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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
By
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.
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ABSTRACT |
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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE HUMAN -GLOBIN locus spans 82 kb on
chromosome 11 and contains a powerful regulatory element, the locus
control region (LCR), located between 5 to 22 kb upstream of the
 -globin gene and composed of a series of DNaseI hypersensitive sites
(5 HS1 to 5).1,2 The importance of this element for
globin gene function became apparent from naturally occuring mutations
that remove the LCR and result in silencing of all the downstream
globin genes and a phenotype of  -thalassemia.3-5
The function of the LCR has been best defined through studies in
transgenic mice.6,7 When human globin gene constructs
lacking the LCR are transferred into the murine genome, the majority of
the transgenes fail to express,8-10 indicating that the
function of such LCR-less constructs is strongly influenced by the
position of integration of the transgene. In contrast, constructs that
link the globin genes with the complete LCR or cassetes containing
combinations of HS sites or individually HS2, or 3 or 4, are
reproducibly expressed in transgenic mice,1,11-13 indicating that a major function of the LCR is to shield the globin genes from the effect of position of integration of the transgene. This
property of the LCR, the provision of position-independent expression
in transgenic mice, has been used in the functional recognition of LCRs
in several other loci.14-16 The LCR also acts as a powerful
globin gene enhancer and is responsible for the abundant synthesis of
globin mRNA in the cells of the erythroid lineage.1 The
current model of the function of the LCR implies that the HSs form a
structure that loops to interact with the promoters of the downstream
globin genes, thus activating globin gene
transcription.17,18
Analyses of the structure-function relationships of the LCR are
optimally performed in transgenic mice carrying constructs containing
the whole human -globin locus. Mice carrying human -globin locus
yeast artificial chromosomes (-YACs) have been used for this
purpose.19-21 Such transgenic mice show correct
developmental regulation of the locus and levels of globin gene
expression that are independent of the position of integration of the
transgene into the murine genome; these results are characteristic of
normal function of the LCR and of the other regulatory elements of the -locus.1,6,7 To ask whether transfer of -YACs into
MEL cells could be used for the analysis of structure-function
relationships of the human -globin locus, we have previously
transferred the -YAC into L-cells by microinjection and then to MEL
cells by LxMEL fusion.22 These L(-YAC)xMEL hybrids
initially expressed , , and globin mRNA but subsequently
switched to predominantly -globin expression. When the -YAC was
transferred by L-cell fusion into GM979 cells (an MEL line expressing
adult as well as embryonic mouse globins), there was continued
-globin expression along with -globin expression, indicating that
the genes of the -YAC respond to the trans-acting factors present in
the GM979 line. These results suggested that transfer of -YACs into
MEL cells could be used for the analysis of structure-function
relationships of the -globin locus.
The purpose of this study was to examine whether direct transfer of a
-YAC into MEL cells (instead of the indirect transfer through the
L-cells) could be used for the analysis of the function of the LCR and
its interactions with the genes of the -globin locus. If this was
the case, YACs transferred into MEL cells could substitute for
transgenic mice in structure-function studies of the LCR. Our results
indicate that the direct transfer of the -YAC into MEL cells is
characterized by globin gene expression that is strongly influenced by
the position of integration of the transgene. However, these position
effects were eliminated when the -YAC was first transferred into
L-cells and subsequently into MEL cells by fusion. We explain these
findings with the hypothesis that correct function of the LCR may
require a stepwise activation that starts in the transcriptional
environment of an uncommitted cell and it is completed when the locus
finds itself in the transcriptional environment of the cells of the
erythroid lineage.
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MATERIALS AND METHODS |
Cell cultures and transfections.
