Inability to enter S phase and defective RNA polymerase II CTD phosphorylation in mice lacking Mat1
2001; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês
10.1093/emboj/20.11.2844
ISSN1460-2075
AutoresDerrick J. Rossi, Anou Londesborough, Nina Korsisaari, Arno Pihlak, Eero Lehtonen, Mark Henkemeyer, Tomi P. Mäkelä,
Tópico(s)RNA Research and Splicing
ResumoArticle1 June 2001free access Inability to enter S phase and defective RNA polymerase II CTD phosphorylation in mice lacking Mat1 Derrick J. Rossi Derrick J. Rossi Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Anou Londesborough Anou Londesborough Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Nina Korsisaari Nina Korsisaari Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Arno Pihlak Arno Pihlak Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Eero Lehtonen Eero Lehtonen Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Mark Henkemeyer Mark Henkemeyer Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9133 USA Search for more papers by this author Tomi P. Mäkelä Corresponding Author Tomi P. Mäkelä Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland HUCH Laboratory Diagnostics, Helsinki University Central Hospital, PO Box 401, 00029 HYKS Finland Search for more papers by this author Derrick J. Rossi Derrick J. Rossi Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Anou Londesborough Anou Londesborough Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Nina Korsisaari Nina Korsisaari Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Arno Pihlak Arno Pihlak Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Eero Lehtonen Eero Lehtonen Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland Search for more papers by this author Mark Henkemeyer Mark Henkemeyer Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9133 USA Search for more papers by this author Tomi P. Mäkelä Corresponding Author Tomi P. Mäkelä Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland HUCH Laboratory Diagnostics, Helsinki University Central Hospital, PO Box 401, 00029 HYKS Finland Search for more papers by this author Author Information Derrick J. Rossi1, Anou Londesborough1, Nina Korsisaari1, Arno Pihlak1, Eero Lehtonen1, Mark Henkemeyer2 and Tomi P. Mäkelä 1,3 1Molecular Cancer Biology Research Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, PO Box 63, 00014 Helsinki, Finland 2Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9133 USA 3HUCH Laboratory Diagnostics, Helsinki University Central Hospital, PO Box 401, 00029 HYKS Finland ‡A. Londesborough and N. Korsisaari contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2844-2856https://doi.org/10.1093/emboj/20.11.2844 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The trimeric Cdk7–cyclin H–Mat1 complex comprises the kinase subunit of basal transcription factor TFIIH and has been shown to function as a cyclin-dependent kinase (Cdk)-activating kinase. Herein we report that disruption of the murine Mat1 gene leads to peri-implantation lethality coincident with depletion of maternal Mat1 protein. In culture, Mat1−/− blastocysts gave rise to viable post-mitotic trophoblast giant cells while mitotic lineages failed to proliferate and survive. In contrast to wild-type trophoblast giant cells, Mat1−/− cells exhibited a rapid arrest in endoreduplication, which was characterized by an inability to enter S phase. Additionally, Mat1−/− cells exhibited defects in phosphorylation of the C-terminal domain (CTD) of RNA polymerase II on both Ser5 and Ser2 of the heptapeptide repeat. Despite this, Mat1−/− cells demonstrated apparent transcriptional and translational integrity. These data indicate an essential role for Mat1 in progression through the endocycle and suggest that while Mat1 modulates CTD phosphorylation, it does not appear to be essential for RNA polymerase II-mediated transcription. Introduction Progression through the cell cycle is mediated by the sequential activation of cyclin-dependent kinases (Cdks), which phosphorylate substrates