J Mol Evol (1996) 43:170–178© Springer-Verlag New York Inc. 1996

Class III POU Genes: Generation of Homopolymeric Amino Acid Repeats
Under GC Pressure in Mammals
Kenta Sumiyama,1 Keiko Washio-Watanabe,1 Naruya Saitou,2 Toshiyuki Hayakawa,1 Shintaroh Ueda1
1
2

Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113, Japan
Laboratory of Evolutionary Genetics, National Institute of Genetics, 1111 Yata, Mishima 411, Japan

Received: 27 December 1995 / Accepted: 10 April 1996

Abstract. The class III POU transcription factor genes
play an important role in the nervous system. Comparison of their entire amino acid sequences disclosed a remarkable feature of particular mammalian class III POU
genes. Alanine, glycine, and proline repeats were present
in the mammalian Brain-1 gene, whereas most of these
repeats were absent in the nonmammalian homologue.
The mammalian Brain-2 gene had alanine, glycine, proline, and glutamine repeats, which were missing in thenonmammalian homologue. The mammalian Scip gene
had alanine, glycine, proline, and histidine repeats, but
the nonmammalian homologue completely lacked these
repeats. In contrast, the mammalian Brain-4 gene had no
amino acid repeats like its nonmammalian homologue.
The mammalian genes containing the characteristic
amino acid repeats had another feature, higher GC content. We found a positive correlation between the GC
content and the amino acid repeat ratio. Those aminoacids were encoded by triplet codons with relatively highGC content. These results suggest that the GC pressure
has facilitated generation of the homopolymeric aminoacid repeats.
Key words: POU — Human Brain-1 — GC pressure— Amino acid repeats

Correspondence to: S. Ueda

Introduction
A large number of homeobox genes have been isolated
and grouped into several families. The POU family geneis identified as a homeobox gene containing a conserved
150–160-amino-acid region termed ‘‘POU domain,’’which was first discovered as a common sequenceamong four transcription factors, Pit-1, Oct-1, Oct-2, andunc-86 (Herr et al. 1988). The POU domain is requiredfor high-affinity binding to the octamer DNA sequence,
which is a well-studied motif present in the enhancers
and the promoters of both ubiquitously and tissuespecifically expressed genes. The POU domain is divided into two regions separated by a short sequencecalled a linker: a classical homeodomain termed ‘‘POUhomeo domain’’ in the carboxy-terminal region and a
specific domain termed a ‘‘POU-specific domain’’ in theamino-terminal region. At present, POU family genes areclassified into five or more classes, based on the homology of the POU domain (for review, see Rosenfeld 1991;
Verrijzer and Van der Vliet 1993; Duboule 1994).
POU family proteins act as transcription factors,
which regulate tissue-specific gene expression at different stages of development. The class III POU members
are especially thought to have an important role in neuralsystem development. The mammalian class III POU
genes (Brain-1, Brain-2, Brain-4, and Scip) are expressedin the central nervous system (He et al. 1989; Le Moine
and Young 1992; Mathis et al. 1992). The Brain-2 geneis strongly expressed in the dorsomedial paracellular region of the paraventricular nucleus in which corticotropin-releasing hormone (CRH) is produced, and its prod-

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uct binds to the DNA element in the promoter region of
the CRH gene (Li et al. 1993). The Scip gene is expressed in specific neurons and myelinating glia, and its
product binds to the promoter of myelin protein P0 gene,a Schwann cell surface adhesion molecule (He et al.
1991). The Brain-4 gene is expressed in rats from embryonic day 11.5 into adulthood (Le Moine and Young
1992) and is considered to be a candidate gene for deafness with fixation of the stapes (De Kok et al. 1995)
Cf1a, a neuron-specific class III POU transcription factor
of fruit fly, binds to the DNA element required for expression of the DOPA decarboxylase gene in selected
dopaminergic neurons (Johnson and Hirsh 1990). POUM1 of silk worm is expressed in neural system as well as
in silk gland (Fukuta et al. 1993). Class III POU genes
were found also in nematode and planarian (Bürglin et al.
1989; Orii et al. 1993).
Through comparison of the amino acid sequencesamong the class III POU genes so far published, we
noticed the presence of amino acid repeats only in certain
genes. Using newly determined complete nucleotide sequences of the human Brain-1 gene, we discovered a
remarkable feature—that characteristic amino acid repeats were present only in the mammalian Brain-1,
Brain-2, and Scip genes and were well conserved in both
position and repeat number among each mammaliangene. It is known that the genome of warm-blooded vertebrates including mammals is a mosaic of very long(more than 200 kilobases) DNA segments, isochores,
whereas the genome of cold-blooded vertebrates is compositionally uniform, and that the base composition ofgenes mainly depends on the GC content of the isochores
harboring the sequences (Bernardi 1993 and references
therein). Nucleotide changes are, therefore, considered to
be under the influence of compositional constraints (GC/AT pressure). As a significant proportion of the nucleotide changes at the third codon position are silent substitutions, it is likely that the GC content at the third codon
position reflects directly the degree of compositional
constraints onto the isochores harboring the sequences.
We found a strong correlation between the repeat numbers of the characteristic amino acids and the GC content
at the third codon position of the class III POU genes.

