(Received for publication, May 15, 1995; and in revised form, July 5, 1995)
From the
We have previously identified a human estrogen-responsive gene, efp (estrogen-responsive finger protein), which encodes a
putative transcription regulator (Inoue, S., Orimo, A., Hosoi, T.,
Kondo, S., Toyoshima, H., Kondo, T., Ikegami, A., Ouchi, Y., Orimo, H.,
and Muramatsu, M.(1993) Proc. Natl. Acad. Sci. U. S. A. 90,
11117-11121). Here, we report isolation of mouse Efp cDNA and its
structure containing three cysteine-rich domains (RING finger and B1
and B2 boxes), a coiled-coil domain, and a C-terminal domain. High
levels of Efp mRNA were detected in uterus, ovary, and placenta by
RNase protection assay. By in situ hybridization
histochemistry the transcripts of efp were also detected in
uterus, mammary gland, ovary, and brain, and the co-localization of Efp
and estrogen receptor mRNA was particularly demonstrated in these
female organs. Moreover, the level of Efp mRNA in uterus and brain,
which are known as target organs for estrogen, was up-regulated in
vivo by 17-estradiol. Furthermore, both the Efp and estrogen
receptor mRNA were stained in the brain vesicles of 11.5-day embryos by
whole mount in situ hybridization. These findings raise the
possibility that efp is an estrogen-responsive gene that
mediates estrogen action in various target organs.
Estrogen plays important roles in the reproductive system as a
sex steroid hormone. It is involved in the growth and development of
female organs such as the uterus and mammary gland. Estrogen receptor
(ER) ()acts as an estrogen-dependent transcription factor
recognizing and binding to specific estrogen-responsive elements (ERE)
in the enhancer region of target genes and regulating their
transcription directly. Thus, estrogen exerts its action on target
organs by regulating target gene products(1, 2) . ER
has also been identified in the central nervous
system(3, 4) , in the skeletal
system(5, 6) , and in the cardiovascular
system(7, 8) , implying some important roles for
estrogen and estrogen responsive genes in a number of nonreproductive
organs. From the clinical point of view, estrogen replacement therapy
is effective to protect postmenopausal women from osteoporosis (9) and coronary heart disease(10) . Furthermore,
estrogen plays critical roles in carcinogenesis and growth of breast
cancers(11) .
In contrast to the wide variety of estrogen action on different organs, tissues, and cells, estrogen-responsive genes that are known so far are relatively few and include vitellogenin(12) , prolactin(13) , pS2(14) , uteroglobin(15) , ovalbumin(16) , progesterone receptor(17) , and lactoferrin(18) . More genes that mediate estrogen action in a number of organs should be present. To identify estrogen-responsive genes, we have developed a method designated ``genomic binding site cloning'' used for isolation of ERE-containing fragments from human genomic DNA(19) . Using one of those fragments, we have cloned a novel estrogen-responsive gene, efp (for estrogen-responsive finger protein)(20) . The predicted human Efp protein had a RING finger motif present in a new family of apparent nuclear proteins including transcription regulators(21, 22) . Human efp contained a consensus ERE sequence at the 3` region that could act as a downstream estrogen-dependent enhancer, and it was up-regulated by estrogen in ER-positive cells derived from mammary gland.
Here, we have identified the mouse homologue of human efp. The predicted mouse Efp showed a high degree of
conservation with human Efp. Interesting differential conservation of
different domains of the protein was also noted. Mouse Efp mRNA was
detected in reproductive organs and in the central nervous system by in situ hybridization histochemistry. Northern blot analysis
indicated that the level of Efp mRNA was up-regulated within 2 h in
uterus and brain by 17-estradiol. Moreover, the co-localized
expression of Efp mRNA with ER mRNA in female reproductive organs and
in brain vesicles of embryos was demonstrated.
Figure 1:
Molecular
structure of the mouse Efp. A, nucleotide and deduced amino
acid sequences of mouse Efp cDNA (N3). The deduced amino acids are
shown below their respective codons. The TAG stop codon (*)
and the polyadenylation signal are underlined. The long
3`-untranslated region contains two (GT)
repeats,
which are also underlined. An imperfect palindromic sequence
of ERE is doubleunderlined. Conserved residues,
including cysteines/histidines, in three cysteine-rich domains that may
be involved in zinc finger-like structures are circled. They
contain the RING finger motif (amino acids 13-53), B1 box (amino
acids 106-142), and B2 box (amino acids 156-185). The
potential coiled-coil domain (amino acids 190-313) is underlined. The potential C-terminal domain (amino acids
462-632) is indicated by a grayline. The
deleted 33 amino acids (amino acids 395-427) resulting in the
short form cDNA are indicated by opentriangles. B, diagrammatic representation and comparison of conserved
domains between mouse and human Efp. The cysteine-rich (RING finger, B1
box, and B2 box domains), coiled-coil domain, and C-terminal domain are
shown as distinctive boxes. The homology of amino acids
between mouse and human Efp is shown below the respective
conserved domains. The number of amino acids in the spacing among the
respective domains is shown in parentheses. C,
genomic Southern blot analysis of efp in mouse and other
species. Cross-species genomic Southern blot analysis shows the
presence of homologue or related genes in human and rat. Lanes1, 2, and 3 contained genomic DNA
digested with EcoRI from human, mouse, and rat, respectively.
