(Received for publication, July 20, 1995)
From the
Ke 6 gene is a newly identified gene located in the major histocompatibility complex and is a candidate steroid dehydrogenase gene because of structural homology and regulatory similarities with mammalian steroid dehydrogenases. We report here the complete nucleotide sequence and intron-exon organization of the Ke 6 gene and cloning of the alternatively spliced Ke 6b transcript. We find that Ke 6 gene expression is down-regulated in pcy mice which is a murine model of polycystic kidney disease (PKD). Thus far, Ke 6 gene expression is down-regulated in all murine models of PKD we have examined. Abnormal steroid metabolism as a possible cause of PKD is discussed.
Polycystic kidney disease (PKD) ()is a major
inherited cause of renal failure in humans. The occurrence of the
autosomal dominant form of the disease is very high with a frequency of
1 in 1,000(1) . Recently, the ADPKD1 gene was isolated
and sequenced, but the nature of the protein it encodes is as yet
unknown(2) . However, it is clear that polycystic kidney
disease is a multigenetic disease, since numerous inherited diseases
occur in both humans and mice which give rise to renal cysts caused by
different genes. In rodents, several genes have been identified e.g. cpk, pcy, jck, cy, bpk, jcpk, TgN737, and Bcl-2 which
give rise to polycystic kidneys(3) . In humans there is a
second gene (ADPKD2) responsible for the dominant form of the
disease, located on chromosome 4(4, 5) . Autosomal
recessive polycystic kidney disease is a rarer, although more
aggressive, form of PKD in man, where the mutation has been mapped to
chromosome 6(6) . There are several other lesser known
heritable diseases, such as Meckle's syndrome, Jeune's
Syndrome, and Von Hipple Lindau Syndrome, where cysts occur in the
kidney by what may be yet other cyst causative genes. Renal cysts can
also occur in long term dialysis patients,. and this form of the
disease has been termed acquired PKD. To date the biochemical and
molecular basis of renal cysts formation is virtually unknown.
We
reported the identification of a new mammalian gene, Ke 6, whose
expression is specifically repressed in two different murine models of
PKD: the cpk and jck mouse(7) . The Ke 6 gene
is encoded within the major histocompatibility complex and is a member
of the superfamily of short-chain alcohol dehydrogenases. The normal
expression pattern of the Ke 6 gene is very high in kidney and liver (7) , the two organs that are most extensively affected in
polycystic kidney disease. We have postulated that the Ke 6 gene may
encode steroid dehydrogenase activity, since it has substantial
homology to specific functional domains of bacterial and mammalian
steroid dehydrogenases(8) . In addition, the pattern of
down-regulation of the Ke 6 gene is remarkably similar to the
11-hydroxysteroid dehydrogenase gene in liver and kidneys of jck and cpk mice(8) . Steroid dehydrogenases
are enzymes that metabolize biologically active forms of hormone to
their inactive keto derivatives. The possibility that Ke 6 is a steroid
dehydrogenase is therefore very exciting, especially since a role of
steroids in cystic malformation of nephrons is well established. We
have postulated that the primary genetic defect in the cpk and jck homozygous mice leads, either directly or indirectly, to
the repression of the Ke 6 gene(7, 8) . The lowered
activity of Ke 6 gene expression could lead to a deficiency of a
steroid metabolic enzyme and consequently result in elevating the
intrarenal steroid levels. The increased concentration of biologically
active steroids is most likely responsible for initiating events that
ultimately result in polycystic kidney disease(8) . Steroids
administered to neonate rodents are known to induce cystic
maldevelopment of nephrons in
vivo(9, 10, 11, 12, 13, 14) ,
and in vitro, in organ cultures of metanephric
kidney(15) . Elevated concentration of steroids could be
detrimental to developing kidney during embryogenesis and during
postnatal differentiation. Even though in numerous studies steroids
have been implicated in forming renal cysts, the specific molecular
mechanism of steroids' action in cyst formation is unknown.
In order to understand the function of the Ke 6 gene and the mechanism of its normal and aberrant regulation in cpk and jck mice, it is essential to characterize the transcriptional unit of Ke 6 gene in greater detail. Characterization of the Ke 6 gene promoter sequence offers the possibility of investigating the interaction of transcription factors and cis-regulatory elements responsible for its high level of expression in kidney and liver and its aberrant regulation in PKD. We report here the cloning and sequencing of an alternatively spliced mRNA Ke 6b and complete sequence determination of the Ke 6 gene and its intron/exon organization. We used RNase protection assay and primer extension analysis to determine the transcriptional initiation site of the gene. Putative regulatory elements in the promoter region were identified by searching the transcriptional regulatory sequence data base at National Center for Biotechnology Information.
