(Received for publication, April 19, 1995; and in revised form, July 11, 1995)
From the
Cytochrome b is an electron transfer
protein unique to neuroendocrine secretory vesicles. The Southern blot
hybridization shows that it is a single copy gene highly conserved
throughout phylogeny. The transcription unit spans approximately 11
kilobases, and heterologous transcription sites are located 404 bases
5` to the translation initiation codon. The sequence of the 5`-flanking
region is GC-rich and lacks a typical TATA box at the usual position.
However, it has a CAAT sequence at -132 and potential recognition
sequences for several transcription factors including SP1, GR-PR-MMTV,
AP4, gERE, JCV repeat, AP2, and NF-
B. Each of the five
transmembrane segments are encoded by five consecutive exons. This
corroborates the five-transmembrane model proposed for human, mouse,
and Xenopus rather than six proposed for bovine. The
cytochrome was found to be highly expressed in colon cancer cell lines,
T cell lymphomas, and K-562 cell lines. However, in B-cell lymphomas
such as Burkitt's and Daudi, the cytochrome b
expression was completely shut down. The results in this report
are the first to demonstrate the structural organization and regulatory
sequences of the cytochrome b
gene encoding an
integral membrane protein of neuroendocrine storage vesicles of
neurotransmitters and peptide hormones. Unexpected results on
cytochrome b
expression in cells of lymphocytic
origin and its complex regulation in tumor cells provide new insights
into cytochrome b
gene regulation.
The integral membrane protein, cytochrome b, was first discovered in catecholamine storage
granules (1) and was subsequently found to be specific to
catecholamine and neuropeptide secretory vesicles of the adrenal
medulla, pituitary gland, and other neuroendocrine
tissues(2, 3, 4, 5, 6, 7, 8) .
This 30-kDa protein comprises 15% of the granule membrane protein (9) and is present in both small synaptic vesicles and large
dense core vesicles (10, 11) where it plays a central
role with ascorbic acid in the biosynthesis of several catecholamine (6, 12) and peptide
neurotransmitters(13, 14) . Among the cytochromes,
cytochrome b
is unique in its localization to
neuroendocrine tissues and appears to be unusual in its enzymatic
mechanisms. Unlike other electron carriers which need other proteins
for its oxidation-reduction cycle, the cytochrome b
does not interact with any other protein. Instead, ascorbic acid
serves as the extravesicular electron donor for cytochrome b
, and semidehydroascorbic acid acts as the
intravesicular electron acceptor, which replenishes the high
intravesicular ascorbic acid concentrations. Ascorbic acid serves as a
cofactor for intravesicular dopamine
-hydroxylase activity in
catecholamine storage granules (15) and peptidyl
-amidating monooxygenase activity in neuropeptide storage
vesicles(14) . Cytochrome b
appears to
be a simple, functionally symmetric electron transfer protein and acts
as a pure electron channel.
Understanding the mechanism of this long
range electron transfer process has been facilitated by the cloning of
bovine cytochrome b which revealed that it has
six transmembrane helices with N- and C-terminal ends of the protein
facing the cytoplasm(16) . Subsequent biochemical analysis
using peptide antibodies raised against N-terminal and C-terminal ends
of the protein and protease digestion suggested that the N terminus was
unavailable to both(17) . In addition, the human cytochrome b
lacked the first putative 22 amino acids at
the N terminus predicted from the bovine sequence to be in the
cytoplasmic side. Based on the sequence comparison and low
hydrophobicity of the second transmembrane domain, combined with the
biochemical evidence, a revised hypothesis was proposed in which the
gene product is likely to be distributed within the membrane with five
transmembrane helices(18) . Subsequent cloning of the mouse (
)and Xenopus(
)cytochrome b
confirmed this structure and further revealed
that it was colocalized to specific tissues with either DBH or
peptidylglycine
-amidating mono-oxygenase. In addition, we found
that cytochrome b
expression was developmentally
regulated in mouse and Xenopus. Furthermore, more recent data
on cytochrome b
expression in different tissues
exhibited uniform distribution in all the neuroendocrine tissues
tested.
In order to better understand the molecular basis of
developmental and tissue-specific cytochrome b expression, it is necessary to characterize the genomic structure
and regulatory elements of the gene. This approach could also help
establish the relationship between this gene and those of other
membrane proteins, thereby providing clues concerning the evolution of
the eukaryotic chromaffin granule. Cloning and sequence analysis of the
regulatory region of the cytochrome b
gene would
provide reagents and information necessary to examine the expression of
a major structural protein of the chromaffin granule. For these
reasons, we have determined the structural organization and promoter
sequence of human cytochrome b
, and studied gene
expression in different cell lineages.