Sizes were estimated using
DNA restricted with HindIII.
Figure 2:
The tissue distributions of efp and ER transcripts. A, RNase protection assay with Efp
and -actin RNA probe. Lane1, M is the HapII-digested pBR 322 as size marker (calibration in bp); lane2, undigested Efp RNA probe; lane3, undigested
-actin RNA probe; lanes4-9, placenta, uterus, ovary, mammary gland, brain,
and liver RNA, respectively. 15 µg of total RNA was used in each
assay. Full-length protected fragments for each probe are indicated. As
an internal control,
-actin RNA probe was included in the Efp
assay. The Efp mRNA was shown to be expressed in uterus, ovary, and
placenta at relatively high levels followed by mammary gland and liver.
The Efp mRNA in brain was detected as a faint band. B, RNase
protection assay with ER and glyceraldehyde-3-phosphate dehydrogenase
RNA probe. Lane1, M is the HapII-digested pBR 322 as size marker (calibration in bp); lane2, undigested ER RNA probe; lane3, undigested glyceraldehyde-3-phosphate dehydrogenase
RNA probe; lane4, yeast total RNA control; lanes5-10, placenta, uterus, ovary, mammary gland,
brain, and liver RNA, respectively. 15 µg of total RNA was used in
each assay. Full-length protected fragments for each probe are
indicated. As an internal control, glyceraldehyde-3-phosphate
dehydrogenase RNA probe was included in the ER assay. The ER mRNA was
shown to be expressed in uterus and ovary at high levels. C,
Western blot analysis shows the existence of Efp. Nuclear extract
prepared from mouse placenta was separated on a 10% SDS-polyacrylamide
gel and electroblotted to polyvinylidene difluoride membrane (Millipore
Corp.). Western blot analysis with anti-mouse Efp antibody (1:10,000)
detects a 70-kDa native protein with the M
predicted from Efp cDNA. Molecular masses are given in
kilodaltons.
In situ hybridization histochemical studies detected transcripts of efp in mouse uterus, mammary gland, ovary, and brain. In uterus, Efp mRNA was localized predominantly over endometrium (Fig. 3A). The highest stain density was found in columnar epithelial cells of uterus, and lower levels were found in stromal cells of the lamina propria (Fig. 3A) and in smooth muscle cells of the myometrium (data not shown). No staining was seen with the sense Efp RNA probe (Fig. 3B). The hybridization signal with antisense ER RNA probe showed the co-localization of Efp and ER mRNA in endometrium (Fig. 3, A and C). Both Efp and ER mRNA were detected in luminal epithelial cells in mammary gland (Fig. 3, D and F), while no staining was found with sense Efp RNA probe (Fig. 3E). In mouse ovary, only granulosa cells but not thecal cells were well stained by antisense Efp RNA probe (Fig. 3G). Antisense ER RNA probe stained the granulosa cells well and also stained a part of the thecal cells (Fig. 3, G and I). No staining was detected with sense Efp RNA probe here too (Fig. 3H). In mouse brain, Efp mRNA was shown widely distributed. The Efp mRNA-containing neurons were found with greater cell densities on the sections of cerebral cortex (Fig. 4A) and hypothalamus including the ventromedial hypothalamic nucleus (Fig. 4, B and D), while no staining was detected with sense Efp RNA probe (Fig. 4C).
Figure 3: In situ hybridization histochemistry of Efp mRNA in reproductive organs. Photographs A-C represent a set of serial uterus sections. D-F represent a set of serial mammary gland sections. G-I represent a set of serial ovary sections. (A, D, and G) are hybridized with the Efp antisense probe. (B, E, and H) are hybridized with the Efp sense probe for the negative control. (C, F, and I) are hybridized with the ER antisense probe. To facilitate orientation, the epithelium (e) and the stroma (s) of uterus and granulosa cells (g) and thecal cells (t) of ovary are indicated. The scalebar indicates 1 µm (A-C) and 100 µm (D-I). The co-localization of Efp and ER mRNA in female organs is shown.