Semiadherent diploid MEL585 cells (a kind gift from W. Wood, Oxford,
UK) were maintained in RPMI 1640 medium (HyClone, Logan, UT)
supplemented with 10% fetal bovine serum (FBS; HyClone). One day
before transfection, 1 × 106 cells were plated in a
35-mm tissue culture dish and, by the time of lipofection, they reached
a confluency of approximately 80%. The adherent LA9 mouse fibroblast
line was purchased from ATCC (Rockville, MD) and maintained in
Dulbecco's modified Eagle's medium (DMEM)-high glucose
medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% FBS. LA9
cells were seeded at 0.5 × 106 cells/35-mm tissue
culture plate the day before transfection. For transfection of the
-YAC, the cells were washed twice with phosphate-buffered saline
(PBS) and resupended in 800 µL of Opti-Mem (GIBCO-BRL). Approximately
100 ng of gel-purified YAC-DNA was mixed with 5 µL of lipofectin
(GIBCO-BRL) in a total volume of 200 µL in OptiMEM medium. Lipid-DNA
complexes were allowed to form for 30 minutes at room
temperature. The mixture was then added to the cells and transfection
was performed for 6 hours in a tissue culture incubator. At the end,
normal medium was added to the cells supplemented with FBS to a final
concentration of 20% and the cells were cultured for another 24 hours.
The cells were then collected, counted, and plated in 24-well plates at a concentration of 20 × 103 cells/mL/well in medium
containing 700 µg/mL of active G418 (GIBCO-BRL) for selection.
Calculation of the transfection efficiency was performed by dividing
the number of G418-resistant colonies with the number of transfected
cells. Because the doubling time for MEL cells is between 16 and 18 hours, the number of transfected cells on transfection day was
calculated from the number of cells initially plated multiplied by two.
Individual G418-resistant colonies were expanded and induced to
terminally differentiate with 3 mmol/L hexamethylene-bis-acetamide
(HMBA) and 10 µmol/L hemin. Induction of differentiation was
performed with cells plated at 2 × 105 cells/mL in
10-mL cultures. Cells were collected for RNA analysis and for globin
staining on the days 3 and 4 of induction, respectively. For
lipofection of the LA9 cells with the -YAC, the same protocol was
applied, but the cells were plated for G418 selection in 100-mm TC
plates and individual G418-resistant colonies were picked up with
cloning rings. The cells were trypsinized in the well and expanded
under selection. L(-YAC)xMEL hybrids were generated as previously
described.23 Briefly, 107 MEL 585 cells
(suspension phenotype) and 5 × 106 G418-resistant
LA-9 cells (adherent phenotype) carrying the -YAC were fused by
polyethyleneglycol-mediated fusion. After fusion and every 2 to 3 days
(depending on the cell growth), G418-resistant suspension cells were
transferred to a new flask and grown under further selection. Parental
MEL cells (G418-sensitive) were killed by G418, whereas parental
L-cells (adherent) were removed each time the suspension cells were
transferred to a new flask. After 2 weeks of selection, the hybrids
were induced to differentiate with HMBA and hemin. Generation of
hygromycin-resistant LA9 cells with an SV-Hygro plasmid was also
performed by lipofection using the same protocol that was used for the
-YAC but with different amounts of DNA (5 µg) and lipofectin (15 µL). The cells were selected with 1 mg/mL hygromycin (GIBCO-BRL) and
a hygromycin-resistant cell pool was used for fusion with the
MEL(-YAC) clones. Fusion between MEL(-YAC)-G418-resistant clones
and LA9-hygromycin-resistant cells was performed as described above
and the hybrids were selected by dual resistance to both hygromycin and
G418. These are referred to as reverse hybrids.
DNA constructs.
The hygromycin resistance gene was isolated as a Sal
I-Sma I fragment from the plasmid pCEP4 (Invitrogen,
Carlsbad, CA). The pSV--Gal plasmid (Promega, Madison, WI) was cut
with Sal I and HindIII to remove the -Gal gene. The
HindIII site was blunted and ligated to the Sal
I-Sma I fragment from plasmid pCEP4. The resulting
construct has the SV40 promoter and enhancer linked to the hygromycin
resistance gene.
Production and purification of the 155-kb -locus YAC.
