MCAP is a conserved bromodomain protein.
A bromodomain is found in a growing number of proteins involved in the regulation of nuclear activities (15
). Recent information on lower eukaryote genomes indicates the presence of additional bromodomain-containing proteins in higher eukaryotes. To identify novel mammalian proteins carrying a bromodomain, we screened mouse cDNA libraries with a 150-bp PCR fragment containing a part of bromodomain sequence. By assembling five overlapping cDNAs obtained by several cycles of screening, we generated a full-length clone of 5,281 bp. The predicted first methionine was identified at nucleotide position +35 preceded by stop codons in all three reading frames. The assembled MCAP cDNA encodes a protein of 1,400 amino acids, which we designated MCAP (mitotic chromosome-associated protein). It has two bromodomains in the N-terminal region and an ET domain in the more C-terminal region, a characteristic feature of the BET subgroup of the bromodomain superfamily (Fig. A) (21
). This subgroup includes the human RING3, Drosophila
FSH, and yeast BDF1/BDF2. Similar to other members of the BET subgroup, the MCAP bromodomains contain a core motif and flanking motifs (14
), which likely form a helical bundle (8
). MCAP carries a stretch of amino acids homologous to the “kinase like motifs” described for RING3 (6
). As shown in Fig. B, four mammalian cDNAs show homology with MCAP: HUNK1, RING3 (Fsrg1), BRDT, and ORFX (24
). MCAP shows highest homology to the uncharacterized human cDNA HUNK1 (y12059) and to cosmids (R28194 and R31546 [GenBank accession no. AC003111
]). The HUNK1 cDNA, however, encodes a protein of 722 amino acids, much smaller than MCAP.
FIG. 1 Amino acid sequence and chromosomal mapping of murine MCAP. (A) Predicted amino acid sequence of MCAP. A single open reading frame containing 1,400 amino acids was derived from a 5,281-bp cDNA. Two bromodomains (BDI and BDII) are shaded (black; core motif; (more ...)
In vitro translation of full-length MCAP cDNA produced a protein of approximately 200 kDa, larger than the deduced molecular mass of 155 kDa. A posttranslational modification or a protein secondary structure may account for the difference (see below). Northern blot analysis revealed a single RNA species of ~6.5 kb, ubiquitously expressed in mouse adult and embryonic tissues and in human cells (not shown).
The murine MCAP gene is mapped to chromosome 17.
Genetic mapping of the murine MCAP gene was done by FISH analysis. A biotinylated 18-kb genomic fragment of MCAP was hybridized to normal, mitogen-stimulated male spleen cells. A clear single hybridization signal was detected on the distal region of band B, chromosome 17, in the vicinity of the complement component 3 (C3) locus (Fig. C). This region is syntenic to human chromosome 19, in which the human homologue HUNK1 has been mapped. It is of note that mouse RING3 (Fsrg1) is also localized on chromosome 17, but within the major histocompatibility complex (2
), located proximal to the C3 locus.
MCAP is a nuclear protein broadly expressed in mouse tissues.
Immunoblot analysis was performed using two antibodies raised against a N-terminal or C-terminal region of MCAP. As shown in Fig. , both antibodies revealed a 200-kDa protein expressed in the nuclear fraction of all mouse tissues tested. MCAP was expressed at the highest levels in spleen and thymus; expression was lower in liver and brain. The antibodies also reacted with a 200-kDa nuclear protein in cultured cells, e.g., mouse P19 and human HeLa cells (Fig. ). In all cases no other bands were detected, in agreement with a single RNA species. The cytoplasmic fractions were devoid of antibody reactivity (not shown; see Fig. ). The antibodies also immunoprecipitated a 200-kDa protein from in vitro-translated MCAP as well as from nuclear extracts of various cells (not shown).
MCAP protein expression detected by immunoblot analysis using anti-MCAP antibody with 10 μg of nuclear extracts from adult mouse tissues, HeLa cells, or P19 cells. S. Intestine, small intestine.
FIG. 4 Localization of MCAP on mitotic chromosomes. (A) Indirect immunofluorescent staining of P19 cells. P19 cells were fixed with paraformaldehyde and stained with antibodies against MCAP (N-MCAP) (a), CBP (c), or Sp1 (e) and counterstained with Hoechst 33342 (more ...) MCAP expression is enhanced by growth-stimulatory signals and repressed by growth-inhibitory signals.