Figure 1:
Structural organization of the human
cytochrome b gene. a, schematic
representation of the cDNA, divided into open bars indicating
5`- and 3`-untranslated regions and hatched bars from I to V showing five transmembrane domains. b,
organization of the human cytochrome b
gene. Filled or open boxes represent exon or 5`- and
3`-untranslated sequences, respectively. Introns and flanking regions
are shown by a thin connecting line. Protein coding exons are
numbered 1-5, an exon encoding only 3`-untranslated
sequence is labeled NC. The relationship between exons and the
position of the transmembrane domain is shown by connecting
vertical lines of the upper half. The location of the
subclones obtained using primer sets A to H are labeled, and their
specific sequences are given in Table 1. c, arrows show portions of the various subclones
sequenced.
Partial nucleotide sequences, including the sequence of the 5` upstream region and less than 200 bp of all introns are shown in Fig. 2. Exons flanked by introns ranged in size from 99 bases (exon 2) to 1374 bases (exon 5) (Fig. 2, Table 2). The intron-exon junction sequences are consistent with the reported 5`-donor (GTGAG) and the 3`-acceptor (C/TAG) consensus sequence (21) (Table 2). The intron splice phase is type 0 (the intron occurs between codons) for intron 3, type 1 (the intron interrupts the first and the second bases of the codon) for introns 1 and 2, and type 2 (the intron interrupts the second and the third base of the codon) for intron 4(22) .
Figure 2:
Partial nucleotide sequence of the human
cytochrome b gene. Nucleotides in exons are
shown in bold letters with the deduced amino acid shown below.
The transcription initiation sites and the end of the human cytochrome b
mRNA is shown in bold type upper
case. The asterisks indicate the two strong transcription
initiation sites. Numbering of the nucleotides begins at 4572 bp before
the start of the transcription site and is located on the left
side, whereas the amino acids are numbered under the protein
sequence and on the right side. Intron boundaries having the
consensus GT and AG dinucleotides are underlined. Also, two
direct Alu repeats (positions 2986 and 8051), ATG, stop codon, and two
dinucleotide CA repeats are underlined.
Figure 3:
Site of transcription initiation as
determined by ribonuclease protection analysis. Human total brain RNA
(20 µg) or yeast tRNA was annealed to a P-labeled
antisense RNA probe (prepared by T7 polymerase transcription) and
digested with RNase A and T1 as described under ``Materials and
Methods.'' The probe fragments protected by total RNA from human
brain was analyzed on 6% polyacrylamide-8 M urea gel
electrophoresis and were compared with the adjacent sequencing
ladder.
Figure 4:
Nucleotide sequence of the 5`-flanking
sequence of the human cytochrome b. Amino acids
encoded by the first exon are indicated below the appropriate codons.
The transcription start sites are indicated in bold, and the
last one is numbered 1. Numbers to the left refer to the
nucleotide position relative to the transcription initiation site.
Sequences similar to consensus sequences for transcription factors (e.g. SP-1, AP-2, AP-4, GR-PR, MMTV, NF-
B, JCV repeat,
PEA-3, H4TF-1, hist, E-alpha box, and HinF-hist) are underlined.
Figure 5:
A, a, Northern blot analysis of
poly(A) RNA from different cancer lines and tissues
from human. The blot was hybridized to the human cytochrome b
coding region. A, b, the
same blot was hybridized to
-actin after stripping. B,
reverse transcriptase-PCR and Southern blot analysis of human
cytochrome b
mRNA. Poly(A)
RNA
from different cancer cell lines was reverse-transcribed and run on a
1.5% agarose gel followed by blotting onto nitrocellulose paper (see
``Materials and Methods''). B, a, agarose
gel electrophoresis of reverse transcriptase-PCR amplified mRNA from
different cancer lines from human. B, b, agarose gel
electrophoresis of reverse transcriptase-PCR amplified mRNA from cancer
cell lines using oligonucleotide primers from a different gene. B, c, Southern blot of the same gel probed with
P-labeled antisense primer from set D in Table 1.
Control lane has the markers.
To understand the molecular basis of the tissue-specific
expression of cytochrome b, we have now cloned
and characterized the genomic sequence of cytochrome b
including the promoter sequences possibly regulating the specific
expression of cytochrome b
. Although the
structure, function, and expression of cytochrome b
protein has been studied in detail, the biological significance
of its uniform expression in neuroendocrine tissues have not been
understood. The results described here, therefore, are of particular
significance in as much as they represent first characterization of the
gene encoding the integral membrane protein of the chromaffin granule
and its differential expression in hematopoietic cells.
Southern
hybridization analysis established that cytochrome b gene is highly conserved (data reviewed, but not included) and
occurs as a single copy of the cytochrome b
gene
in the human genome, spanning approximately 11 kb, and is split into
five exons. The coding sequence of the gene is 753 bp, representing
7.1% of the gene, and perfect agreement between the nucleotide
sequences of the chromosomal gene exons and the cDNA sequences was
observed. The introns interrupt the cytochrome b
protein coding sequence in such a way that many of the protein
segments are revealed as products of individual exons. A similar
correspondence has been observed in other highly hydrophobic proteins
such as human myelin(27) , human T cell-specific proteolipid
protein(28) , and human vacuolar ATPase(29) . Studies
on the structure of the bovine cytochrome b
have
been interpreted to indicate that the protein might traverse the
membrane six times with N- and C-terminal ends facing the cytoplasm.
