(Received for publication, October 20, 1995; and in revised form, January 18, 1996)
From the
With sequence homology to the SV40 T antigen-binding domain of the retinoblastoma protein (Rb), p107 and p130 constitute two additional members of the Rb family. To explore the potential function of p130 in mouse development, we cloned the full-length mouse cDNA for p130 and characterized p130 mRNA expression in mice. The deduced mouse p130 protein sequence shares a higher degree of similarity with mouse p107 than with mouse Rb. In adult mice, p130 mRNA is found in all tissues examined. Levels of p130 mRNA vary among different adult tissues, with the highest level in testis. Within testis, p130 mRNA is found predominantly in Leydig cells. Additionally, p130 expression in testis correlates with sexual maturation, suggesting p130 is important for the development of testis and, in particular, Leydig cells. In situ hybridization shows that in post coitus day 12.5 and 14.5 mouse embryos, distribution of p130 mRNA is quite uniform with the exception of a few tissues. Little differences in mRNA levels of either p130 or p107 were found between normal and Rb-deficient embryos, suggesting that p130 and p107 are expressed independently of Rb. Our data are consistent with the hypothesis that p130 and p107 do not compensate for the loss of Rb and support the view that p130 is related to, yet distinct from, the RB gene.
The retinoblastoma protein (Rb) family is comprised of Rb, p107, and p130(1) . The gene for human P130 has been cloned through its association with E1A(2) , cyclins, and CDK2(3) , and sequence homology to RB and P107(4) . Structurally, P130 contains the SV40 T antigen-binding (T-binding) domain characteristic of the RB protein family, which is composed of subdomains A and B, separated by a spacer region. The protein sequences of P130 and RB are conserved in subdomains A and B and exhibit little similarity outside this region. The P107 protein sequence, on the other hand, shares homology with the P130 sequence over its entire span. The primary structures of the three human proteins suggest that P130 is more closely related to P107 than to RB(2, 3, 4) .
Like Rb and p107,
overexpression of p130 suppresses cell growth in
vitro(5, 6) . A major target of the Rb family
members in inhibiting cell proliferation is the transcriptional factor
E2F(7) , which regulates expression of many genes important for
S phase progression such as DNA polymerase , dihydrofolate
reductase, and thymidine kinase(8, 9) . The Rb family
proteins bind to E2F and repress E2F's function as a
transcriptional activator. The T-binding domain of the Rb family
proteins plays a primary role in their function. Through this domain,
Rb, p107, and p130 interact with E2F and other cellular factors. Viral
oncoproteins such as SV40 T antigen, adenovirus E1A, and papilloma
virus E7 inhibit the function of p130, p107, and Rb by association with
the same domain of those proteins, disrupting complexes formed between
cellular factors and the Rb family proteins (1) .
Despite some shared functional and structural features, p130 possesses certain functions distinct from Rb. The retinoblastoma gene (RB) is frequently mutated in tumors(1) . Mutations in the P130 gene, on the other hand, have yet to be documented in human primary tumors. p130 also differs from Rb in its respective binding efficiencies toward distinct E2F family members(10, 11, 12) . In the glioblastoma cell line T98G, overexpression of p130, but not Rb, inhibits cell growth(6) . These observations implicate functional differences between p130 and Rb.
The ubiquitous expression of Rb suggests Rb may function in all cell types(13, 14, 15) . However, only a limited spectrum of human tumors contains RB mutations. In the Rb-deficient mouse embryos, abnormalities are observed in specific cell types such as developing neurons, erythroblasts, and lens epithelial cells(16, 17, 18, 19) . The limited spectrum of human tumors with RB mutations and the limited phenotype of RB nullizygous embryos raise the question whether there is functional redundancy among p107, p130, and Rb, such that p130 and p107 could compensate for the loss of Rb in certain cell types. This compensation hypothesis was proposed in an in vitro study(20) . In the Rb-deficient skeletal myoblast cell line CC42, p107 levels were elevated compared with C2C12 cells, a myogenic cell line with normal RB. It was then suggested that the up-regulation of p107 partially fulfilled the requirement of Rb for skeletal muscle cell differentiation in vitro. Because skeletal muscle tissue in the Rb-deficient mouse embryos was grossly normal, it is of interest to determine if p107 or p130 acts to compensate for the loss of Rb in skeletal muscle in vivo.
In this study, to examine the functions of p130 in vivo, we cloned the mouse cDNA for p130 and characterized its expression during mouse development. In addition, we also addressed the question whether p107 and p130 acted to compensate for the loss of Rb in the Rb-deficient mouse embryos, conferring grossly normal phenotypes to certain tissues.