In addition, we find that Ke 6 gene expression is down-regulated in mice carrying the pcy mutation. The pcy mouse is another murine form of PKD which manifests in homozygous juveniles. Ke 6 is down-regulated in all murine models of PKD, congenital and juvenile, that we have examined so far. This indicates that Ke 6 most likely represents a critical steroid metabolic enzyme in normal kidney function whose reduction can be deleterious not only to post-natal development of nephrons but also for the maintenance of healthy renal tubules.
Figure 6: Restriction map, subclones, and sequence strategy of genomic loci for Ke 6 and Ke 4 genes. a, horizontal line represents a 4.93-kb KpnI-BamHI genomic fragment containing both Ke 4 and Ke 6 genes. Vertical lines indicate restriction sites as noted above or below the lines. b, horizontal arrows indicate the direction and extent of sequencing. c, subclones generated from this region for sequencing purposes are denoted by horizontal lines. Restriction enzymes used to generate subclones are noted at the end of the lines, and size is indicated by the length of line. A 460-bp AccI-SmaI subclone used for the RNase protection experiment in Fig. 8is indicated by an asterisk and heavier line. d, direction of transcription of Ke 4 and Ke 6 genes is indicated by heavy horizontal arrows.
Figure 8:
Ribonuclease protection anala, a
463-bp AccI-SmaI genomic subclone which extends 367
bp upstream of the first nucleotide in Ke 6a (clone 13.3.1) codon used
for RNase protection experiment is depicted in the first line. The arrows indicate the direction and extent of transcripts
generated from Ke 4 and Ke 6 genes corresponding to the sequence within
this subclone. The subclone in pBluescript SK+ vector was
linearized at BstEII or at StuI in order to
synthesize antisense RNA probes directed by T3 polymerase. Lengths of
RNA probes are indicated below. The shaded box in BstEII and StuI RNA probes indicates the region
corresponding to the first exon of Ke 6 gene as determined from the
cDNA clone of Ke 6a (13.3.1). The open box in the BstEII probe indicates the region corresponding to a 35-base
region from the 3` end of the Ke 4 transcript. b,
approximately 2.5 10
cpm of each probe was
hybridized with 2 µg of poly(A)
RNA isolated from
mouse kidney or spleen. As controls, the same probes were hybridized
with cRNA synthesized from Ke 6a cDNA (clone 13.3.1) or with tRNA
((-)RNA). After hybridization and RNase treatment the protected
fragments were analyzed by electrophoresis on a 6% polyacrylamide gel
containing 7 M urea and visualized by autoradiography. The arrowheads indicate the position of three protected bands of
57, 63, and 73 bp in kidney and spleen poly(A)
RNA
lanes corresponding to Ke 6 transcripts with both RNA probes. 35 bases
of the protected RNA band corresponding to the 3` end Ke 4 mRNA is seen
with the BstEII probe in kidney and spleen poly(A)
RNA lanes.
Figure 1:
Down-regulation of Ke 6 gene expression
in murine models of PKD. 6 µg of poly(A) RNA from
kidneys and liver of +/+, pcy/pcy,jck/jck,
and cpk/cpk mice were used for Northern blot analysis. There
was no gross morphologic difference in any organs besides kidney in
homozygote pcy, jck or cpk mice. RNA was run
on a 1% agarose, 2.2 M formaldehyde gel and blotted onto nylon
membranes. [
-
P]dCTP labeled Ke 6a cDNA was
used as a probe for hybridization at 65 °C in Church's buffer
for 16-20 h, and the membrane was washed in 0.1
SSC, 0.1%
SDS at 50 °C and exposed to x-ray film. Equal loading of RNA was
confirmed by ethidium bromide staining intensity and by hybridizing the
membrane with [
-
P]dCTP-labeled 18 S cDNA
probe.
Figure 2:
Ke 6
is a single copy gene. Genomic DNA isolated from C57Bl/J6 mouse was
digested with BamHI, EcoRI, HindIII, and PstI and analyzed by Southern blotting. The membrane was
hybridized with Ke 6a cDNA and washed in 0.2 SSC, 0.1% SDS at
50 °C.
