions that can be drawn are completely consistent with those presented in our previous work [8] but the inclusion of the probable complete set of bHLH from a vertebrate strengthens these conclusions.

Most families (43/44) contain genes from protostomes (fly and/or nematode) and deuterostomes, indicating that these families were already present in the last common ancestor of both protostomes and deuterostomes, that is, of all bilaterians. The fact that most families contain both protostome and deuterostome genes also suggests that there was no addition of new bHLH types in the corresponding lineages, and therefore no important diversification of the ancestral repertoire. A single family contains vertebrate members and no fly or worm genes. This may represent the emergence of new bHLH types in the vertebrate lineage, or alternatively a loss of ancestral types in both fly and nematode. The analysis of bHLH genes from molluscs or annelids might help to settle this question. It is now widely believed that the Bilateria (the triploblastic metazoans) are composed of three main lineages: deuterostomes (which include vertebrates and echinoderms) and protostomes, which themselves include two large groups, the ecdysozoans (for example, arthropods and nematodes) and the lophotrochozoans (for example, annelids, molluscs and flatworms) (reviewed in [16]). Therefore, the finding of ortholog genes in vertebrates and lophotrochozoans but not in fly and nematode would strongly suggest that gene loss(es) has (have) occurred in the ecdysozoan lineage.

Similarly, the case of families that contain vertebrate and either worm or fly genes is best explained by gene losses that occurred, inside the ecdysozoan clade, in either lineage after the arthropod/nematode divergence. This occurred in the fly lineage for very few families (4/44), suggesting the existence of a strong pressure to maintain the entire bHLH repertoire. The much larger number of families (13/44) that have vertebrate and fly members but no nematode representative suggests that extensive bHLH gene losses have occurred in the worm lineage. Strikingly, the worm lacks the important cellular and developmental regulator Myc. A similar absence of important developmental regulators, such as Hedgehog, Toll/IL-1 and JAK/STAT pathway elements has also been reported in the nematode [27]. In addition, a large number of nematode genes (6/39) cannot be clearly assigned to specific families (orphan genes). This is probably due to the high divergence rate reported for nematode genes in general [40,41] and which we found within our specific data set ([8] and data not shown).

Interestingly, however, some nematode sequences have diverged very little from their fly or mouse counterparts. These include the few functionally characterized C. elegans bHLH genes that show overall functional conservation with their vertebrate and/or fly orthologs; for example, the C. elegans orthologs of twist and myoD are involved in muscle formation [42,43], and the orthologs of atonal and NeuroD (lin-32 and cnd-1) have a role in nervous-system development [44,45]. The genetic control of developmental processes such as neurogenesis and myogenesis relies on small sets of interacting genes (syntagms) [46]. The function of syntagms crucially relies on specific molecular interactions among their members, hence imposing strong structural constraints on them and preventing structural diversification (for discussion on syntagms and evolution, see [47]). This may explain why such networks are strongly conserved throughout metazoan evolution [48,49] and why nematode genes involved in such networks have been subject to special constraints.

Duplication of bHLH genes in vertebrates

An extensive increase of bHLH family complexity has occurred in vertebrates: the most frequent number of different bHLH genes per family is one in fly (30/44) and worm (27/44), and two in human (14/44; but 20/44 human families do in fact contain more than two genes). Most bHLH families (32/44), as with other gene families, have more members in vertebrates than in other phyla (Table 1). Of these families, 14 (32%) contain four or more vertebrate genes (Table 1) and hence may reveal the occurrence of two whole-genome duplications (the 2R hypothesis) in early vertebrate evolution. In the most popular version, this is thought to have occurred by one duplication at the root of the vertebrates and a second in the Gnathostomata lineage, after its divergence from Agnatha (reviewed in [50]).

Several recent analyses, however, tend to refute (at least, do not support) this hypothesis (reviewed in [51]). For example, the current mammalian gene number estimations based on the human draft sequence, ESTs and comparisons with other vertebrates propose that the human genome would contain no more than 35,000 genes; that is, about twice the number of fly and worm [12]. Consistent with this, many gene families in vertebrates have fewer than four genes. This might, however, result from gene loss during or after the rounds of duplication [50]. In addition, phylogenetic analyses of gene families that comprise four members cast doubt on the 2R hypothesis.

As pointed out by Hughes [52], the presence of four members in a vertebrate gene family by itself does not support the genome duplication hypothesis. Support may only come from families whose phylogenetic tree shows a topology of the (AB) (CD) form, that is, two pairs of two closely related paralogs [52]. Hughes [52] discussed the phylogenies of 13 protein families important in development, and found that only one of them shows an (AB) (CD) topology. Similar results were recently obtained by Martin [53] and Hughes et al. [54] on several other families with much more rigorous phylogenetic tests. These results have led to the alternative hypothesis that the abundance of duplicated genes in vertebrates compared to invertebrates may be due to a high rate of local duplications, rather than entire genome duplications (reviewed in [51]). The analysis of additional gene families may help to discriminate between these hypotheses. Phylogenetic trees of the 14 bHLH families that contain four or more members do not clearly show such (AB) (CD) topologies (see Additional data). We have, however, to note that the phylogenies inside families often have only poor resolution and it is therefore difficult to draw firm conclusions from them. Nevertheless, our data clearly do not support the 2R hypothesis.