However, subsequent biochemical analysis using antibodies raised
against N- and C-terminal peptides along with topological orientation
suggested that N-terminal is not facing the cytoplasm. Comparative
amino acid sequences derived from human, mouse, and Xenopus,
along with low hydrophobicity and unusual amino acid composition of the
second transmembrane segment, suggested that bovine is 22 amino acids
shorter, and a model with five transmembrane segments was
proposed(18) . The analysis of the cytochrome b
gene in the present study corroborates the
above view in that five transmembrane segments occur in five different
exons, and thus each exon encodes a potential unit of the cytochrome b
gene. The two histidine residues His-91 and
His-160, which form putative ligands to heme, are separated into
different exons. In addition, the amino acid residues Try, Gly, and
Phe, which form the heme binding pocket, are in exon 4 along with
His-160.
Inspection of the genomic sequences in the vicinity of the
transcription start sites indicates the absence of an apparent TATA box
in this region. The 5`-flanking region includes the dinucleotide CpG in
its recognition sequence, suggesting that the cytochrome b promoter is G + C-rich. These features
place the cytochrome b
gene in an emerging group
of tissue-specific genes with TATA-less and G + C-rich promoters,
which also includes other genes differentially expressed in T
lymphocytes such as CD7 (30) and Thy-1(31) . The
present results also provide initial information as to what factors
potentially regulate the expression of human cytochrome b
. Analysis of the transcription factor binding
sites in the human gene promoter suggests that seven potential
signaling pathways could regulate cytochrome transcription: SP-1,
involved in maintaining appropriate basal transcription
levels(32) ; AP-2, which mediates induction involving cyclic
AMP(33) ; AP-4, whose function is unknown(34) ;
glucocorticoid and progesterone receptor, which binds to the promoter
of mouse mammary tumor virus (MMTV)(35) ; MRE, a putative metal
regulatory factor which induces transcription in response to heavy
metals(36) ; NF-
B, which regulates gene expression via
cytokines (37) ; and the JCV repeat, a sequence specific to
human polyoma virus which is highly neuro-oncogenic in
hamsters(38) . Another feature in the cytochrome b
gene that could be related to tissue- or
development-specific or cell type-specific expression is the presence
of regulatory elements such as PEA-3, H4TF-1, hist, E-alpha H box, and
HinF-hist(20, 39, 40) , along with the
heterologous transcription initiation sites. One example of
tissue-specific mRNAs with alternative 5`-untranslated sequences is the
different transcripts that arise from the
-amylase gene in
salivary gland and pancreas(19) . Since cytochrome b
is known to be specifically expressed in
neuroendocrine tissues and in this study differentially expressed in
hematopoietic tumor cells, additional experimentation is needed to
elucidate the elements responsible for the regulation of tissue- and
cell-specific expression.
We have demonstrated previously that
cytochrome b is constitutively expressed in all
the neuroendocrine tissues studied, at high levels in brain, placenta,
lung, and pancreas(18) . As a first step toward investigating
the biological function of cytochrome b
, the
expression patterns of the corresponding transcripts were analyzed in a
wide variety of cancer lines using reverse transcriptase-PCR and
Northern blot analysis. Significant levels of cytochrome b
mRNA expression were detected in colon cancer
cell lines, T cell lymphomas, and undifferentiated cell lines such as
K-562 compared to peripheral blood leukocyte. In addition, the
cytochrome b
expression was completely shut down
in B cell lymphomas. These findings suggest that it may play a
specialized role in growth and differentiation pathways of the
hematopoietic cells. The differential expression of cytochrome b
mRNA confirmed by reverse transcriptase-PCR
and Northern blot analysis in hematopoietic cell lines will provide an
excellent model system for identification of cis-acting
elements required for gene expression in cells from T and B lymphomas.
Cytochrome b plays a pivotal role in
regenerating ascorbate in neuroendocrine vesicles for dopamine
-hydroxylase and peptidyl
-amidase, and, hence, understanding
the regulation of cytochrome b
may yield
insights for development and neuroendocrine disorders. Because it
constitutes 15% of the membrane protein in chromaffin granules,
cytochrome b
may have evolved concurrently with
the formation of the vesicles. The studies reported here provide key
surprising findings on the expression of cytochrome b
in hematopoietic cells. Thus, it is obvious that the cytochrome b
expression is not exclusive to neuroendocrine
cells. Further studies in the process of tumorigenesis will give clues
about the biological function of cytochrome b
in
the newly identified cells. Cloning of the human cytochrome b
gene now enables us to use molecular genetic
techniques to further study its expression patterns, gene regulation,
and function, particularly its role in processes such as cell
differentiation, development, and carcinogenesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29460[GenBank], U29461[GenBank], U29462[GenBank], U29463[GenBank], U29464[GenBank], and U29469[GenBank].