Materials and Methods
Cloning of the Human Class III POU Genes. The class III POU probe
was prepared by degenerate PCR using total brain cDNA from rat
embryo as a template DNA. The primers were 58-CCGAATTCACCTCTGACGACCTGGA-38 and 58-CCAAGCTTGGGTCATGCT(T/C)TT(T/C)TC(T/C)TT(T/C)TG-38, which were designed toamplify the conserved POU domain region. PCR product was digested
with restriction enzymes EcoRI and HindIII and cloned into aphagemid vector. A phagemid clone containing the POU domain of therat Brain-2 gene was obtained, and its insert DNA was used as a probe
for screening the human genomic library. Recombinant phage clones

Table 1. List of genomic DNA (g), cDNA (c), and amino acid (a)sequences employed in this study
Species

Gene

Reference

Human

Brain-1 (g)Brain-2 (c)Brain-4 (c)Scip (c)Brain-1 (g)Brain-2 (g)Brain-4 (g)Scip (c)Brain-2 (a)Brain-4 (c)Scip (c)XLPOU1 (c)XLPOU2 (c)XLPOU3 (c)ZFPOU1 (c)Cfla (c)POU-M1 (c)DJPOU1 (c)ceh6 (a)This study
Schreiber et al. 1993de Kok et al. 1995
Faus et al. 1994
Hara et al. 1992
Hara et al. 1992
Hara et al. 1992
Meijer et al. 1990
Li et al. 1993
Le Moine and Young 1992
Monuki et al. 1990
Agarwal and Sato 1991
Witta et al. 1995
Baltzinger et al. 1992
Matsuzaki et al. 1992
Billin et al. 1991
Fukuta et al. 1993
Orii et al. 1993Bürglin et al. 1989

Mouse

Rat

Frog

Zebrafish
Fruit fly
Silk worm
Planarian
Nematode

containing the human class III POU genes were isolated, and their
nucleotide sequences were determined by the dideoxynucleotide chain
termination method. Other DNA manipulation procedures were performed using standard protocols (Sambrook et al. 1989).
Sequence Analysis. The class III POU genes employed here werefrom five vertebrates and four invertebrates as listed in Table 1: human(Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), frog
(Xenopus laevis), zebrafish (Brachydanio rerio), fruit fly (Drosophilamelanogaster), silk worm (Bombyx mori), planarian (Dugesia japonica), and nematode (Caenorhabditis elegans). The amino acid sequence of the human Brain-1 was deduced from the nucleotide sequence of genomic DNA determined in the present study. Amino acidsequences of human Brain-2, Brain-4, and Scip (also termed Oct-6 orTst-1); mouse Scip; rat Brain-4 and Scip; frog XLPOU1, XLPOU2, andXLPOU3; zebrafish XFPOU1; fruit fly Cf1a; silk worm POU-M1; andplanarian DJPOU1 are deduced from their cDNA sequences, while
those of mouse Brain-1, Brain-2, and Brain-4 are from genomic sequences. As for rat Brain-2 and nematode ceh6, only their amino acidsequences are reported. These sequence data were obtained from
DDBJ/EMBL/GenBank, PIR and SWISS-PROT databases. These
amino acid sequences were initially aligned using a multiple sequencealignment program in CLUSTAL V (Higgins et al. 1992) and were
further visually adjusted to increase similarity. Phylogenetic trees wereconstructed for 144 amino acid residues of the conserved POU domain
containing POU-specific, linker, and POU-homeo domains using theneighbor-joining method (Saitou and Nei 1987), because the sequencehomology outside the POU domain was too low to align their entire
amino acid sequences among all the class III POU genes. The bootstrap
probabilities were obtained by resampling 1,000 times using
CLUSTAL V.