The 150-kb -locus YAC (clone A201F4)24 was retrofitted
with two I-PpoI sites at the vector-insert junctions using two
retrofitting vectors.25 The selectable marker PGKneo that
confers resitance to G418 was inserted at the right vector arm. This
YAC is 5 kb larger in size from the original clone and will be referred
to as Ppo-155 -YAC. The YAC was purified according to the
earlier protocol, with slight modifications.19,21
Preparative agarose blocks with high molecular weight DNA from the
yeast carrying the 155-kb -YAC were fractionated by pulsed field gel
electrophoresis (PFGE) on a 0.5% agarose MP gel (Boehhringer Mannheim,
Indianapolis, IN) in 0.5× TBE under the following conditions: 200 V, 60 seconds switch at 12°C for 18 to 24 hours. The slice that
contained the -YAC DNA was located on the gel by cutting and
staining separately the marker lanes at the edge of the gel. The
agarose slice was then cut together with a slice containing one of the
yeast chromosomes. To concentrate the -YAC DNA, the agarose slice
with the -YAC and the yeast chromosome slice were rotated vertically
relative to their original electrophoresis migration and subjected to
electrophoresis in 4% low-melting point agarose (LMPA; NuSieve GTG;
FMC, Rockland, ME) in 0.5× TBE at 47 V for 16 hours. The yeast
lane was stained to determine the relative migration of the -YAC DNA
into the LMPA gel. The slice with the YAC DNA was equilibrated in 40 to 100× volume of transfection buffer (10 mmol/L Tris, 250 mmol/L EDTA, 100 mmol/L NaCl) for 1 hour. The agarose slice was then melted at
68°C for 10 minutes, transferred in a 42.5°C water bath, and
digested with agarase (New England Biolabs, Beverly, MA) overnight with
2 U of the enzyme per 100 mg of melted gel. The DNA concentration of
the YAC was estimated by standard agarose electrophoresis with a
 HindIII marker and integrity was determined by PFGE. The DNA was diluted to 2 ng/µL in transfection buffer and filtered through a
0.22-mm Acrodisk (Gelman Sciences, Ann Arbor, MI) before transfection.
Structural analysis of the transfected Ppo-155 -YAC.
Transfected MEL cells were washed twice in ice-cold PBS and resupended
at 3 × 107 cells/mL of PBS. An equal volume of 2%
low temperature melting agarose (LTMA; Seaplaque GTG agarose; FMC) was
added to the cells and plugs were cast. The plugs were incubated in LDS
solution (1% lithium dodecyl sulfate, 100 mmol/L EDTA, 10 mmol/L
Tris-HCl) at 37°C for 1 hour, followed by a second incubation
overnight with fresh solution. After two 30-minute washes in 0.2% NDS
(0.2% lauryl sarcosinate, 100 mmol/L EDTA, 2 mmol/L Tris, pH 9.5) and three 30-minute washes in Tris EDTA (TE), pH 8.0, the
plugs were stored at 4°C in TE. For structural analysis, portions
of the agarose plugs were equilibrated with 1× I-PpoI
buffer in a volume of 0.2 mL and digested with 360 U of I-PpoI
(Promega) overnight. A total of 12 digested agarose slices were
fractionated on an agarose gel by PFGE. The DNA was then transferred by
capillary blot onto a nylon membrane (Hybond N+; Amersham, Arlington
Heights, IL). Individual strips that represent the lanes of the agarose gel were cut from the blot and hybridized separately to 12 probes that
span the entire -globin locus. The probes used were as follows: BamHI-HindII 5HS5, Stu I-Sph I
1.1-kb 5HS4, 0.7-kb Pst I 5HS3, 1.9-kb
HindIII 5HS2, Dra I-HindIII 5HS1,
3.7-kb EcoRI -globin gene, 2.4-kb EcoRI
A globin gene, 1.0-kb EcoRV  , 2.1-kb
Pst I -globin gene, 0.9-kb EcoRI-BamHI
-globin gene, 1.4-kb Xba I DF-10 (3HS1), and a 1.9-kb Bgl II HPFH3 (a gift from N. Anagnou, University of Crete,
Crete, Greece). All fragments were radiolabeled using a Decaprime II random labeling kit (Ambion, Austin, TX). After hybridization and
washing, the blot was reassembled by aligning the individual strips and
autoradiographed. The hybridization profile reflects the extent of the
locus for each YAC copy.
Southern analysis and copy number determination.
