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Correct function of the locus control region may require passage through a nonerythroid cellular environment
G Vassilopoulos1, P A Navas, E Skarpidi
1Divisions of Medical Genetics and of Hematology, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
Blood
|January 13, 1999
Summary
The beta-globin locus control region (LCR) requires an uncommitted cell environment for proper activation. Transferring the beta-globin locus YAC directly into erythroid cells resulted in variable gene expression, unlike transfer via non-erythroid cells.
Area of Science:
- Gene Regulation
- Molecular Biology
- Hematopoiesis
Background:
- The beta-globin locus control region (LCR) is crucial for regulating beta-globin gene expression during development.
- Previous studies showed normal LCR function when a beta-locus YAC was transferred through L-cells into MEL cells.
Purpose of the Study:
- To investigate whether direct transfer of a beta-globin locus YAC into MEL cells allows for studies of LCR function.
- To determine the role of the L-cell environment in LCR activation and globin gene regulation.
Main Methods:
- Lipofection of a 155-kb beta-globin locus YAC into MEL 585 cells.
- Pulsed field gel electrophoresis (PFGE) to detect intact YACs.
- Fusion of L-cell YAC clones with MEL cells.
Main Results:
- Direct lipofection of beta-YAC into MEL cells led to >97-fold variation in globin mRNA levels, indicating position-dependent integration effects.
- Transferring the beta-YAC through L-cells first resulted in fetal-like globin gene transcription in L-cells.
- Subsequent fusion with MEL cells yielded LxMEL hybrids with only 2.5-fold variation in globin mRNA, demonstrating position-independent expression and normal LCR function.
Conclusions:
- Direct transfer of naked beta-YAC DNA into erythroid cells impairs LCR function.
- The L-cell environment is necessary for proper LCR activation, suggesting a multistep process involving non-erythroid and erythroid factors.
- LCR activation may involve initial binding of ubiquitous factors in hematopoietic stem cells, followed by erythroid-specific factors in progenitors.