Results and Discussion
Human Brain-1 Gene
We isolated several independent clones of the humangenomic DNAs containing the class III POU genes withthe reduced washing condition using 0.15 M sodium

172

Fig. 1. Nucleotide sequence of the human Brain-1 genomic DNA and its deduced amino acid sequence. Translation start site (ATG) is predicted
in concordance with that of the mouse Brain-1 gene (Hara et al. 1992). The POU-specific and POU-homeo domains are indicated with shadowed
boxes. Asterisk indicates stop codon.

chloride and 0.015 M sodium citrate at 55°C. Theseclones were identified by partially determining their
nucleotide sequences as the Brain-1, Brain-2, and Scip
genes. Entire coding sequences of the human Brain-2,
Brain-4, and Scip genes have already been published,
whereas only the amino acid sequence of the POU domain was reported for the human Brain-1 gene. Therefore, we determined the complete nucleotide sequence ofthe human Brain-1 gene on both strands and deduced its
amino acid sequence (Fig. 1), based on the similarities tothe mouse Brain-1 gene (Hara et al. 1992). There was adiscrepancy of a single amino acid residue between theprevious sequence by He et al. (1989) and the present
sequence: Position 433 of figure 1 was serine in He et al.

(1989), whereas that was alanine in the present study.
This discrepancy can be accounted for by polymorphism.
However, the alanine residue of this position is conserved among the Brain-1 homologues of the distantly
related species (mice and zebrafish). This alanine residue
is also highly conserved among the class III POU genes.
We therefore adopted our sequence for the basis of thecomparison in the present study.
The human Brain-1 gene was intronless and GC rich
(73.9% in the entire coding region), especially in theamino-terminal region of the coding sequence, like themouse Brain-1 gene (Hara et al. 1992). This region encoded alanine, glycine, and proline repeats, and thesecharacteristic amino acid repeats were well conserved

173

Fig. 2. Sequence alignment within the Brain-1 homologues. h, m, and z indicate human, mouse, and zebrafish, respectively. The POU-specificand POU-homeo domains are indicated with shadowed boxes. Gaps are represented by hyphens.

between humans and mice not only in position but also in
repeat number.

Amino Acid Repeats Characteristic to
Mammalian Genes
Whole amino acid comparison showed high sequencesimilarity among the human and mouse Brain-1 and thezebrafish ZFPOU1 genes, as shown in Fig. 2. The humanand the mouse amino acid sequences were almost identical with each other. Variations in the Brain-1 between
humans and mice were single amino acid difference inpositions 10, 17, and 158, one gap involving two residues
in positions 84–85, and two gaps caused by length variations in repeat number of amino acid repeats in positions
30–43 (14 and 13 glycine repeats in humans and mice,
respectively) and positions 298–303 (six and four glycine repeats in humans and mice, respectively). In contrast, the zebrafish ZFPOU1 showed a striking featurequite different from the mammalian homologues (Brain1): absence of some homopolymeric amino acid repeats(sequences without interruptions in the run of a singleamino acid residue). Those missing repeats were alanine
repeats in positions 191–206, glycine repeats in positions
30–43, 243–248, and 298–303, and proline repeats inpositions 141–146 and 176–181.
There was the same situation in the Brain-2 homologues as that in the Brain-1 homologues. The mammalian Brain-2 genes also had several homopolymericamino acid repeats like the mammalian Brain-1 genes.

However, the nonmammalian Brain-2 homologue (frog
XLPOU3) lacked all the amino acid repeats consisting of
alanine (positions 173–177), glycine (positions 68–88),
proline (positions 241–247), and glutamine (positions
129–151) in mammals, as shown in Fig. 3A.
In the Scip homologues, the situation was the same as
those in the Brain-1 and Brain-2 homologues (Fig. 3B).
The mammalian genes were highly similar to each otherthroughout the entire coding region including amino acidrepeats such as alanine, glycine, proline, and histidine
repeats (positions 27–37, 80–85, 113–118, 176–181,
449–454, 458–463, and so on), whereas the frog homologue (XLPOU1) had no amino acid repeats. In addition,
an intraspecific length variation of alanine repeats in positions 27–37 both in humans and mice was found. In
humans Faus et al. (1994) found nine repeats, while wefound 11 repeats (data not shown). In mice there wereeight and nine repeats (Meijer et al. 1990; Suzuki et al.
1990; Zimmerman et al. 1991; Hara et al. 1992).
In contrast to the Brain-1, Brain-2, and Scip genes, themammalian Brain-4 genes so far reported have no characteristic amino acid repeats. Therefore, there were nogaps between the mammalian and nonmammalian homologues (Brain-4 an XLPOU2), as shown in Fig. 3C.