critical for advancing the cell cycle. Cdk activity is regulated by cyclin binding, Cdk inhibitors, proteolysis, localization and phosphorylation (reviewed in Morgan, 1997). Several cell cycle Cdks require phosphorylation of a conserved threonine residue within the T-loop for full activity (reviewed in Kaldis, 1999). The enzymes that catalyze this activation are known as Cdk-activating kinases or Caks. Biochemical purification of an activity from mammalian and Xenopus lysates capable of activating cdc2 in vitro (Solomon et al., 1992) led to the identification of a trimeric complex of the MO15/Cdk7 kinase together with cyclin H and a third subunit termed Mat1 (ménage-à-trois; reviewed in Nigg, 1996). In vitro, the mammalian Cdk7–cyclin H–Mat1 trimer can phosphorylate and activate Cdc2/Cdk1, Cdk2, Cdk3, Cdk4 and Cdk6 in complex with their cognate cyclin partners (reviewed in Kaldis, 1999). Shortly after their identification in Cak fractions, Cdk7–cyclin H–Mat1, along with the homologous complex Kin28–Ccl1–Tfb3 in budding yeast Saccharomyces cerevisiae, were found to be components of the nine-subunit basal transcription factor TFIIH (Feaver et al., 1994; Mäkelä et al., 1995; Serizawa et al., 1995; Shiekhattar et al., 1995). TFIIH provides several catalytic functions in mediating both basal transcription by RNA polymerase II (pol II) and transcription-coupled nucleotide excision repair (see Tirode et al., 1999, and references therein). These include the helicase activity afforded by the XPB and XPD subunits, which are involved in promoter melting prior to transcription initiation, in addition to the kinase activity provided by Cdk7–cyclin H–Mat1. TFIIH kinase activity is believed to be directed primarily at Ser5 of the repeated heptapeptide consensus sequence YSPTSPS (Gebara et al., 1997; Hengartner et al., 1998; Sun et al., 1998; Trigon et al., 1998), which comprises the C-terminal domain (CTD) of the large subunit of pol II. Phosphorylation of the CTD during pol II-mediated transcription has been shown to be critical in the recruitment of proteins necessary for proper processing of the nascent transcript (McCracken et al., 1997; Hirose and Manley, 1998; Hirose et al., 1999; Misteli and Spector, 1999; Komarnitsky et al., 2000; Rodriguez et al., 2000). The functions of Mat1 in the trimeric Cdk7–cyclin H–Mat1 kinase and as a subunit of TFIIH are not well understood. Mat1 enhances complex formation of cyclin H and Cdk7 in the absence of Cdk7 T-loop phosphorylation (Devault et al., 1995; Fisher et al., 1995; Tassan et al., 1995; Martinez et al., 1997). Several studies have suggested a role for Mat1 in the control of substrate specificity whereby Mat1 shifts the kinase activity of dimeric Cdk7–cyclin H from Cdk substrates towards CTD substrates in vitro (Inamoto et al., 1997; Rossignol et al., 1997; Yankulov and Bentley, 1997). Cdk7–cyclin H–Mat1 has been shown to have a wide range of additional in vitro substrates, and phosphorylation of several of these appears to be Mat1 dependent and is likely to occur within the context of TFIIH. These include p53 (Ko et al., 1997), pRb (Wu et al., 2001), the POU domains of the Oct factors (Inamoto et al., 1997), retinoic acid receptor-α (Rochette-Egly et al., 1997) and estrogen receptor-α (Chen et al., 2000). Primary sequence analysis indicates that Mat1 contains two conserved structural domains: a canonical RING finger domain and a coiled-coil domain. Structure–function mapping of Mat1 has suggested that the N-terminally located RING finger domain is associated with TFIIH-mediated transcriptional activation (Busso et al., 2000). The central coiled-coil domain is involved in establishing contacts with other TFIIH subunits, primarily with the XPD helicase (Busso et al., 2000). C-terminal sequences appear to be sufficient to mediate the assembly of Cdk7–cyclin H–Mat1 trimers (Tassan et al., 1995; Busso et al., 2000). The two principal pathways in which Cdk7–cyclin H–Mat1 are suggested to act, namely Cdk activation and pol II-mediated