Inability to enter S phase and defective RNA polymerase II CTD phosphorylation in mice lacking Mat1

Abstract

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.

Fig. 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.

Fig. 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).

Fig. 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).

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 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 of cells used for quantitation are indicated on the left for MEFs and on the right for trophoblasts.

Fig. 8. Endocycle arrest in Mat1^–/– trophoblast giant cells. (A) DNA synthesis in Mat1^–/– blastocysts. BrdU incorporation for 16 h of wild-type (wt) and Mat1^–/– (–/–) blastocysts visualized by α-BrdU immunofluorescence and co-stained with Hoechst (400× magnification). (B) DNA synthesis in Mat1^–/– trophoblasts. BrdU incorporation for 46 h of wild-type (wt) and Mat1^–/– (–/–) trophoblasts visualized as in (A) (200× magnification).