Wide Variation of GC Content in the Class III POU
Gene GroupA high GC content was another characteristic feature forthe mammalian Brain-1, Brain-2, and Scip genes, in ad-

174Fig. 3. Sequence alignment within the homologues of the class III POU genes. A Brain-2, B Scip and C Brain-4. h, m, r, and f indicate human,
mouse, rat, and frog, respectively. The POU-specific and POU-homeo domains are indicated with shadowed boxes. Gaps are represented byhyphens.

dition to an extraordinary structure containing homopolymeric amino acid repeats. Table 2 shows the GC
content at the first, second, and third codon positions of
all the class III POU genes, together with those of the
POU domain alone. A great variation in the GC content
was observed especially at the third codon position,

where GC pressure is directly reflected. The lowest GC
content at the third position was 23.0% for the planarian
DJPOU1, while the highest one was 91.4% for the human Brain-1. There was a wide variation of the GC content at the third position only among the mammaliangenes, ranging from 64.6% for the rat Brain-4 to 91.4%

175

Fig. 3.

Continued.

for the human Brain-1. This wide variation among the
mammalian class III POU genes was not interspecific but
intergenic.
To the contrary, the zebrafish ZFPOU1 and the frog
XLPOU1 and XLPOU3 genes showed relatively lower
GC content. There is a wide variation at the third codon
position among genes of warm-blooded vertebrates including mammals (from 40% through nearly 100%),

Table 2.

whereas the GC content in the other organisms waswithin a narrow range (Bernardi 1993 and references
therein). Therefore, difference of the GC content between the nonmammalian genes (zebrafish ZFPOU1,
frog XLPOU3, and frog XLPOU1) and their mammalian
homologues (Brain-1, Brain-2, and Scip) might reflect
the difference in genome structure between the warmand cold-blooded vertebrates.

GC contents (%) of class III POU genes
POU domain

Species

Entire coding region

Gene

First

Second

Third

First

SecondThird

Brain-1
Brain-2
Brain-4
Scip

55.6
50.0
50.0
56.9

38.9
39.6
38.9
38.9

95.8
84.0
76.4
96.5

73.3
70.5
59.4
76.7

57.1
49.6
46.7
56.5

91.4
86.3
72.7
89.4

Brain-1
Brain-2
Brain-4
Scip

55.6
49.3
49.3
57.6

38.9
39.6
38.9
38.9

82.6
79.2
63.9
91.7

73.0
70.2
58.6
77.3

56.9
49.3
46.7
56.4

83.3
77.8
65.8
84.7

Brain-4
Scip

49.3
57.6

38.9
38.9

63.2
90.3

58.6
77.0

46.7
56.6

64.6
83.9

XLPOU1XLPOU2XLPOU3

46.5
48.6
46.5

38.2
38.2
41.0

68.1
56.9
72.9

56.2
55.0
57.7

42.4
44.2
46.2

67.6
61.6
71.0

ZFPOU1

50.0

38.2

72.2

58.5

47.9

65.7

Cf1a

56.3

38.9

84.7

65.1

48.2

74.0

POU-M1

53.5

38.9

74.3

63.4

47.7

78.1

DJPOU1

39.6

38.2

25.7

37.3

36.4

23.0

Human

Mouse

Rat

Frog

Zebrafish
Fruit fly
Silk worm
Planarian176

Correlation Between GC Content and Amino Acid
Repeat Ratio

Fig. 4. Correlation between the GC content (%) and the amino acidrepeat ratio (AARR). The genes employed here were human Brain-1(H1), mouse Brain-1 (M1), zebrafish Brain-1 homologue, ZFPOU1(Z1), human Brain-2 (H2), mouse Brain-2 (M2), frog Brain-2 homologue, XLPOU3 (F2), human Scip (HS), mouse Scip(MS), rat Scip
(RS), frog Scip homologue, XLPOU1 (FS), human Brain-4 (H4),mouse Brain-4 (M4), rat Brain-4 (R4), frog Brain-4 homologue,
XLPOU2 (F4), fruit fly Cf1a (D), and silk worm POU-M1 (B). Triangles and circles represent the mammalian and nonmammalian class
III POU genes, respectively. Since the planarian DJPOU1 containing
no amino acid repeats showed an extraordinary GC content (23.0%), it
was excluded from this figure and calculation of correlation coefficient.