transcription, are both essential for the viability of the cell. Studies have shown that homologous molecules in non-mammalian species are essential genes, and thus a clear dissection of the functions of Cdk7–cyclin H–Mat1 in a physiological setting has proven difficult (Valay et al., 1995; Faye et al., 1997; Larochelle et al., 1998). Compounding this problem is the fact that it has become apparent that differences in the actual in vivo capacities of homologous kinases from different species exist. Notably, the Kin28–Ccl1–Tfb3 complex of S.cerevisiae has been shown to function only in TFIIH-mediated transcription (Cismowski et al., 1995; Valay et al., 1995; Holstege et al., 1998) while Cdk activation is provided by a separate monomeric kinase named Cak1/Civ1 (Espinoza et al., 1996; Kaldis et al., 1996; Thuret et al., 1996; Kimmelman et al., 1999). In Schizosaccharomyces pombe, on the other hand, both the Cdk7-related Mcs6 kinase complex and a single-subunit kinase Csk1 activate Cdks in vivo (Hermand et al., 1998; Lee et al., 1999; Hermand et al., 2001). To date, the best in vivo evidence that Cdk7–cyclin H–Mat1 functions as a Cak in metazoan species comes from experiments in Drosophila that utilized a temperature-sensitive allele of Cdk7 (DmCdk7) to show that activation of mitotic Cdk–cyclins was impeded at the restrictive temperature (Larochelle et al., 1998). However, a separate study utilizing a dominant-negative allele of DmCdk7 was unable to detect defects in Cak activity and instead described transcriptional defects (Leclerc et al., 2000). Although genetic studies in mammalian systems have not yet been reported, the recent biochemical characterization of a potential budding yeast Cak1/Civ1 homolog from mammalian cells (Nagahara et al., 1999; Kaldis and Solomon, 2000) has suggested that mammalian Cdk activation in vivo could be mediated by a single-subunit Cak in addition to, or perhaps in place of, Cdk7–cyclin H–Mat1. These putative kinases are unlikely to represent close sequence homologs of Cak1 based on the lack of evident Cak1 homologs in currently available mammalian databases. Thus the actual in vivo functions of the mammalian Cdk7–cyclin H–Mat1 kinase remain elusive. In this report, we have attempted to add some solvency to this issue by generating a loss-of-function allele of Mat1 in mouse to address whether the Cdk7–cyclin H–Mat1 kinase functions in pol II-mediated transcription, cell cycle progression or both. Results Targeted disruption of the murine Mat1 gene Mat1 genomic sequences were isolated from a 129-Sv library using a full-length human MAT1 cDNA as a probe. Restriction analysis and sequencing of several overlapping clones revealed that a single exon of Mat1 spanning 153 nucleotides of the cDNA (nucleotides 242–394 in DDBJ/EMBL/GenBank accession No. U35249) had been isolated. This exon encodes 51 N-terminally located amino acids encompassing most of the RING finger domain. A replacement-type targeting vector (Figure 1A) was constructed and electroporated into embryonic stem (ES) cells. Three clones were isolated (of 800 screened) that were confirmed to be targeted correctly by Southern blotting with both 5′ and 3′ external probes (Figure 1B). Targeted cells were then injected into BL/6 blastocysts and several of the resulting chimeras were found to transmit the targeted allele through the germline (Figure 1C). Figure 1.Generation of a Mat1 null allele. (A) Partial genomic structure of the murine Mat1 gene encompassing the targeted exon (nucleotides 242–394, amino acids 31–81) and target vector used to disrupt the locus. Upon homologous recombination into the murine genome, the PGK-neomycin resistance cassette of the target vector replaces 3 kb of genomic sequences between the HindIII (H) and KpnI (K) restriction sites flanking the targeted exon. The HSV-TK cassette was used as a negative selection marker. An introduced SacI restriction site (Sc1*) was used in screening for targeted events with both 5′ and 3′ probes. (B) Southern blotting analysis