Data in Fig. suggested that MCAP expression correlated with the presence of proliferating cells in tissues. To examine whether MCAP expression is linked to cell proliferation, we first tested mitogen-stimulated lymphocytes. Mouse spleen cells were stimulated by bacterial LPS or ConA, a B-cell- or T-cell-specific mitogen, respectively, and MCAP expression was tested by RT-PCR and immunoblot assays. As shown in Fig. A and B, untreated lymphocytes expressed very low levels of MCAP, as the majority of cells were quiescent. However, within 6 h of treatment with either mitogen, MCAP expression was markedly increased at both RNA and protein levels (Fig. A and B). The protein levels reached maximum at 6 h and persisted until 24 h. A slight increase in untreated cells at 12 and 24 h was presumably due to activation of some cells by serum factors. Expression of TFIIB, tested as a control, remained at a constant level in these cells (Fig. B). To assess a relationship between MCAP expression and DNA synthesis, we examined the kinetics of [3H]thymidine ([3H]TdR) incorporation. As seen in Fig. C, [3H]TdR incorporation was at a background level 6 and 12 h after stimulation when MCAP protein expression was already at maximum. An increase in [3H]TdR uptake was detected only at 24 h, after which levels remained high for an additional 24 h. Thus, the onset of DNA synthesis lagged behind that of MCAP expression. These results indicate that MCAP is induced by mitogen stimulation during the G0/G1 transition in lymphocytes, prior to the entry into S phase.
FIG. 3 MCAP expression is linked to cell growth. (A) Induction of MCAP RNA in mitogen-stimulated lymphocytes. Spleen cells were stimulated by indicated mitogens for 6 h, and MCAP transcripts were detected by semiquantitative RT-PCR. HPRT transcripts were tested (more ...)
Next, we evaluated MCAP expression in an opposite situation, where cell growth was arrested by growth factor deprivation. Immunoblot analysis was performed with 32D myeloid progenitor cells that underwent growth arrest upon IL-3 withdrawal (47
) (Fig. D). In the presence of IL-3, MCAP was expressed at high levels in 32D cells (lane 1); within 6 h after IL-3 withdrawal, expression was completely extinguished (lane 2). MCAP expression resumed when IL-3 was added back to the medium (lanes 3 and 4).
We also tested MCAP expression in RA-treated P19 embryonal carcinoma cells, which underwent growth inhibition concurrent with the induction of differentiation (19
). As seen in Fig. E, MCAP levels were high in rapidly proliferating, untreated P19 cells, but steadily decreased during 4 days of RA treatment. These results indicate that MCAP expression is regulated by growth-stimulatory and growth-inhibitory signals in opposite ways.
MCAP localizes on noncentromeric regions of mitotic chromosomes.
Consistent with the Western blot data above, indirect immunofluorescent staining of P19 cells detected MCAP in the nucleus but not in the cytoplasm (Fig. A). During interphase MCAP was uniformly distributed in the nucleus with the exception of nucleoli. However, in mitotic cells, MCAP was detected almost exclusively on condensed chromosomes (Fig. A, a and b). Chromosomal localization of MCAP was likewise detected on mitotic HeLa cells and 32D cells (not shown). In well-spread metaphase preparations (Fig. B), almost the entire length of chromosomes was intensely stained with anti-MCAP antibody. However, MCAP staining was distinctly absent from the centromeres, which showed brighter DNA staining than the rest of chromosomes (Fig. B, inset).
In mammalian cells, a number of transcription factors and regulatory proteins are displaced from chromosomes during mitosis, which coincides with global transcriptional repression (29
). In accordance, we found that both Sp1 and CBP, a DNA-binding transcription factor and general coactivator, respectively, were dispersed into the cytoplasm during mitosis, while they were localized in the nucleus during interphase (Fig. A, c to f). We noted that several other transcription factors are also dispersed into the cytoplasm during mitosis in P19 cells (not shown). These results indicate that MCAP is held onto mitotic chromosomes during when many regulatory factors are released into the cytoplasm.
MCAP staining during mitosis.
Figure A shows immunostaining of MCAP at different stages of mitosis. In prophase when heterochromatic regions of chromosomes began to condense, MCAP was evenly distributed in the nucleus. In the subsequent prometaphase, MCAP localization to the chromosomes became evident as chromosomal condensation intensified. At this point, MCAP showed little residual staining elsewhere in the cell. In metaphase, MCAP staining remained on fully condensed chromosomes that were assembled on the equatorial plate and attached to the spindles. In anaphase and telophase when sister chromatids separated, MCAP was still detected on the segregating chromosomes. Thus, MCAP-chromosome association becomes visible following the onset of early chromosomal condensation and persists until the end of mitosis.
