Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome

Abstract

The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the rational design of nonviral episomal vectors for mammalian cells. In this paper, we demonstrate that a tetramer of a 155-bp minimal nuclear scaffold/matrix attached region DNA module linked to an upstream transcription unit is sufficient for replication and mitotic stability of a mammalian episome in the absence of selection. Fluorescence in situ hybridization analyses, crosslinking with cis-diammineplatinum(II)-dichloride and chromatin immunoprecipitation demonstrate that this vector associates with the nuclear matrix or scaffold in vivo by means of specific interaction of the nuclear scaffold/matrix attached region with the nuclear matrix protein SAF-A. Results presented in this paper define the minimal requirements of an episomal vector for mammalian cells on the molecular level.

Fig. 1.

Vectors used in this study are pGFP-C1 (Clontech) (A), pEPI-eGFP (B) (16), pEPI-RSV (C), pDiMAR (D), pTetMAR (E), and pMARS (F). For vector construction, see Methods. The regions are color-coded as follows. Pink box, CMV promoter; green arrow, GFP and eGFP; red box, S/MAR; gray box, multiple cloning site; orange box, SV40 polyadenylation sequence; blue box, SV40 origin of replication/promoter; yellow arrow, kanamycin/G418 resistance gene; orange box, herpes simplex virus thymidine kinase polyadenylation sequence; dark-red box, pUC origin of replication.

Fig. 2.

Southern analyses of DNA isolated from CHO cells transfected with pEPI-RSV (A), pDiMAR (B), and pTetMAR (C). Hybridization was done as described in Methods. The hybridization pattern of one representative clone is shown for each construct. Lanes: M, DNA Marker SMART-Ladder (Eurogentec, Brussels); P, pEPI-RSV (A), pDiMAR (B), or pTetMAR (C) plasmid DNA as a control linearized by digestion with BglII (A) or HindIII (B and C); 1, total DNA digested with BglII (A) or HindIII (B and C); 2, Hirt extract digested with BglII (A) or HindIII (C); 3, undigested DNA from a Hirt extract.

Fig. 3.

Binding of pMARS in the cell. (A) Southern analyses of DNA isolated from CHO cells transfected with pMARS. Hybridization was done as described in Methods. The hybridization pattern of one representative clone is shown. (B) Rescue experiment in E. coli with Hirt extract from CHO cells transfected with pMARS. (C) Northern analysis of total RNA isolated from CHO cells transfected with pMARS. Hybridization was done as described in Methods. The hybridization pattern of one representative clone is shown. Lanes: M, DNA Marker SMART-Ladder (Eurogentec); P, pMARS (400 ng in B) plasmid DNA as a control linearized by digestion with HindIII; 1, total DNA digested with HindIII (A), DNA isolated from one bacterial clone digested with HindIII (B), or total RNA hybridized with a ³²P-labeled eGFP probe (C); 2, Hirt extract digested with HindIII; 3, undigested DNA from a Hirt extract.

Fig. 4.

Binding of pMARS to the nuclear matrix. (A) FISH analyses of CHO cells transfected either with pDiMAR (Left) or with pMARS (Right) were done as described in Methods. (Scale bar, 10 μm.) (B) PCR analyses from DNA bound to HAP after crosslinking of transfected CHO cells by using 250 ng of DNA as a template. Lanes: 1, supernatant from pGFP-C1-transfected cells; 2, supernatant from pEPI-eGFP-transfected cells; 3, supernatant from pDiMAR-transfected cells; 4, supernatant from pMARS-transfected cells; 5, DNA bound to HAP from pGFP-C1-transfected cells; 6, DNA bound to HAP from pEPI-eGFP-transfected cells; 7, DNA bound to HAP from pDiMAR-transfected cells; 8, DNA bound to HAP from pMARS-transfected cells; 9, control PCR with 100 pg of pEPI-eGFP vector as a template.

Fig. 5.

Binding of pMARS to SAF-A. (A) In vitro pull-down assay with pDiMAR as a template and without competitor DNA (lane 1), with a 100-fold excess of E. coli competitor DNA (lane 2), and with a 10,000-fold excess of E. coli competitor DNA (lane 3) or with pTetMAR as a template and without competitor DNA (lane 4), with a 100-fold excess of E. coli competitor DNA (lane 5), and with a 10,000-fold excess of E. coli competitor DNA (lane 6). (B) PCR analyses from DNA (Top) and Western analyses (Bottom) with an anti-SAF-A antibody from proteins isolated after in vivo crosslinking of transfected CHO cells and immunoprecipitation. As an internal control, the endogenous minimal S/MAR of the dhfr-locus (29) was amplified (30 cycles) from the immunoprecipitates (Middle). (Top) PCR analysis of DNA isolated from an immunoprecipitate of pGFP-C1- (lane 1), pDiMAR- (lane 2), pTetMAR- (lane 3), or pEPI-eGFP-transfected (lane 4) cells. (Middle) Amplification of the minimal S/MAR of the dhfr locus. (Bottom) Western analysis of immunoprecipitates by using an anti-SAF-A antibody. Lanes correspond to Top.