gb-2002-3-6-research0030 GBJ Research <p>Phylogenetic analysis of the human basic helix-loop-helix proteins</p> Ledent Valérie Paquet Odier Vervoort Michel vervoort@cgm.cnrs-gif.fr

Belgian EMBnet Node - Service de Bioinformatique, Université Libre de Bruxelles, Département de Biologie Moléculaire, Rue des Professeurs Jeener et Brachet 12, B-6041 Gosselies, Belgium

Evolution et Développement des protostomiens, Centre de Génétique moléculaire - UPR 2167 CNRS, 1 Ave de la terrasse, 91198 Gif-sur-Yvette Cedex, France

 Genome Biology 1465-6906 2002 3 6 research0030.1 research0030.18 http://genomebiology.com/2002/3/6/research/0030 10.1186/gb-2002-3-6-research0030 12093377 29 11 2001 7 3 2002 15 4 2002 30 5 2002 2002 Ledent et al., licensee BioMed Central Ltd

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

The basic helix-loop-helix (bHLH) proteins are a large and complex multigene family of transcription factors with important roles in animal development. This article identifies 39 bHLH genes in Caenorhabditis elegans, 58 in Drosophila melanogaster and 125 in human, in 44 orthologous families in total. Genes from these families seem to have been present in the last common ancestor of worm, fly and human.

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.

 Evolution Bioinformatics Genetics Genome studies

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].

<p>Table 1</p>

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&#38;#8224;

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].

<p>Figure 1</p>

Phylogenetic relationships and high-order grouping of the bHLH families

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.

 <p>Figure 2</p>

Some examples of phylogenetic relationships among human and mouse bHLH

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.

<p>Table 2</p>

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

</