of targeted ES cell lines. SacI-digested genomic DNA from wild-type (wt) and targeted ES cell line 6.85 yielding predicted 5.5 kb (5′ probe) and 3.2 kb (3′ probe) bands in addition to a 10.5 kb wild-type band. (C) PCR genotyping of DNA isolated from tail cuts of F1 animals resulting from crossing a chimeric male (ES cell line 6.85) to wild-type females. A 190 bp wild-type band and a 310 mutant band are amplified with primer pairs M7–M10 and N4–M10 shown in (A). (D) Western blot of targeted ES cell lines. Total cell lysates from a wild-type ES cell (wt) and targeted ES cell line 6.85 western blotted with α-Mat1. Download figure Download PowerPoint Splicing around targeted exons has been demonstrated with other loci and in this case could lead potentially to the production of an in-frame Mat1 transcript. To investigate whether a truncated protein might be synthesized by the targeted allele, we performed western blot analysis on total lysates obtained from ES cells heterozygous for the targeted allele using an antibody against Mat1 (Figure 1D). No truncated Mat1 proteins were observed in the ES cell lysates, suggesting that the targeted allele represents a null allele of Mat1. Disruption of Mat1 leads to early embryonic lethality Mat1 heterozygous (Mat1+/−) intercrosses were set to ascertain the viability of Mat1 null homozygotes (Mat1−/−). PCR genotyping analysis of 104 adult offspring indicated that while both Mat1+/+ and Mat1+/− animals were observed at the expected frequencies, no homozygote null animals were obtained (Table I). This demonstrated that disruption of Mat1 leads to embryonic lethality. Dissection and genotyping of embryos at embryonic day E10.5, E9.0 and E7.5 of development also failed to identify viable homozygotes. In contrast, blastocysts at E4.0 showed close to Mendelian ratios for all genotypes, indicating that lethality probably occurred after implantation but before gastrulation (Table I). Indeed, the observation of resorbing embryos and empty decidua in roughly Mendelian numbers at later times of gestation suggests that Mat1−/− embryos die shortly after implantation. Table 1. Genotypes resulting from Mat+/− intercrosses +/+ +/− −/− Resorbed Total 3 weeks 31 73 0 − 104 E7.5–E10.5 13 30 0 19 62 E3.5 14 31 15 − 61 As Mat1 is an essential gene in S.cerevisiae, deletion of mammalian Mat1 might also be expected to compromise cell viability (Faye et al., 1997). Thus it was surprising that early embryogenesis would proceed in a genetically Mat1 null background. We reasoned that survival of Mat1−/− embryos to the implantation stage of development might be due to maternally provided Mat1. In order to test this hypothesis, embryos obtained from Mat1+/− intercrosses were isolated at eight-cell, 16-cell and blastocyst stages and immunostained with an anti-Mat1 antibody. Immunofluorescence analysis demonstrated decreasing Mat1 signal in approximately one-quarter of the embryos as development proceeded such that by the blastocyst stage, Mat1 immunoreactivity was barely observable (Figure 2). Subsequent PCR analysis revealed that embryos with diminishing signal genotyped as Mat1−/− (Figure 2 and data not shown). The timing of the lethality in Mat1−/− embryos therefore probably reflects the depletion of maternal Mat1 protein below threshold levels required to sustain an essential function. Figure 2.Depletion of maternal Mat1 protein in Mat1−/− pre-implantation embryos. α-Mat1 immunofluorescence analysis of embryos derived from Mat1+/– intercrosses isolated at the eight-cell (A), 16-cell morula (B) or blastocyst (C) stage of development. The genotypes indicated on top were determined by PCR genotyping. Nuclei of blastocysts depicted in (C) were visualized by staining DNA with Hoechst 33342 (D). Download figure Download PowerPoint Interestingly, Mat1−/− blastocysts were phenotypically indistinguishable from wild-type controls, demonstrating that Mat1−/− cells have the developmental capacity to differentiate into both inner cell mass (ICM) and trophectodermal