|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 703-712
Correct Function of the Locus Control Region May Require Passage
Through a Nonerythroid Cellular Environment
George Vassilopoulos,
Patrick A. Navas,
Evangelia Skarpidi,
Kenneth R. Peterson,
Chris H. Lowrey,
Thalia Papayannopoulou, and
George Stamatoyannopoulos
From the Divisions of Medical Genetics and of Hematology, Department
of Medicine, University of Washington, Seattle, WA; and Dartmouth
Medical School, Hanover, NH.
The function of the  -globin locus control region (LCR) has been
studied both in cell lines and in transgenic mice. We have previously
shown that when a 248-kb -locus YAC was first microinjected into
L-cells and then transferred into MEL cells by fusion, the YAC loci of
the LxMEL hybrids displayed normal expression and developmental
regulation.To test whether direct transfer of a -globin locus
(-YAC) into MEL cells could be used for studies of the function of
the LCR, a 155-kb -YAC that encompasses the entire -globin locus
was used. This YAC was retrofitted with a PGK-neo selectable marker and
with two I-PpoI sites at the vector arm-cloned insert
junctions, allowing detection of the intact globin loci on a single
I-PpoI fragment by pulsed field gel electrophoresis (PFGE). The
Ppo-155 -YAC was used to directly lipofect MEL 585 cells. In
7 -YAC MEL clones with at least one intact copy of the YAC, the
levels of total human globin mRNA (ie,  +  + ) per copy of integrated -YAC varied more than 97-fold between clones.
These results indicated that globin gene expression was strongly
influenced by the position of integration of the -YAC into the MEL
cell genome and suggested that the LCR cannot function properly when
the locus is directly transferred into an erythroid cell environment as
naked -YAC DNA. To test whether passage of the -YAC through
L-cells before transfer into MEL cells was the reason for the
previously observed correct developmental regulation of human globin
genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by
lipofection. Three clones carried the intact 144-kb I-PpoI
fragment and transcribed the human globin genes with a fetal-like
pattern. Subsequent transfer of the YAC of these L(-YAC) clones into
MEL cells by fusion resulted in LxMEL hybrids that synthesized human
globin mRNA. The variation in human -globin mRNA (ie, + + ) 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
raises the possibility that normal activation of the LCR requires
interaction with 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 and completed with the binding of erythroid type of factors
in the committed erythroid progenitors.
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. Harju-Baker, F. C. Costa, H. Fedosyuk, R. Neades, and K. R. Peterson
Silencing of A{gamma}-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the -566 GATA Site
Mol. Cell. Biol.,
May 15, 2008;
28(10):
3101 - 3113.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Blau, C. F. Barbas III, A. L. Bomhoff, R. Neades, J. Yan, P. A. Navas, and K. R. Peterson
{gamma}-Globin Gene Expression in Chemical Inducer of Dimerization (CID)-dependent Multipotential Cells Established from Human {beta}-Globin Locus Yeast Artificial Chromosome ({beta}-YAC) Transgenic Mice
J. Biol. Chem.,
November 4, 2005;
280(44):
36642 - 36647.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Li, D. W. Emery, H. Han, J. Sun, M. Yu, and G. Stamatoyannopoulos
Differences of globin transgene expression in stably transfected cell lines and transgenic mice
Blood,
April 15, 2005;
105(8):
3346 - 3352.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Harrow, J. U. Amuta, S. R. Hutchinson, F. Akwaa, and B. D. Ortiz
Factors Binding a Non-classical Cis-element Prevent Heterochromatin Effects on Locus Control Region Activity
J. Biol. Chem.,
April 23, 2004;
279(17):
17842 - 17849.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Bottardi, A. Aumont, F. Grosveld, and E. Milot
Developmental stage-specific epigenetic control of human {beta}-globin gene expression is potentiated in hematopoietic progenitor cells prior to their transcriptional activation
Blood,
December 1, 2003;
102(12):
3989 - 3997.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Vadolas, H. Wardan, M. Orford, L. Voullaire, F. Zaibak, R. Williamson, and P. A. Ioannou
Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human beta -globin locus in erythropoietic cells
Blood,
December 1, 2002;
100(12):
4209 - 4216.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Anguita, J. A. Sharpe, J. A. Sloane-Stanley, C. Tufarelli, D. R. Higgs, and W. G. Wood
Deletion of the mouse alpha -globin regulatory element (HS -26) has an unexpectedly mild phenotype
Blood,
November 15, 2002;
100(10):
3450 - 3456.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Skarpidi, G. Vassilopoulos, Q. Li, and G. Stamatoyannopoulos
Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin
Blood,
July 1, 2000;
96(1):
321 - 326.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Kaufman, C. T.N. Pham, and T. J. Ley
Transgenic Analysis of a 100-kb Human beta -Globin Cluster-Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome
Blood,
November 1, 1999;
94(9):
3178 - 3184.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Li, D. W. Emery, M. Fernandez, H. Han, and G. Stamatoyannopoulos
Development of Viral Vectors for Gene Therapy of beta -Chain Hemoglobinopathies: Optimization of a gamma -Globin Gene Expression Cassette
Blood,
April 1, 1999;
93(7):
2208 - 2216.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