FIG. 5 Fine timing of MCAP chromosome staining. (A) P19 cells were stained with anti-MCAP antibody, Hoechst 33342, and anti-β-tubulin antibody. Arrows in prophase indicate condensing chromosomes. At this stage, MCAP distribution is uniform over the entire (more ...)
The above results raised the possibility that MCAP selectively localizes to the region of chromosomes that condense relatively late. Chromosomal condensation proceeds in a nonrandom, spatiotemporal order which can be monitored by the timing of histone H3 phosphorylation (16
). Histone H3 phosphorylation starts first at the pericentromeric heterochromatin and then extends to other parts of the chromosomes. We compared the timing of histone H3 phosphorylation with that of MCAP-chromosome association. As shown in Fig. B, a prominent dot-like staining of phospho-histone H3 was seen in G2
and prophase nuclei, corresponding to the sites of early chromosomal condensation. Staining of MCAP at those stages remained diffuse throughout the nuclei, without specifically colocalizing with phospho-histone H3. MCAP staining matched that of phospho-histone H3 only after the latter spread to the rest of the chromosomes, which began in prometaphase. These results, together with the absence of MCAP on the centromeres (Fig. B), suggest that MCAP predominantly associates with the late-condensing regions of the chromosomes rather than the early-condensing heterochromatic regions.
Localization of GFP-MCAP on mitotic chromosomes.
Chromosomal association of the endogenous MCAP observed above prompted us to investigate localization of an exogenously expressed MCAP. HeLa or NRK cells were transfected with a construct containing GFP-MCAP, and GFP distributions were analyzed in a series of z sections, which were reconstructed to three-dimensional images (Fig. A). Similar to the endogenous MCAP, GFP-MCAP was detected in the interphase nucleus as fine grains distributed evenly from the periphery to the center, except for nucleoli (Fig. A, left). On the other hand, during mitosis, GFP-MCAP signals smoothly outlined the condensed chromosomes (Fig. A, right). These observations confirm that MCAP is uniformly distributed in the nucleus during interphase and associates with chromosomes during mitosis.
FIG. 6 (A) Three-dimensional reconstruction of GFP-MCAP localization in living cells. Three-dimensional images were reconstructed with serial z sections of HeLa or NRK cells to visualize the distribution of MCAP-GFP in the interphase (left) and in mitosis (right). (more ...) FRAP and biochemical analysis.
To test whether MCAP is a stable component of chromatin, we used FRAP (10
). This method has been used to measure mobility of intracellular molecules by the recovery of fluorescent signals after brief laser irradiation. Several previous papers on studies using this method reported that chromatin in interphase nuclei is relatively immobile and may be anchored as a defined structure (1
). On the other hand, recent reports on photobleaching of the high-mobility-group proteins and glucocorticoid hormone receptor (31
) indicate that chromatin-bound proteins can recover relatively rapidly after bleach. Thus, if MCAP is very strongly immobilized on chromatin, its exchange might be slow. FRAP analysis was performed with GFP-MCAP transfected in HeLa cells (used for Fig. ). To compare the mobility of MCAP with that of a known chromatin component, histone H2B-GFP was analyzed in parallel. A 4-μm2
strip through the nucleus was photobleached, and recovery of fluorescence into this area was recorded until the intensity reached a stable plateau. As shown in Fig. B, GFP-MCAP fluorescence was reduced to background levels immediately after photobleaching but recovered ~86% of its intensity within 1 min. By contrast, histone H2B-GFP did not recover any fluorescence over this period. These results indicate that while histone H2B, a stable component of chromatin is immobile, the majority of GFP-MCAP is capable of exchanging with a half-life of ~4 s within the interphase nucleus. The relatively small but significant immobile fraction (~14%) of GFP-MCAP may represent a more tightly chromatin-bound pool of the protein.