Conclusions

We identified the probable full complement of bHLH in three different metazoans that are representative of the two major subdivisions of the animal kingdom, the protostomes (C. elegans and D. melanogaster) and the deuterostomes (humans). Most of these genes belong to one of 44 orthology families. Most of these families (43/44) have protostome and deuterostome members, and must therefore have been represented in their common ancestor before the Cambrian radiation which saw the emergence of all present-day phyla, and many extinct ones. Morphologically, these ancestors (also called Urbilateria [38]) were probably coelomates with antero-posterior and dorso-ventral polarity, rudimentary appendages, some form of metamerism, a heart, sense organs such as photoreceptors and a complex nervous system [55]. Genetically, they possessed numerous homeobox genes (among which are at least seven Hox genes [56]), several intercellular signaling pathways (TGF-β, Hedgehog, Notch, EGF), at least four Pax genes [25], and 38 C2H2 zinc-finger proteins [57]. Our analysis suggests that their genome contained at least 43 different bHLH genes. The functional conservation that is often observed between protostome and deuterostome orthologs indicates that some of the developmental functions associated with the present-day genes were already established in Urbilateria, further indicating the genomic and developmental complexity of these ancient ancestors.

Materials and methods

BLAST searches

The full set of bHLH sequences in the fly, worm, and yeast were obtained mostly by BLASTP searches [13] against the new releases of the complete genomic sequences of C. elegans [58], D. melanogaster [59], and S. cerevisiae [59]. Mouse bHLH genes were obtained by BLASTP searches [13] against the more recent versions of the non-redundant database at NCBI [59] and the Sanger protein databases [60]. In addition, we retrieved and analyzed all the bHLHs from these organisms that are listed in the SMART database [14,15,61]. The comparison with the lists of bHLHs found in the SMART database and published by other groups [20,27,62,63] strongly suggests that we retrieved the full set of bHLH genes present in the fly, yeast, and worm genomes, as well as all the cloned mouse bHLH genes to date.

TBLASTN searches were done at the NCBI on the human genome [64] and at the Doe Joint Genomic Institute (University of California and the US Department of Energy) for the pufferfish and sea squirt genomes [65]. We used as query two different sequences (usually one from mouse and one from fly or worm) of each of the families we defined previously [8]. Searches were done at two stringencies, E < 1 and E < 0.01, with all other parameters set to default. The BLAST searches detected some sequences that display only low overall similarity with the query, or similarities only to a part of the bHLH domain. We checked these sequences by hand and found that in all cases they did not correspond to bona fide bHLH domains. We hence did not include these sequences in our subsequent analyses. During the course of our work, four different successive drafts of the human genome have become available. The data presented in this paper come from the third version (April 2001). Careful examination of the fourth version (July 2001) did not give additional data. A final check has been done on the latest release (version 6) in November 2001 with no significant changes, except that some contigs have been renamed and two sequences were no longer found. We do not include these two sequences (which were closely related duplications of existing bHLH genes) as they may represent artifacts of the genome sequence assembly process. We cannot exclude the possibility, however, that they are bona fide bHLH genes that were no longer detected as a result of limitations of the current version of BLAST (see Results and discussion).

Phylogenetic analyses

Protein alignments were made using ClustalW [66] with no adjustment of the default parameters and were subsequently edited and manually improved in Genedoc Multiple Sequence Alignment Editor and Shading Utility (Version 2.6.001) [67]. The evaluation of percentage conservation of residues in multiple sequence alignments was done using the Blosum62 Similarity Scoring Table [68]. Only the bHLH motif (determined as in [69]), plus a few flanking amino acids, was used in most of our analyses because the remaining parts of proteins from independent clades are either not homologous or have diverged so much that the alignments are meaningless. The facilities of the Belgian EMBnet Node [70] were used for sequence analysis using Genedoc software and for most of the protein alignments using ClustalW.