Figure 4: In situ hybridization histochemistry of Efp mRNA in brain. Photograph A represents a frontal section of cerebral cortex. B-D represent frontal sections of hypothalamus. A, B, and D are hybridized with the Efp antisense probe. C is hybridized with the Efp sense probe for the negative control. An arrow indicates the ventromedial hypothalamic nucleus, and 3V indicates the third ventricle. In a higher magnification (D), Efp mRNA is significantly expressed in neurons. The scalebar indicates 1000 (A-C) and 100 µm (D).
To examine estrogen responsiveness of the
mouse efp gene in vivo, the effect of estrogen
administration on the amount of Efp mRNA was studied in uterus and
brain. By Northern blot analysis, a 6.0-kilobase transcript of mouse efp was detected in these organs, and its amount was increased
by subcutaneous injection of 17-estradiol (500 µg/kg) to 2.5
times in uterus and 2.0 times in brain as early as 2 h, using
-actin mRNA as an internal standard and then returned to normal
level by 4-6 h (Fig. 5).
Figure 5:
Mouse Efp mRNA was regulated by estrogen
in uterus and brain. Northern blot analysis shows that the level of Efp
mRNA is elevated at 2 h in uterus and brain after subcutaneous
injection of 17-estradiol (500 µg/kg) into 3-week ICR female
mice. The level of Efp mRNA is decreased 4 and 6 h
thereafter.
Figure 6:
Detection of Efp mRNA in mouse embryos
during gestation. The photograph represents biochemical
detection of the efp transcript in the heads of mouse embryos
during the gestation. The RNase protection assay was performed with Efp
and -actin RNA probe. Lane1, M is the HapII-digested pBR 322 as size marker (calibration in bp); lane2, undigested Efp RNA probe; lane3, undigested
-actin RNA probe; lane4, yeast total RNA control; lanes5-9, 10.5-, 12.5-, 14.5-, 16.5-, and 18.5-day
embryo RNA, respectively. 15 µg of total RNA was used in each
assay. Full-length protected fragments for each probe are indicated. As
an internal control, the
-actin RNA probe was included in the Efp
assay. The transcript of efp was detected from day 10.5 to day
18.5 in the heads of embryos.
Figure 7: In situ hybridization histochemistry of Efp mRNA in mouse embryos. Photographs A-C represent the lateral view of 11.5-day whole mount embryos. D-E represent a set of serial sagittal sections of the head in embryo, and the regions of telencephalon and lateral ventricle are indicated. A and D are hybridized with the Efp antisense RNA probe. B and E are hybridized with the Efp sense RNA probe. C and F are hybridized with the ER antisense RNA probe. te, telencephalon; ms, mesencephalon; mt, metencephalon; lv, lateral ventricle. Scalebar, 1000 µm (D-F).
The
seven RING finger containing proteins (i.e. the human PML or
Myl protein(31, 32, 33, 34) , the
mouse T18 protein(35) , the mouse Rpt-1 regulatory
protein(36) , the human Rfp protein and the related Ret fusion
protein(37) , the human 52-kDa SS-A/Ro
autoantigen(38, 39) , XNF7 from Xenopus(40) , and PWA33 from the newt Pleurodeles
waltl(41) are known to have B box domains so far. All of
these B box-containing proteins have a coiled-coil domain present
immediately carboxyl-terminal to the B boxes(34, 40) .
Amino acid comparison of proteins with putative coiled-coil domain
shows that there is little sequence identity except for mouse and human
Efp. However, there is conservation of heptad repeats of hydrophobic
amino acids over this region. PML could form a complex with
PML-RAR (presumably as a heterodimer) by the coiled-coil domain
comprised of heptad repeats(34) . The coiled-coil structure
could be required for dimer formation. Amino acid comparison of
proteins with the putative C-terminal domain shows the existence of
conserved residues(38, 42) . Excepting the PML, T18,
and Rpt-1, the RING-B box-containing subfamily has a C-terminal domain,
which is highly conserved, though nothing is known about its function.
A number of proteins having the RING finger motif are involved in regulating gene expression. For example, Rpt-1 is a down-regulator of the interleukin-2 receptor and human immunodeficiency virus type 1 genes(36) . The Rfp is proposed to be a transcription regulator in spermatogenesis(37) . XNF-7 is a putative transcription regulator expressed maternally in Xenopus laevis(40) . PWA33 is associated with the nascent transcripts on the lampbrush chromosome loops and likely to be a regulatory protein during early development (41) . These data suggest that Efp may also be a transcription regulator, although rigorous proof awaits more experimentation.