For Northern blot analysis, mRNA was separated by electrophoresis in agarose gels containing formaldehyde. RNA transfer and hybridization were performed as described by Sambrook et al.(21) .
In the
study of p130 expression in mouse embryos, the p130 sense and antisense probes were 50-mer oligonucleotides
corresponding to nucleotides 3146-3195 of p130 cDNA. They were
labeled at the 3` end with [-
P]dATP and TdT
to a specific activity of approximately 10
cpm/µg and
purified through an NENsorb column (DuPont NEN). In the study involving
mouse testes, the p130 sense and antisense RNA probes were
labeled with digoxigenin-11-UTP. Hybridization signals were visualized
following the manufacturer's suggestions (Boehringer Mannheim),
using an alkaline phosphatase-conjugated secondary antibody.
Figure 1: Nucleotide sequence of mouse p130 cDNA and the deduced protein sequence.
Like their human counterparts, mouse p130 shares a higher degree of similarity with mouse p107 than with mouse Rb (Fig. 2). The A and B subdomains of mouse p130 are conserved with those of mouse Rb and p107. Little similarity exists in the N termini, the C termini, and the spacer regions of mouse p130 and Rb. On the other hand, the N termini of mouse p130 and p107 are well conserved, suggesting that this region is important for the function of p130 and p107.
Figure 2: Schematic diagram showing mouse protein sequence similarities between p130 and Rb, and between p130 and p107. N and C represent N terminus and C terminus, respectively. A, S, and B represent the A region, the spacer region, and the B region of the T-binding domain, respectively. The A domains include amino acids 367-573 of mouse Rb(14) , 413-612 of mouse p130, and 384-583 of mouse p107(26) . The B domains include amino acids 630-765 of mouse Rb(14) , 822-1020 of mouse p130, and 779-942 of mouse p107(26) . The sizes drawn do not reflect the actual scale of the three proteins.
Figure 3: Expression of mouse p130 and mouse Rb in adult mouse tissues, determined by RNase protection assays. Antisense RNA probes to mouse p130, Rb, and GAPDH were hybridized together to mRNA samples. The sizes of the probes are shown at the left. Protected fragments of the three genes are marked at the right side of the gel with arrows. At the bottom is a shorter exposure of the same gel showing protected GAPDH contents. The amounts of mRNA used were normalized to GAPDH and were in the µg range. bp, base pairs.
Figure 4:
Developmental expression of p130 mRNA in
testis. A, Northern blot of p130. mRNA was obtained
from testes of 5-day, 8-day, 15-day, 25-day, and 83-day-old mice. The P-labeled DNA probe corresponded to nucleotides
2865-3221 of p130 cDNA. 2 µg of each mRNA was loaded onto the
gel. The sizes of the two bands hybridized to p130 were
marked. B, levels of
-actin mRNA present in the filter.
The filter used in A was rehybridized to a
-actin
probe.
To identify the cell types in testis that express mRNA for p130, we performed in situ hybridization on adult testes. Intense cytoplasmic staining was found in the interstitial Leydig cells that were located between seminiferous tubules (Fig. 5). Thus the Leydig cell population is the predominant source of p130 mRNA in testis.
Figure 5:
In situ hybridization of p130 on testes from 2-month-old mice with antisense (A and B) and sense (C and D) p130 probes.
Microscopic magnifications are 10 2.5 for A and C and 40
1.25 for B and D. The RNA probe
encompassing p130 cDNA nucleotides 2865-3221 was labeled with
digoxigenin-UTP. Hybridization signals were visualized as described
under ``Experimental Procedures.'' St, seminiferous
tubule; Ld, Leydig cell.
Figure 6: Expression of p130 mRNA in mouse embryos from gestation day 9.5 to 16.5. mRNA was extracted from whole embryos, and its p130 contents were examined by RNase protection assay. Probes against p130 and GAPDH were added together to the hybridization mixtures.
Figure 7: In situ hybridization of p130 on RB heterozygous (RB +/-) and RB nullizygous (-/-) mouse embryos. A, E12.5, RB +/-; B, E12.5, RB -/-; C, E14.5, RB +/-; D, E14.5, RB -/-; E, E14.5, RB +/-. Antisense p130 probes were used in A, B, C, and D. In E, in situ hybridization was performed with sense p130 probes as a negative control. Abbreviations: co, colon; h, heart; hb, hindbrain; ie, inner ear; k, kidney; lu, lung; lv, liver; sc, spinal cord; tg, trigeminal ganglion. RB +/- embryos are grossly normal and phenotypically indistinguishable from wild type embryos.