Distance trees were constructed with the neighbor-joining (NJ) algorithm [71] using PAUP 4.0 [72] based on a Dayhoff PAM 250 distance matrix [73]. The resultant trees were bootstrapped (1,000 bootstrap replicates) to provide information about their statistical reliability. Bootstraps were made with PAUP 4.0, parameters set to default values. Given the large number of sequences (> 300), we were unable, because of computer calculation limitations, to perform maximum-parsimony (MP) and maximum-likelihood (ML) analyses on the multiple alignment that contains all sequences. We made several additional alignments that include only those bHLH sequences that belong to a particular high-order group (Figure 1) [8]. NJ, MP and ML trees were constructed from these alignments and were fully congruent with the NJ trees constructed from the general alignments. The MP analysis was performed using PAUP 4.0 with the following settings: heuristic search over 100 bootstrap replicates, MAXTREES set up to 1,000 due to computer limitations, other parameters set to default values. Maximum likelihood (ML) was done using TreePuzzle 4.0.2 [74]. The ML was performed using the quartet-puzzling tree-search procedure with 25,000 puzzling steps, using the Jones-Taylor-Thornton (JTT) model of substitution [75], the frequencies of amino acids being estimated from the data set [74], with an uniform rate of substitution. The trees were displayed with the Tree view program (version 1.5) [76], saved as PICT files, converted into JPEG files using Graphic Converter, and then annotated using Adobe Photoshop and Adobe Illustrator.

Additional data files

Additional data files are available with the online version of this paper as follows:

Multiple alignments (in rich text format) on which the trees displayed in the figures are based.

A list of all bHLH sequences from human in rich text format

Multiple alignments for each family of bHLH proteins: the Achaete-Scute a family, the Achaete-Scute b family, the AHR family, the AP4 family, the ARNT family, the Atonal family, the Beta3 and Oligo families, the Bmal family, the Clock family, the E12/E47 family, the Extramacrochaete family, the Enhancer of split family, the Fig alpha family, the Hairy family, the Hand family, the Hey family, the Mad and Mnt families, the Max family, the Mesp family, the Mist family, the MITF family, the Myc family, the Myod family, the MyoR family, the Net family, the NeuroD family, the Neurogenin family, the NSCL family, the Paraxis family, the PTF a and b families, the SCL family, the SRC family, the SREBP family, the TF4 and Mlx families, the Trh, Hif, and Sim families, the Twist family, and the USF family.

Representative phylogenetic trees (NJ trees bootstrapped 1,000 times to provide statistical support to the nodes, usually rooted with a sequence from a closely related family. In a few cases, closely related families are shown in the same phylogenetic tree) of each family of bHLH proteins: the Achaete-Scute a family, the Achaete-Scute b family, the AHR family, the ARNT family, the Atonal family, the Beta3, Mist, and Oligo families, the Bmal family, the Clock family, the E12/E47 family, the Extramacrochaete family, the Enhancer of split family, the Fig alpha family, the Hairy family, the Hand family, the Hey family, the Mad and Mnt families, the Max family, the Mesp family, the MITF family, the Myc family, the Myod family, the MyoR family, the Net family, the NeuroD family, the Neurogenin family, the NSCL family, the Paraxis family, the PTF a and b families, the SCL family, the SRC family, the SREBP family, the TF4 and Mlx families, the Trh, Hif, and Sim families, the Twist family, and the USF family.

Species name abbreviations are as in the figure legends and as below: Av, Asteris vulgaris; AVIM, avian myelocytomatosis virus CMII; Bb, Branchiostoma belcheri; Bf, Branchiostoma floridae; Bm, Bombyx mori (domestic silkworm); Caebr, Caenorhabditis briggsae; Cc, Ceratitis capitata; Cp, Cynops pyrrhogaster; Cs, Cupiennius salei; Cyca, Cyprinus carpio; Ds, Drosophila simulans; Dv, Drosophila virilis; Dy, Drosophila yakuba; Hr, Halocynthia roretzi; Hv, Hydra vulgaris (Hydra attenuata); Ilo, Ilyanassa obsoleta; Jc, Juonia coenia (Precis coenia) (peacock butterfly); Kl, Kluyveromyces lactis; Lv, Lytechinus variegatus (green urchin); Nv, Notophtalmus viridens; Ol, Oryzias latipes (Japanese medaka); Om, Oncorhyncus mikis; pc, Podocorine carnea; Pv, Patella vulgata (common limpet); Rn, Rattus norvegicus; Sb, Spermophilus beecheyi; Sc, Saccharomyces cerevisiae (baker's yeast); Sp, Schizosaccharomyces pombe; Spu, Strongylocentrotus purpuratus (purple urchin); St, Silurana tropicalis; Tc, Tribolium castaneum; Tricho, Trichinella spiralis

Declarations

Acknowledgements

We thank Robert Herzog, Marc Colet and André Adoutte for support. We are grateful to Lionel Christiaen who made us aware of the Takifugu and Ciona genome projects, and to Marc Colet and Robert Herzog for comments on the manuscript. This work has been supported by the Federal Office for Scientific, Technical and Cultural Affairs (V.L.) and the Centre National de la Recherche Scientifique, the Institut Français de la Biodiversité, and the Université Paris-Sud (M.V.).

Authors’ Affiliations

(1)
Belgian EMBnet Node - Service de Bioinformatique, Université Libre de Bruxelles, Département de Biologie Moléculaire
(2)
Evolution et Développement des protostomiens, Centre de Génétique moléculaire

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