Correct function of the locus control region may require passage through anonerythroid cellular environment

The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that w hen a 248-kb beta-locus YAC was first microinjected into L-cells and then t ransferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids disp layed normal expression and developmental regulation. To test whether direc t transfer of a beta-globin locus (beta-YAC) into MEL cells could be used f or studies of the function of the LCR, a 155-kb beta-YAC that encompasses t he entire beta-globin locus was used. This YAC was retrofitted with a PGK-n eo selectable marker and with two I-Ppol sites at the vector arm-cloned ins ert junctions, allowing detection of the intact globin loci on a single I-P pol fragment by pulsed field gel electrophoresis (PFGE). The Ppo-155 beta-Y AC was used to directly lipofect MEL 585 cells. In 7 beta-YAC MEL clones wi th at least one intact copy of the YAC, the levels of total human globin mR NA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones. These results indicated that globin gene expr ession was strongly influenced by the position of integration of the beta-Y AC into the MEL cell genome and suggested that the LCR cannot function prop erly when the locus is directly transferred into an erythroid cell environm ent as naked beta-YAC DNA. To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously ob served correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clo nes carried the intact 144-kb I-Ppol fragment and transcribed the human glo bin genes with a fetal-like pattern. Subsequent transfer of the YAC of thes e L.(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids th at synthesized human globin mRNA. The variation in human beta-globin mRNA ( ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment rais es the possibility that normal activation of the LCR requires interaction w ith the transcriptional environment: of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells end completed with the bind ing of erythroid type of factors in the committed erythroid progenitors. (C ) 1999 by The American Society of Hematology.