Figure 8: Comparison of p130 (A), p107, and Rb (B) mRNA levels in E13.5 wild type and RB nullizygous mouse embryos by RNase protection assays. Mouse genotypes are shown above the gel, under the bracket. mRNA was extracted from the head, trunk, limb, and liver of the embryos and used in the RNase protection assays. The antisense RNA probes for p130 and RB were the same as in Fig. 3. Antisense p107 probe corresponded to mouse p107 cDNA nucleotides 2532-2690(26) . GAPDH antisense RNA probes were added together with p130 (A) or p107 and RB probes (B) to the hybridization mixtures. Protected bands were present in the Rb-deficient embryos because the antisense RB probe used encompassed a region 5` to exon 20, the exon that was targeted by homologous recombination.
In this study, we cloned the cDNA for mouse p130 and characterized p130 mRNA expression in mouse development. Similar levels of p130 and p107 mRNA were observed between RB nullizygous embryos and normal embryos in tissues that are not affected by RB mutations, suggesting that the limited phenotypic defects seen in RB nullizygous mouse embryos could not be due to compensation by p107 or p130. Our results indicate that p130 and p107 mRNA levels are regulated independently of Rb in embryogenesis.
Sequence comparison of the three mouse proteins Rb, p107, and p130
reveals that mouse p130 is more closely related to p107 than to Rb.
Conservation between p130 and Rb is limited to the A and B subdomains,
while p130 and p107 are conserved in their entire length. Similar
conclusions on their three human counterparts were also
drawn(2, 3, 4) . The sequence information
suggests p130 and p107 may serve unique functions and do not overlap
entirely with Rb functions. This view is reinforced by several
observations. First, p130 and p107 both associate with E2F-4, whereas
Rb associates with E2F-1, E2F-2, and E2F-3(11, 25) .
Second, the RB gene is mutated in many tumors. In contrast, no
mutations in the P107 gene or the P130 gene have been
reported in human primary tumors(1, 7) . Third, we did
not observe an apparent compensation of p130 and p107 for loss of Rb during mouse development. Fourth, mice
deficient in p130 or p107 were viable, ()in contrast to the
embryonic lethality caused by RB gene mutations.
Schneider et al.(20) reported that the Rb-deficient mouse skeletal myoblast cell line CC42 cells expressed elevated levels of p107 compared with C2C12 cells, a myogenic cell line with wild type RB(20) . The authors proposed that the increased levels of p107 acted to assume certain functions of Rb to allow myogenesis to occur. In our study, we did not detect elevated mRNA levels of p107 and p130 in the skeletal muscles of RB nullizygous mouse embryos. The discrepancy in observations made by us and Schneider et al.(20) could reflect differences between in vivo and in vitro conditions.
The expression of p130 in testis is of interest. Testis was the one adult tissue that expressed the highest levels of p130 mRNA. A significant portion of p130 mRNA in testis was from Leydig cells. Additionally, expression of p130 was greatly increased in sexually mature mice relative to prepubescent mice. Kim et al.(26) observed the highest level of mouse p107 mRNA in adult testis. We compared levels of p130, p107, and Rb mRNA in adult mouse testes with 9-day-old mouse testes. Levels of p107 and Rb mRNA, unlike p130 mRNA, were only modestly higher in adult mouse testes (data not shown). Our data suggest p130 may play important functions in testis development and, in particular, Leydig cells.
A 4.7-kb band and a 2.1-kb band were seen in the p130 Northern blots using testis mRNA. The two transcripts, which were also present in other adult tissues (data not shown), were likely the results of alternative splicing, given the precedence of alternative spliced forms of Rb and p107 mRNA(14, 26) . The 2.1-kb transcripts hybridized to p130 probes in this study would be too small to encode the full-length p130 protein. The functional significance of the shorter transcripts of the three mouse RB family genes is currently unknown.
Observations made in this study support the notion that the functions of Rb and p130 are distinct. The exact roles of p130 in vivo are not yet clear and cannot be simply extrapolated from those of Rb. The molecular cloning of mouse p130 cDNA and characterization of p130 mRNA expression pattern should facilitate future studies on the function of p130.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U50850[GenBank].
Note Added in Proof-During the review process of the manuscript, a study describing the murine p130 cDNA was published (Pertile, P., Baldi, A., De Luca, A., Bagella, L., Virgilio, L., Pisano, M. M., and Giordano, A.(1995) Cell Growth & Differ.6, 1659-1664). There are minor sequence differences, most likely due to an inclusion of an additional small exon in our cDNA. The 10 amino acids encoded in our cDNA but not in the published cDNA are present in human p130.