Figure 3:
Ke 6 gene gives rise to two transcripts. 4
µg of poly(A) RNA from a normal C57BL/6 mouse
kidney and spleen was run on a formaldehyde-agarose gel and blotted for
Northern analysis. The membrane was hybridized to Ke 6a cDNA and washed
with 0.1
SSC, 0.1% SDS at 50
°C.
Figure 4: Restriction map of Ke 6b cDNA reveals a longer BsmI fragment.
Figure 5: Comparison of the predicted amino acid sequence of Ke 6a and Ke 6b protein. The amino acid number of each protein is indicated in the beginning and at the end of each line.
Figure 7: Intron-exon organization and complete nucleotide sequence of Ke 6 gene. a, open boxes represent Ke 6a exons, black box represents intron 8, and stippled boxes represent exons for the previously described Ke 4 gene. Numbers under the boxes indicate exon numbers for Ke 6a and Ke 6b transcripts. Heavy lines indicate relative lengths of Ke 6a and Ke 6b transcripts. b, exon sequences are represented in capital letters, introns and 3`- and 5`-flanking sequences are indicated in lowercase letters. Nucleotides are numbered in the right margin relative to +1 which marks the start of Ke 6a cDNA. Initiation of translation is proposed to occur at nucleotide +11. Amino acids and exon numbers are indicated in the right margin. The alcohol dehydrogenase superfamily signature motif is underlined. The Ke 6b mRNA-specific sequence is represented in bold letters. Codons which are split by exon-intron junctions are marked by a dot. The carboxyl-terminal Ke 6a amino acid number, which is different from Ke 6b due to alternative splicing, is presented within parenthesis. The poly(A) addition signal is italicized.
The alternative splicing pattern that gives rise to the spleen-specific Ke 6b transcript is due to failure of splicing of a 170-bp intron between exons 8 and 10 (Fig. 7). In contrast, the Ke 6a mRNA is created by excision of this intron which results in a smaller size message. This difference in splicing pattern is responsible for the deduced amino acid sequence difference between the two proteins at the carboxyl ends (Fig. 5). Within the Ke 6b-specific exon sequence (Fig. 7, boldface sequence) is a protein translational termination codon that gives rise to a longer 3`-untranslated region for the Ke 6b transcript (300 bases) compared with the 3`-untranslated region for the Ke 6a transcript (175 bases).
Approximately 2.5 10
cpm of probe was hybridized
to in vitro transcribed Ke 6a cRNA (positive control), tRNA
(negative control(-) RNA), kidney poly(A)
RNA,
and spleen poly(A)
RNA for RNase protection assay. In
all experiments, the positive control yields a major protected fragment
of 57 bases, corresponding to the beginning of Ke 6a cDNA at position
-10 (Fig. 8b, cRNA lane). The BstEII and StuI RNA probes yield common protected
fragments of about 73, 63, and 57 bases, which represent Ke 6
transcripts having heterogeneous 5` ends. The previously cloned Ke 6a
cDNA (13.3.1) represents a full-length clone whose 5` end corresponds
to a start site at position -10 matching that represented by the
57-bp fragment. The 73- and 63-bp protected fragments represent mRNAs
that are slightly longer than the 13.3.1 cDNA at position -16 and
-26. The 35-bp protected fragment seen in kidney and spleen lanes
with the BstEII-specific probe represents a portion protected
by Ke 4 mRNA. Results of the RNase protection assay indicate that the
Ke 6 gene has multiple start sites of transcription.
Primer extension analysis utilizing an antisense 27-mer complementary to bp +27 to +1 of the Ke 6a cDNA also revealed multiple transcription initiation sites, ranging from position -9 to -26 bp upstream of the predicted translational initiation codon (Fig. 9). The size range of the extension products are consistent with the result obtained with the RNase protection assay. However, with primer extension analysis the existence of more heterogeneity in the 5` end of Ke 6 mRNAs is apparent than that observed with RNase protection analysis. This could be because some of the mRNAs differ in size by 1 or 2 bp, which is resolved by sequencing gel analysis of the primer extension experiment and which may not be distinguishable with RNase protection assay. The three major bands seen in the primer extension analysis gel are at positions -9, -16, and -26. This phenomenon of multiple initiation sites of transcription is not uncommon in genes lacking a TATA sequence, since the TATA sequence plays a role in the accurate positioning of RNA polymerase II for the initiation of transcription(28, 29) .