lineages. TUNEL analysis of blastocyst stage embryos derived from Mat1+/− intercrosses showed no differences in either the ICM or trophectoderm cells of Mat1−/− and wild-type controls (data not shown). This indicated that pre-implantation embryos severely depleted of Mat1 protein stores were not committed rapidly to an apoptotic cell fate at this stage. Mat1 is required for the survival of mitotic but not post-mitotic lineages To examine more directly the role of Mat1 in cell proliferation and differentiation, we followed the development of pre-implantation embryos derived from Mat1+/− intercrosses in culture. Regardless of genotype, most embryos hatched from the zona pelucida and formed blastocyst outgrowths, which were maintained in culture for 7 days. Both Mat1+/− and Mat1+/+ outgrowths were characterized by the establishment of a rapidly proliferating cluster of ICM-derived cells growing on a base of trophectoderm-derived trophoblast giant cells that had attached to the culture dish (Figure 3A and B). After 4–5 days in culture, a subpopulation of cells, presumably representing endodermal lineages by morphology, was often observed to differentiate from the ICM and migrate away from the wild-type outgrowths. Figure 3.Cultured Mat1−/− embryos give rise to post-mitotic but not mitotic lineages. (A) Phase contrast micrographs of Mat1+/+ (left panel) or Mat1−/− (right panel) blastocyst outgrowths at day 6 in culture (10.5 days post-coitum). Trophoblast giant cells (T) and inner cell mass (ICM) cells (absent in the Mat1−/− outgrowth) are indicated. Endodermal lineages that differentiate from wild-type ICM have migrated outside the field of this micrograph. (B) Summary of cell types identified in blastocyst outgrowth. Trophectoderm, ICM and endodermal cell types were scored from outgrowths derived from Mat1+/− intercrosses after 7 days in culture. PCR genotyping as described in the text was used to determine the indicated genotype. (C) Establishing immunofluorescence as a genotyping tool. PCR-genotyped Mat1+/+ (wt) or Mat1−/− (−/−) outgrowths were analyzed following Mat1 antibody immunofluorescence and Hoechst 33342 staining (200× magnification). Download figure Download PowerPoint In contrast, the Mat1−/− embryos gave rise to outgrowths with trophectodermal cells yet failed to develop proliferative ICM cells and, as a consequence, endodermal derivatives (Figure 3A and B). Cells derived from the ICM of the blastocyst would often be observed associated with the remnants of the zona pelucida in Mat1−/− outgrowths, but these cells were never noted to proliferate. Hoechst staining of these cells revealed condensed, fragmented nuclei, suggesting that the Mat1−/− ICM cells underwent apoptosis during the first few days in culture (data not shown). The trophoblast cells established in the Mat1−/− outgrowths, on the other hand, were indistinguishable from wild-type counterparts by light microscopy over the first few days in culture (Figure 3A). However, as the cells were maintained further in culture, the Mat1−/− trophoblast cells were noted to be smaller than the controls. We determined that this was not a secondary phenotype resulting from the absence of ICM lineages in the Mat1−/− outgrowths, as control outgrowths dissected of their ICM early in culture developed giant cells comparable to those with an intact ICM. In order to address the function of Mat1 beyond embryonic lethality, we took advantage of the blastocyst outgrowth system as a means of generating a population of trophoblast giant cells on coverslips (20–40 cells per outgrowth). Genotyping of outgrown cells was accomplished by staining with Mat1 antibodies (Figure 3C), which in all cases analyzed was in agreement with PCR genotyping of ICM cells dissected away from wild-type outgrowths. It should be noted that genotyping by immunofluorescence does not distinguish between Mat1+/+ and Mat1+/−, and so outgrowths genotyped in this manner are referred to collectively as wild-type (Mat1wt). Mat1 specifically regulates the