Fig. 5. A phylogenetic tree of the class III POU genes. The humanOct-1 was used as an outgroup. Numbers on the branches represent
bootstrap probabilities (%). The probabilities are shown only for thosegreater than 50%. Since the POU domain amino acid sequences werehighly conserved, those of Brain-1, Brain-2, Brain-4, and Scip were

The mammalian Brain-1, Brain-2, and Scip genes had
both the high content of characteristic amino acid repeats
and the high GC content. We thus investigated whether
there was a positive correlation between them or not.
There were no amino acid repeats within the POU domain and the GC content at the third codon position of
the POU domain was nearly equal to that at the third
codon position of the entire region (see Table 2). Thus
we used the GC content at the third codon position of the
POU domain so as to make an independent comparison
with the amino acid repeat ratio possible.
We defined the amino acid repeat ratio (AARR) in %as follows:
AARR 4 NRAA/NAA × 100(1)

where NRAA is the total number of identical amino acidresidues consecutive more than three, and NAA is thetotal number of amino acid residues compared. As shownin Fig. 4, we found a clear positive correlation between
GC content and AARR (correlation coefficient was0.82).
One possible explanation for this correlation is that

identical among their respective homologues of all the mammals compared in this study, as well as between fruit fly Cf1a and silk worm
POU-M1, shown as vertical lines in this figure. Asterisks indicate thetheoretically expected values, because of identical amino acid sequences.

177

the GC pressure has caused generation of amino acidrepeats during the vertebrate evolution. There is a wide
variation of the GC content among genes of warmblooded vertebrates, whereas cold-blooded vertebrate
genes show the relatively lower GC content within anarrow range (Bernardi 1993 and references therein). In
fact, the class III POU genes from cold-blooded vertebrates (amphibians and fish) having low GC content had
few repeats, whereas those from warm-blooded vertebrates (mammals) showed two phases opposite to eachother: the higher GC-content genes containing a numberof amino acid repeats such as Brain-1, Brain-2, and Scip,
and the lower GC-content genes containing very few
amino acid repeats such as Brain-4. Moreover, aminoacid residues constituting the repeats common to themammalian Brain-1, Brain-2, and Scip were alanine, glycine, and proline, whose codons were GC rich (GCN,
GGN, and CCN, respectively).
Figure 5 shows a phylogenetic tree among the class III
POU genes constructed using the conserved POU domain sequences. The phylogenetic relationship among
Brain-1, Brain-2, Brain-4, and Scip was not clear because of low bootstrap probabilities (less than 50%) forthe corresponding clusters. But it is likely that Brain-1,
Brain-2, Brain-4, and Scip genes emerged before divergence among vertebrates. This suggests that the ancestralclass III POU gene contained no or few amino acid repeats and that generation of homopolymeric amino acidrepeats encoded by GC-rich trinucleotides occurred independently in the lineages of particular class III POU
genes (Brain-1, Brain-2, and Scip) under the GC pressure. Those events occurred at least after emergence of
mammals, probably after the divergence between coldand warm-blooded vertebrates. It might result from thelocation effect in the mosaic genome of warm-blooded
vertebrates (Bernardi 1993 and references therein). The
human and mouse Brain-4 genes are mapped on Xq21,
an evolutionarily conserved region of the X chromosome(Douville et al. 1994). Based on the above assumption,
the mammalian Brain-4 gene retaining the ancestral feature of the class III POU gene (no or few amino acidrepeats) might be confined in the lower GC region of the
X chromosome. DXS995 has a relatively lower GC content (43.7%), which locates 20 kb distal to the Brain-4
gene in humans (Weissenbach et al. 1992; Huber et al.
1994; de Kok et al. 1995). The other mammalian class III
POU genes with the homopolymeric amino acid repeats
encoded by GC-rich trinucleotides and relatively higher
GC contents locate on other chromosomes (Xia et al.
1993; Atanasoski et al. 1995).
Amino acid residues constituting the repeats of theclass III POU genes were alanine, glycine, proline, glutamine, and histidine. Since alanine-, glycine-, proline-,and glutamine-rich sequences are identified as transcriptional activation domains (Mitchell and Tjian 1989;
Duboule 1994 and references therein), occurrence of the

GC-rich sequences encoding these characteristic aminoacid residues could influence the transactivation ability.
The occurrence of such amino acid repeats in the mammalian transcription factors must facilitate diversification of gene regulation mechanisms in the central
nervous system and might generate novel brain organizations including neocortex. Interestingly, after the
emergence of warm-blooded mammals the amino acidrepeats of the class III POU genes have been well conserved both in amino acid residue and in their repeat
numbers. These suggest that evolution of the neural system has been influenced by the GC pressure, at least in
mammals. Sequence data of the class III POU genes
from reptiles and birds could provide further evidence
for generation of the homopolymeric amino acid repeats
under GC pressure.
Acknowledgments. This study was supported by grants from the
Ministry of Education, Science, Sports, and Culture of Japan, The
Inamori Foundation, and NIG Cooperative Research Program.

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