Several RING finger-containing proteins are
implicated in cell transformation. For example, PML produces a fusion
protein with the retinoic acid receptor in acute
promyelocytic
leukemia(31, 32, 33, 34) . T18 is a
transforming mouse fusion protein with the B-raf proto-oncogene(35) . Human Rfp fused with the ret proto-oncogene acquires transforming activity(37) . Mouse bmi-1 cooperates with the myc oncogene in lymphoma
development(43, 44) . Freemont et
al.(45, 46) report that two oncogenes, c-cbl(47) associated with lymphoma and mdm-2(48) , which forms a complex with p53 protein and
inhibits its transactivation, also contain a RING finger motif.
Recently, the BRCA1 gene that was identified as a
tumor-suppressor gene for the early-onset breast cancer and ovarian
tumor by linkage analysis has been cloned(49) . Interestingly,
the BRCA1 gene also had the RING finger motif and
was localized in the chromosome 17q 21.3 locus close to 17q 23.1, where
human Efp was localized(30) . Efp is a member of the RING-B
box-containing subfamily that includes PML and T18, which raises an
interesting possibility that the Efp may be involved in cell
transformation or make a fusion protein related to oncogenesis.
The co-localization of Efp and ER
mRNA in a number of tissues supports the idea that efp is an
estrogen-responsive gene in vivo in these organs. Previous in situ hybridization histochemistry of ER (3) and I-labeled estrogen autoradiography (4) showed
that ER mRNA was widely distributed among neurons of rat brain, with
the greatest density in medial preoptic and ventromedial nuclei of
hypothalamus. The presence of Efp mRNA-containing neurons in the
ventromedial hypothalamic nucleus may suggest that Efp is involved in
the regulation of reproductive behavior or other behaviors through
estrogen.
Northern blot analysis showed that the expression of mouse Efp mRNA was up-regulated by estrogen within 2 h in uterus as well as in brain in vivo. Human efp was also transcriptionally regulated by estrogen at a short response time (within 2 h) in ER-positive cells derived from mammary gland(20) . Mouse efp and human efp contain ERE sequences in the 3`-untranslated region. An ERE in mouse efp is an imperfect palindromic sequence (AGGGCAGGGTGACCT) (Fig. 1A), but it is known that an imperfect palindromic ERE sequence can actually function (e.g. in the cases of pS2 (14) and prolactin(13) ). This imperfect palindromic ERE of mouse efp might act as a downstream estrogen-dependent enhancer just like the ERE of human efp. Further analysis is required to establish the role of this sequence.
In embryo, Efp mRNA was detected in the head during the gestation by RNase protection assay and also detected throughout the developing brain vesicles by in situ hybridization histochemistry. ER mRNA was also detected in the heads of 10.5- and 12.5-day embryos by the reverse transcriptase-polymerase chain reaction method and in the heads of 14.5-18.5-day embryos by RNase protection assay (data not shown), although the level of ER mRNA in 10.5- and 12.5-day embryos was shown to be rather low. The expression of Efp and ER mRNA during embryogenesis may be related to the regulation of brain development in terms of sexual dimorphism, etc.
In a mouse ER gene targeting model(57, 58) , the female homozygote was found to be infertile, having a hypoplastic uterus, hyperemic cystic ovary, and decreased skeletal mineralization, and showed abnormal sexual behavior. A germ line mutation of the ER gene in humans, resulting in estrogen insensitivity syndrome, was also identified recently(59) . The affected male patient was tall, because of the failure of epiphyseal closure, and had low bone mineral density despite otherwise normal pubertal development. These studies suggest that ER is indispensable at least for female fertility, maturation of the female genital tract, bone maturation, and normal epiphyseal closure, although the disruption of the ER gene is not necessarily lethal.
In the hierarchy of estrogen action that we have postulated(20) , it is assumed that Efp is a candidate for the estrogen-responsive transcription factor. The short response time (2 h) of efp to estrogen in vivo is in line with this prediction.
It is possible that Efp is involved in physiologic actions of estrogen (e.g. maturation of reproductive organs during secondary sexual development, menstrual cycle, pregnancy, bone maturation, reproductive behavior, and/or carcinogenesis). The mouse targeting model of efp that is now under way would help clarify these points. The data presented here could be utilized in future studies of gene targeting as well as transgenic expression. Furthermore, we believe that the isolation of more estrogen-responsive genes and the analyses of their functions would be essential to understand the mechanism of diverse estrogen actions in various organs.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D63902[GenBank].