steady-state levels of Cdk7 and cyclin H Mat1 has been suggested to act as an assembly factor for the Cdk7–cyclin H–Mat1 trimer (Devault et al., 1995; Fisher et al., 1995; Tassan et al., 1995). We therefore wanted to address whether or not the expression of Cdk7 and cyclin H was affected in Mat1−/− cells. In order to do this, outgrown trophoblast cells were subjected to immunofluorescence with antibodies specific for Cdk7 and cyclin H (Figure 4A). Analysis of wild-type cells revealed strong nuclear staining of both Cdk7 and cyclin H (Figure 4A). Mat1−/− cells, however, consistently revealed that the signals for both Cdk7 and cyclin H were severely diminished (Figure 4A). In order to be sure that this was not due to epitope masking in the Mat1−/− cells, immunofluorescence with additional antibodies directed at Cdk7 and cyclin H was performed and found to confirm our initial observations (data not shown). Figure 4.Mat1 regulates steady-state expression of Cdk7 and cyclin H. (A) Steady-state levels of Cdk7 and cyclin H in Mat1−/− cells. Wild-type (wt) or Mat1−/− (−/−) blastocyst outgrowths were analyzed by immunofluorescence with antibodies against Cdk7 or cyclin H as indicated, and co-stained with Hoechst 33342 (400× magnification). (B) Immunofluorescence analysis of wild-type (wt) and Mat1−/− (−/−) blastocyst outgrowths with the indicated antibodies and co-stained with Hoechst 33342. Download figure Download PowerPoint In S.cerevisiae, strains harboring temperature-sensitive alleles of the Mat1 homolog (TFB3) exhibit severely compromised transcription upon shifting to the non-permissive temperature (Faye et al., 1997). We therefore wanted to determine whether the diminished levels of Cdk7 and cyclin H were specific to Mat1 loss or instead a manifestation of a more general transcriptional deregulation. Steady-state transcription can be assayed indirectly by monitoring protein expression, particularly when assaying the levels of proteins with a rapid turnover. We therefore analyzed cells derived from blastocyst outgrowths for expression of several proteins with variable turnover rates by immunofluorescence. These included proliferating cell nuclear antigen (PCNA), p53, Cdk2, cyclin E, cyclin D1, Cdk6 and the large subunit of pol II (Figures 4B and 5A). We found that the expression of all of these proteins was comparable in both Mat1−/− and Mat1wt cells. These observations suggest that the loss of mammalian Mat1 does not deregulate transcription or translation globally and that Mat1 specifically regulates the steady-state levels of Cdk7 and cyclin H in vivo. Figure 5.Mat1 modulates CTD phosphorylation. (A) Immunofluorescence analysis of wild-type (wt) and Mat1−/− (−/−) blastocyst outgrowths with RNA polymerase II large subunit antibody 8WG16 co-stained with Hoechst (400× magnification). (B and C) Immunofluorescence analysis as in (A) using the monoclonal antibody H14 recognizing pol II phosphorylated on Ser5 of the CTD heptapeptide repeat [400× (B) and 1000× (C) magnification]. (D and E) Immunofluorescence analysis as in (A) using the monoclonal antibody H5 recognizing pol II phosphorylated on Ser2 of the CTD heptapeptide repeat [400× (D) and 1000× (E) magnification]. Note the intense localization of Ser2 to discrete foci in the Mat1−/− micrograph. Download figure Download PowerPoint Mat1 modulates pol II CTD phosphorylation Many studies have implicated the CTD of the large subunit of RNA polymerase II as a substrate of TFIIH kinase activity. We therefore wanted to determine whether the phosphorylation status of the CTD was affected in Mat1−/− cells. This was accomplished by immunofluorescence analysis utilizing monoclonal antibodies recognizing specific phospho-epitopes of pol II (Figure 5). Using an antibody (8WG16) that is specific for non-phosphorylated Ser2 of the CTD but that also recognizes partially phosphorylated pol II (Thompson et al., 1989), we were able to visualize the majority of cellular pol II. We found that both Mat1wt and Mat1−/− nuclei immunostained