The potent enhancer activity of the polycythemic strain of spleen focus-forming virus in hematopoietic cells is governed by a binding site for Sp1 in the upstream control region and by a unique enhancer core motif, creating an exclusive target for PEBP/CBF

Abstract

The polycythemic strain of the spleen focus-forming virus (SFFVp) contains the most potent murine retroviral enhancer configuration known so far for gene expression in myeloerythroid hematopoietic cells. In the present study, we mapped two crucial elements responsible for the high activity of the SFFVp enhancer to an altered upstream control region (UCR) containing a GC-rich motif (5-GGGCGGG-3) and to a unique enhancer core (5-TGCGGTC-3). Acquisition of these motifs accounts for half of the activity of the complete retroviral enhancer in hematopoietic cells, irrespective of the developmental stage or lineage. Furthermore, the UCR motif contains the major determinant for the enhancer activity of SFFVp in embryonic stem (ES) cells. Using electrophoretic mobility shift assays, we show that the UCR of SFFVp, but not of Friend murine leukemia virus, is targeted by the ubiquitous transcriptional activator, Sp1. The core motif of SFFVp creates a specific and high-affinity target for polyomavirus enhancer binding protein/core binding factor (PEBP/CBF) and excludes access of CAAT/enhancer binding protein. Cotransfection experiments with ES cells imply that PEBP/CBF cooperates with the neighboring element, LVb (the only conserved Ets consensus in the SFFVp enhancer), and that the Sp1 motif in the UCR stimulates transactivation through the Ets-PEBP interaction. Putative secondary structures of the retroviral enhancers are proposed based on these data.

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

“…Thus, we here have evaluated the suitability of A2UCOEcontaining self-inactivating (SIN) lentiviral vector constructs to stabilize transgenic hCDD expression in murine iPSCs and ESCs prior to and following hematopoietic differentiation. In contrast to primary HSCs, in which long-term transgene expression has been achieved with internal viral or housekeeping promoters, such as the SFFV [27] or EFS [28] promoter, in our studies, these elements on their own failed to promote stable transgene expression in iPSC/ESCs or their hematopoietically differentiated progeny. In contrast, incorporation of the A2UCOE sequence into the respective vector constructs allowed for robust and sustained hCDD expression in naïve pluripotent cells and their hematopoietic progeny and effectively protected these cells from Ara-C toxicity.…”

Host cis -Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

“…It has been postulated that transcriptional promoters and enhancers act to suppress PEV by antagonizing repression by flanking chromatin (3,16,45,46,69). Significantly, several of the transcription factors that bind to the MESV or Mo-MuLV enhancer domain are not present or are present at only low levels in ES cells, thus preventing enhancer function (8,20,61). We therefore postulate that MESV provirus lacks the appropriate enhancer elements necessary to antagonize the repressive activity of chromatin in ES cells.…”

AML1-ETO Inhibits Maturation of Multiple Lymphohematopoietic Lineages and Induces Myeloblast Transformation in Synergy with ICSBP Deficiency

“…Both virus titers and eGFP expression levels were reduced in infected cells expressing the FMEV/AE construct as compared with the control vector FMEV/GFP ( Fig. 1 B), which may be partly due to AML1-ETO transcriptional repression of the vector via a pivotal CBF-binding site in the retroviral LTR (51). Western blot analysis of protein extracted from FMEV/ AE-infected cells confirmed the presence of the AML1-ETO protein with the same molecular weight as that found in Kasumi-1 cells, a cell line established from a t(8;21) AML patient (49) ( Fig.…”