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, 287 (10), 7051-62

Serine Hydroxymethyltransferase Anchors De Novo Thymidylate Synthesis Pathway to Nuclear Lamina for DNA Synthesis

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Serine Hydroxymethyltransferase Anchors De Novo Thymidylate Synthesis Pathway to Nuclear Lamina for DNA Synthesis

Donald D Anderson et al. J Biol Chem.

Abstract

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

Figures

FIGURE 1.
FIGURE 1.
Schematic of folate-mediated one-carbon metabolism in the cytoplasm and nucleus. One-carbon metabolism is required for the de novo synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Folate-activated one-carbon units are shown in boldface type. The de novo thymidylate pathway is sumoylated and translocates to the nucleus during S phase. MTHFD1, methylenetetrahydrofolate dehydrogenase; MTR, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; DHF, dihydrofolate.
FIGURE 2.
FIGURE 2.
SHMT1 is a nuclear lamin-associated protein. A, both lamin A/C and lamin B1 co-immunoprecipitations from benzonase-treated nuclear extracts contained SHMT1 even in high salt conditions (650 mm NaCl), whereas neither contained TYMS or DHFR even under low salt conditions (150 mm NaCl). B, pNTAPb-SHMT1 and pNTAPb empty vectors were transfected into HeLa cells, and tandem affinity purification was performed using streptavidin and calmodulin resins. SHMT1, TYMS, and DHFR were detected in pNTAPb-SHMT1 transfections in samples that were not treated with nucleases. C, the DNA dependence of SHMT1, TYMS, and DHFR interactions were determined by SHMT1 immunoprecipitation in purified nuclear extracts treated with or without benzonase. TYMS and DHFR only immunoprecipitated with SHMT1 in samples lacking benzonase treatment. D, the interaction of SHMT1, lamin B1, and DHFR was investigated by confocal microscopy following transfection of cDNAs encoding GFP-DHFR, SHMT1-YFP, and mKate-lamin B1 fusion proteins in cells blocked in G1 (30 μm lovastatin), S phase (1 mm hydroxyurea), and G2/M (100 ng/ml nocodazole) phases of the cell cycle. Co-localization of DHFR and SHMT1 with lamin B1 is concomitant with nuclear localization of DHFR and SHMT1 during S and G2/M but not during G1 phases of the cell cycle. IP, immunoprecipitation.
FIGURE 3.
FIGURE 3.
SHMT1 and TYMS form linear lamin B1 co-localizing structures independent of nucleoside availability. The cDNAs encoding YFP-SHMT1, TYMS-GFP, and mKate-lamin B1 fusion proteins were transfected into cells cultured in medium containing nucleosides (α-MEM), medium without nucleosides (DMEM), and α-MEM that lacks either deoxyuridine (dU) or thymidine (dT) and visualized by confocal microscopy. The co-localization of SHMT1 and TYMS with lamin B1 was independent of nucleosides in the culture medium.
FIGURE 4.
FIGURE 4.
SHMT1 and SHMT2 are required for TYMS and lamin B1 co-localization. A, cDNAs encoding GFP-TYMS, mKate-lamin B1 fusion proteins, and siRNAs, including scrambled (MOCK), SHMT1, SHMT2, or both SHMT1 and SHMT2, were transfected into HeLa cells. Cells were blocked in S phase using hydroxyurea (2 mm) and visualized with confocal microscopy. For each siRNA treatment, 100 cells were counted. The presence of DHFR and lamin B1 co-localizing structures occurred in 95 ± 4, 14 ± 4.5, 75 ± 5.7, and 0 ± 0.6% of cells transfected with scrambled, SHMT1, SHMT2, and both SHMT1 and SHMT2 siRNAs, respectively. Error is expressed in S.D. (n = 3). B, immunoblotting was performed on siRNA-treated samples to ensure knockdown of SHMT1 and SHMT2. GAPDH was visualized as a loading control.
FIGURE 5.
FIGURE 5.
Dominant negative SHMT1 (DN2-SHMT1) localizes with lamin B1 and enhances SHMT1 activity for de novo thymidylate biosynthesis. A, cDNAs encoding mKate-lamin B1 and either YFP-SHMT1 or YFP-DN2-SHMT1 fusion proteins were expressed in HeLa cells, and co-localization of the proteins was determined by confocal microscopy. Both SHMT1 and DN2-SHMT1 colocalize with lamin B1. B, the effect of tetracycline (Tet)-inducible expression of DN2-SHMT1 on SHMT1 activity in de novo thymidylate biosynthesis was determined in SH-SY5Y cells. Cellular protein and DNA were isolated from cells cultured for 8 days in defined medium containing l-[2,3,3-2H3]serine. Isotopic enrichment of the one carbon derived from l-[2,3,3-2H3]serine into cellular protein pools was determined by the detection of labeled Met and dehydroalanine (DHA). Dehydroalanine is formed from serine during the derivatization procedure. Enrichment of [2,3,3-2H3]serine in thymidine (dT) was determined by analysis of nuclear DNA. All values are expressed as the percentage of l-[2,3,3-2H3]serine-derived carbons that contain two deuterium atoms (CD2) in the target compound (the ratio of carbons containing two deuterium atoms in the target compound divided by the total number of carbons that contain one or two deuterium atoms × 100). Two independent experiments were performed with duplicate measurements for each sample, and identical values were obtained within each experiment. The results from one experiment are shown. The data demonstrate that tet-inducible expression of DN2-SHMT1 decreased SHMT1 specific activity in homocysteine remethylation to methionine in the cytoplasm by 60%, whereas DN2-SHMT1 expression enhances SHMT1 specific activity in de novo thymidylate biosynthesis by 45% C, immunoblotting was performed on SH-SY5Y-DN2-SHMT1 cells in a tet-inducible system. The SH-SY5Y-DN2-SHMT1 cells exhibited increased levels of total SHMT1 in response to tetracycline. These cells were used for isotopic tracer studies. GAPDH was used as a loading control.
FIGURE 6.
FIGURE 6.
The de novo dTMP synthesis pathway is associated with episomal DNA in tandem chromatin immunoprecipitations. HeLa cells were transfected with pBABE-Puro-SV40 LT vector and selected for using puromycin. Clones were isolated and immunoblots performed against SV40 LT to ensure that clones were expressing SV40 LT (D). Tandem ChIP assays were performed as described under “Materials and Methods” using antibodies directed against PCNA and then antibodies directed against non-immune IgG, SHMT1, TYMS, or DHFR. Three separate SV40 LT-expressing clones were subjected to tandem ChIP in duplicate. Error is expressed as S.E. (error bars). Regions of the pBABE-Puro-SV40 LT vector were probed for the presence of SHMT1, TYMS, or DHFR using real-time PCR (A–C). All three enzymes precipitated regions of the vector, including the large T antigen coding region (A), SV40 origin of replication (B), and downstream region of the SV40 origin (C). The highest occupancy was observed around the SV40 origin.

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