comparably throughout the nucleus, excluding nucleolar compartments (Figure 5A). This observation indicated that expression of pol II was unaffected by the loss of Mat1. Phosphorylation of Ser5 of the heptapeptide repeat was visualized using an antibody (H14) specific for this phospho-epitope (Patturajan et al., 1998). Immuno staining of Mat1 wild-type cells with H14 revealed fine punctate immunoreactivity throughout the nucleus (Figure 5B and C). Individual cells of an outgrowth often showed varying degrees of intensity of H14 signal, with some cells exhibiting very intense staining (Figure 5B and C). In contrast, Mat1−/− cells revealed a considerable decrease in H14 immunoreactivity (Figure 5B and C). Although some degree of variability in H14 staining was also noted in Mat1−/− cells, we never observed any cells exhibiting the high levels of Ser5 immunoreactivity that had been noted in wild-type cells. We then looked at the phosphorylation status of Ser2 using an antibody (H5) specific for this phospho-epitope (Patturajan et al., 1998). As had been observed with H14 staining, Mat1wt cells exhibited a finely particulate staining throughout the nucleoplasm that, unlike the H14 staining, did not vary significantly from one cell to another (Figure 5D and E). Immunostaining of Mat1−/− cells with H5 revealed a dramatic decrease in signal when compared with Mat1wt control cells (Figure 5D and E). Additionally, in ∼30% of the Mat1−/− cells (Figure 5D and E; 67 of 231 cells scored), we observed intense H5 immunoreactivity localizing to 20–30 discreet nuclear domains. A similar staining pattern in Mat1wt control cells was only noted in a single nucleus of several hundred scored. Functional de novo transcription and translation in Mat1−/− cells The apparent discrepancy between our observation that multiple proteins were detected at wild-type levels and the observed reduction in pol II CTD phosphorylation prompted us to assay more directly the ability of Mat1−/− cells to engage in de novo transcription and translation. To this end, a cytomegalovirus (CMV) promoter-driven green fluorescent protein (GFP) expression plasmid was microinjected into outgrown cells at day 6.0 in culture (equivalent to 10.5 days post-coitum). Injected cells were returned to an incubator and assayed for GFP expression by fluorescence microscopy the following day. Regardless of genotype, all microinjected cells exhibited strong expression of the GFP protein (Figure 6). This result showed that Mat1−/− cells were comparable with control cells in their ability to engage in de novo transcription and translation of the CMV-GFP expression plasmid. Figure 6.De novo transcription and translation in Mat1−/− cells. Fluorescence microscopy analysis of GFP expression in wild-type (wt) and Mat1−/− (−/−) cells 1 day after microinjection with pEGFP-N2 plasmid counterstained with Hoechst (200× magnification). Download figure Download PowerPoint Mat1 is required for S phase entry in trophoblast giant cells DNA synthesis in trophoblast giant cells occurs by endoreduplication whereby successive rounds of G and S phases proceed without intervening mitosis (Barlow et al., 1972; Gardner, 1983). As a result, endocycling giant cells acquire vast quantities of DNA in their nuclei, thereby becoming polyploid (Varmuza et al., 1988). Throughout our analysis of outgrown cells, it was noted that Mat1−/− nuclei were consistently smaller in size and stained less intensely with the DNA stain Hoechst 33342 than control nuclei (compare Mat1−/− and Mat1wt nuclei in Figures 3,4,5,6 and inset in Figure 7). These observations suggested that the Mat1−/− cells had reduced amounts of DNA in their nuclei. Figure 7.Defective endoreduplication in Mat1−/− trophoblast giant cells. DNA content in mouse embryo fibroblasts (MEFs), wild-type (wt) and Mat1−/− (−/−) blastocyst outgrowths. Quantitation was performed from digital images of Hoechst-stained nuclei (such as those shown in the inset). Numbers o
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