To further investigate MCAP-chromatin association, we examined the solubility of endogenous MCAP by differential salt extractions. Asynchronously growing HeLa cells or those synchronized to M phase were extracted by buffer containing increasing NaCl concentrations. The presence of MCAP in the soluble and insoluble fractions was tested by immunoblot analysis (Fig. C). With the lowest salt concentration (100 mM NaCl), approximately half of MCAP was present in the soluble fraction, with the rest in the insoluble fraction. But with higher NaCl concentrations (200 and 300 mM), most of MCAP was in the soluble fraction. The profile of salt solubility was essentially the same for asynchronous and mitotic cells. As expected, the general transcription factor TFIIB was found in the soluble fraction, while histone H3 was in the insoluble fraction at all NaCl concentrations tested. These results are in agreement with FRAP data above, and indicate that MCAP loosely interacts with chromatin during interphase as well as mitosis. Consistent with these findings, MCAP did not exhibit a strong binding affinity for double-stranded or single-stranded DNA in vitro.
Microinjection of anti-MCAP antibody inhibits cell cycle progression to mitosis.
As an initial step to delineate the function of MCAP, we studied the effect of anti-MCAP antibody microinjection on cell cycle progression in HeLa cells. Cells synchronized by double-thymidine block were released and allowed to proceed through cell cycle. The diagram in Fig. A shows the timing of microinjection and an example of cell cycle profiles monitored during the experiments by FACS analysis. Anti-MCAP IgG was injected into the nuclei when cells were at S or in G2. Normal IgG from preimmune sera was injected as a control. In each experiment, IgG was injected into 25 to 40 nuclei. Cells were then allowed to proceed in culture until they reached mitosis. After being fixed, the cells were stained with anti-rabbit antibody coupled to biotin-streptavidin-Cy3 to distinguish injected cells from uninjected ones and with Hoechst 33342 to detect mitotic cells. Although some cells died soon after injection due to physical shock or injury, about 70% survived until the end of experiments. Table shows the number of cells that successfully reached mitosis in four separate experiments. In the control groups where cells were injected with preimmune IgG, approximately 40% of cells were in mitosis as judged by Hoechst staining, irrespective of whether IgG was injected in S or G2 phase. In contrast, almost no mitotic cells were observed in the groups injected with anti-MCAP IgG regardless of growth phase (S or G2), indicating that anti-MCAP antibody inhibited entry into mitosis. Figure B shows injected IgG and DNA in the cells. In the preimmune IgG-injected group (Fig. B, a and b), three cells were in anaphase/telophase and one was in metaphase. An uninjected cell which was in metaphase was used as a control. In the group injected with anti-MCAP IgG (Fig. B, c and d), four injected cells had a large interphase nucleus, indicative of cells in G2, but none in mitosis. These cells did not even exhibit an early sign of mitosis, as evidenced by the lack of condensing chromosomes, consistent with the idea that MCAP antibody inhibited the entry into mitosis. Although some cells escaped synchronization, ~40% of uninjected cells were in mitosis, similar to the control groups. The paucity of mitotic cells in the anti-MCAP IgG-injected groups was unlikely to be due to a delay in mitosis, because no mitotic cells with anti-MCAP antibody stain were detected when cells were cultured for additional 4 h and mitotic cells were monitored every hour (not shown). It was not due to the acceleration of G2/M phase either, since no newly divided cells with antibody stain were detected in the MCAP antibody-injected groups.
FIG. 7 Anti-MCAP antibody injection inhibits the entry into mitosis. (A) Diagram of microinjection experiments. HeLa cells were synchronized by double-thymidine block and released. The lower panel represents a typical cell cycle profile monitored by FACS analysis. (more ...)
TABLE 1 Injection of anti-MCAP IgG inhibits G2-Mtransitiona
It was of importance to assess whether injection of anti-MCAP IgG into S-phase cells abolished ongoing DNA synthesis. To address this question, cells injected with anti-MCAP IgG were pulse-labeled with BrdU for 1 h. Cells were allowed to continue growth as described above. BrdU uptake was monitored by anti-BrdU monoclonal antibody. Shown in Fig. C (row a) is an example of a cell injected with anti-MCAP IgG. This cell, while arrested prior to mitosis, incorporated BrdU. BrdU staining was absent in the nucleoli, similar to that of normal, uninjected cells (Fig. C, row c). These results show that anti-MCAP IgG, when injected into S-phase cells, inhibited mitosis without completely abrogating ongoing DNA synthesis. Results with G2 cells indicate that anti-MCAP antibody interfered with mitotic entry; however, an additional possibility that anti-MCAP antibody interferes with the completion of DNA replication cannot be excluded.