An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

Abstract

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.

Fig. 1. (A) Restriction maps of pEPI-1 and pGFP-C1. Arrows indicate the positions of primers used for PCR analysis. (B) South-western analysis after nuclear fractionation using either pEPI-1 or pGFP-C1 as a probe. Only binding to fractions 2 (soluble nuclear proteins) and 5 (nuclear matrix) is shown. Lanes 1 and 2, binding of pEPI-1 to fractions 2 and 5, respectively; lanes 3 and 4, binding of pGFP-C1 to fractions 2 and 5, respectively.

Fig. 2. PCR analysis from DNA bound to HAP or the supernatant after crosslinking of CHO cells transfected with either pEPI-1 or pGFP-C1. (A) PCR analysis of HAP-bound DNA. Lane 1, molecular weight marker; lane 2, control PCR using 1 ng of pEPI-1 vector DNA as a template; lane 3, PCR analysis from pEPI-1 transfected cells using 10 ng of DNA as a template; lane 4, PCR analysis from pGFP-C1 transfected cells using 10 ng of DNA as a template; lane 5, as lane 4 but using 100 ng of DNA as a template. (B) PCR analysis of the supernatant from pEPI-1 transfected cells using 10 ng of DNA as a template. Designations are as in (A).

Fig. 3. South-western analysis of HAP-bound proteins and proteins of the supernatant after crosslinking with cis-DDP using either pEPI-1 or pGFP-C1 as a probe. Lane 1, pEPI-1 bound to supernatant proteins; lane 2, pEPI-1 bound to HAP-bound proteins; lane 3, pGFP-C1 bound to supernatant proteins; lane 4, pGFP-C1 bound to HAP-bound proteins. Lanes 1–4, binding was performed in the presence of a 10-fold excess of competitor DNA. Lane 5, as lane 2; lane 6, as lane 4. Lanes 5 and 6, binding was performed in the presence of a 100-fold excess of competitor DNA. Lane 7, western analysis of HAP-bound proteins using an anti-SAF-A antibody. Conditions for western and south-western analyses are described in the Methods.

Fig. 4. (A) Western analysis using an anti-SAF-A antibody from proteins isolated from immunoprecipitates with an anti-SAF-A antibody of HAP-bound DNA–protein complexes after cis-DDP crosslinking of cells transfected with either pEPI-1 or pGFP-C1. Lane 1, total nuclear proteins from untransfected CHO cells; lane 2, proteins isolated from pEPI-1 transfected cells; lane 3, proteins isolated from pGFP-C1 transfected cells. (B) PCR analysis after immunoprecipitation with an anti-SAF-A antibody of HAP-bound DNA–protein complexes after cis-DDP crosslinking of cells transfected with either pEPI-1 or pGFP-C1. Lane 1, molecular weight marker; lane 2, control PCR using 1 ng of pEPI-1 vector DNA as template; lane 3, PCR analysis of immunoprecipitate from 5 × 10⁷ pEPI-1 transfected cells; lane 4, PCR analysis of immunoprecipitate from 5 × 10⁷ pGFP-C1 transfected cells. (C) Western analysis with an anti-SAF-A antibody of proteins after immunoprecipitation with non-specific antibodies (anti-Oxytricha βTP). Lane 1, proteins from the supernatant after immunoprecipitation; lane 2, proteins from immunoprecipitate. A similar result was obtained when immunoprecipitation was performed with an anti-rabbit IgG.