Figure 9:
Primer extension analysis.
Poly(A) RNA (4 µg) from mouse kidney (K)
and spleen (S) was hybridized with a 5` end-labeled
oligonucleotide complementary to Ke 6a cDNA from +27 to +1.
The primer was extended with reverse transcriptase and analyzed by
electrophoresis on an 8% polyacrylamide, 7 M urea gel. Lanes A, C, G, and T, sequencing reaction was done
using the same primer. The numbers on the right indicate the position of the extended products from the start of
the initiator codon (+1) which is indicated in bold.
Similar results obtained by the above two method indicates that the Ke 6b mRNA is identical to the Ke 6a mRNA at the 5` end. This is further evidence that the two mRNAs differ only at the 3` end due to splicing differences within exon 8.
The Ke 6 gene is a single copy gene encoded within the mouse
MHC on chromosome 17 identified by positional cloning
techniques(7) . It is located approximately 25 kb telomeric to
the class I H-2K gene. This region of the mouse chromosome 17, where
the MHC and the t-complex region are located, make it an important
region to identify new genes. The tcl-w5, an early acting
embryonic lethal mutation within the t-complex region is
recombinationally inseparable from the H-2K gene(30) .
Therefore, the Ke 6 gene, in addition to other genes identified in this
region, is a candidate for the tcl-w5 mutation. The search for
new genes in the MHC has led to the discovery of several new genes
which encode proteins of diverse functions. Human and mouse genes in
the MHC were determined to be co-linear in their
organization(31) . The Ke 6 gene, because of its positional
similarity and 86% sequence homology to the Ring 2 gene, ()is most likely the mouse homolog of this human gene.
There is only a 234-bp gap between the 3` end of the previously identified Ke 4 gene (32) and the 5` end of the Ke 6 gene. Since both genes are transcribed in the same direction, it is possible that the span which encodes the 3`-untranslating region of the Ke 4 region encompasses the promoter region for Ke 6 gene. The close proximity of transcription units in this location of the MHC indicates that for some unapparent evolutionary benefit this region is highly enriched with genes.
The genomic organization of Ke 6 and other
genes encoding steroid metabolic enzymes are not similar to each other.
Human genes for the 11-hydroxysteroid dehydrogenase(27) ,
3
-hydroxysteroid dehydrogenase/
isomerase(33) , and 17
-hydroxysteroid dehydrogenase (34) which have been cloned all have unrelated exon/intron
organization and also all vary in length.
We report that the Ke 6
gene is down-regulated in a third murine model of PKD: the pcy mouse (Fig. 1). Thus far, Ke 6 is repressed in all murine
models of PKD that we have investigated: cpk, jck(7) , and pcy mice (this report). This
suggests that Ke 6 has a critical role in the normal functioning of the
kidney and that molecular events leading to its repression in kidney
may be responsible for the development of renal cysts. In the cpk homozygote, the Ke 6 gene is down-regulated to the same extent in
the liver as in the kidney, even though the liver of affected animals
appears perfectly normal by morphology and by histological
examination(7) . The repression of Ke 6 in liver suggest that an aberration in the regulation of expression of Ke 6
gene in the kidney does not occur as a consequence of a
diseased organ, but rather it occurs before the onset of
pathogenesis(7) . The Ke 6a protein sequence is homologous to
bacterial and mammalian steroid dehydrogenases and also to human
prostaglandin dehydrogenase (8) . Similarity of Ke 6a protein
to the dehydrogenases is high within specific functional domains of
these enzymes: the NAD binding domain and the dehydrogenase active site
domain. Furthermore, we find that the rat 11-hydroxysteroid
dehydrogenase has a similar pattern of aberrant regulation as the Ke 6
gene in older cpk heterozygote liver and in young cpk homozygote liver(8) . Therefore the structural homology
and regulatory expression pattern similarity to a known steroid
dehydrogenase strongly suggest that Ke 6 itself is a steroid
dehydrogenase(8) . Incidentally, the gene for a steroidogenic
enzyme, the 21-steroid hydroxylase gene, is also encoded within the
MHC.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34072[GenBank].