Genomic DNA was isolated by standard procedures26 and
digested overnight with EcoRI. Ten micrograms of the digested
product was fractionated on a 0.8% agarose gel and transferred by
alkaline capillary blot on a nitrocellulose membrane (ZetaProbeGT;
Bio-Rad, Hercules, CA). The blot was hybridized to the pPN201
probe27 that consists of the human HS3 core 780-bp
Pst I fragment linked to a murine 544-bp BamHI Thy1.1
cDNA fragment that served as the endogenous murine two-copy control. To
correct for any differences in the specific activity between the two
DNA fragments, the plasmid pPN201 was digested with Sma
I/Sca I to yield a 2.6-kb HS3 and a 1.7-kb Thy1.1
fragment. Twenty picograms of the plasmid digest was run along with the
genomic digests in the same gel. The value of the HS3 to Thy1.1 ratio
in the plasmid lanes was used to multiply the Thy1.1 genomic signal;
this corrected value, divided by two, was then compared with the HS3
signal. All of the signals were quantitated on a phosphoimager
(Molecular Dynamics, Sunnyvale, CA). -YAC copies were calculated by
comparing the human 5HS3 with the mouse two-copy Thy1.1 gene on
a conventional Southern analysis. Fragmented YACs that hybridized to
the HS3 probe in the PFGE analysis were subtracted so that only intact
copies were accounted in the per copy gene expression.
RNA analysis.
Total cellular RNA was isolated by the method of Chomczynski and
Sacchi.28 Human globin mRNA was analyzed by
RNase protection assay with probes that gave protected fragments from
the second exon of the respective genes (the size of the protected band
is in parentheses): pT7(205), pT7A(170), and
pT7(188). Mouse RNA was analyzed with a murine pT7 (128) probe
from exon I; a T7-actin(217) probe (a gift from Dr S. Sakiyama, Chiba Cancer Research Institute, Nitoya, Japan) was used
as a gel-loading control. The probes were synthesized with T7 RNA
polymerase (Promega). Two micrograms of RNA was hybridized overnight in
a 47°C oven with 106 cpm from each radioactive probe;
after digestion with a cocktail of RNases (Ambion, Austin, TX), the
protected fragments were separated on 6% polyacrylamide-8 mol/L urea
gel and autoradiographed. The signals were quantitated by phosphorimage
analysis.
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RESULTS |
Construction of the Ppo-155 -YAC.
We have previously shown that transfer of a 248-kb -YAC in
transgenic mice is associated with structural rearrangements that can
have detrimental effects on the analysis of structure-function relationships of the -like globin gene expression.29,30
Conventional Southern blots for examining the presence of sequences of
the -globin locus are not adequate for determining the structure of
the -YAC, because different parts of the -globin locus may be
contained in different fragments. These fragments will give positive
hybridization signals providing false information that the -globin
locus of the YAC is intact. For structural analysis of the 248-kb
-YAC, the structure of a 140-kb Sfi I fragment that contains
most of the -globin locus is analyzed in detail.29,30 Using this approach, Sfi I-digested agarose plugs containing
murine genomic DNA are subjected to PFGE, the gel is blotted, and each individual lane of the blot is probed for sequences extending from the
5HS3 to the 3 of the -globin locus. Upon reassembly of
the blot and autoradiography, the structure of each locus copy can be
reconstructed; any deletions or rearrangements of the -globin locus
contained in the Sfi I fragment become readily visible.
A 150-kb -YAC was identified and has been used for production of
transgenic mice.21 This YAC contains the entire -globin locus; it has the same 5 end as the 248-kb -YAC but a shorter 3 end that extends 23 kb downstream of 3HS1 relative to
the 109 kb of the 248-kb -YAC. This 150-kb -YAC displays correct developmental control of globin gene expression in transgenic mice.30,31 Although per copy expression has been reported
to range threefold between transgenic lines, this YAC is considered to
display position-independent expression.30 Because of its smaller size, this -YAC may be less prone to
rearrangement32; thus, it may be preferable to the 248-kb
-YAC for gene transfer experiments in cell lines. However, unlike
the 248-kb -YAC, it lacks one of the two Sfi I sites that
flank most of the -globin locus (it lacks the 3 SfiI
site downstream of 3HS1); therefore, it is not useful for
detailed structural analysis. To facilitate its use, two I-PpoI
sites were introduced at the junctions of the pYAC4 vector sequence and
the globin insert (Fig 1A). Digestion with
I-PpoI yields a 144-kb fragment that contains the entire globin
insert. A selectable marker (PGK-neo) was concommitantly retrofitted in
the right vector arm to allow selection of the transfected cells with
G418.
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| Fig 1.
Structural analysis of the Ppo-155 -YAC
tranferred into MEL cells by lipofection. (A) Diagram of the
Ppo-155 -YAC. The 144-kb I-Ppo-I fragment used to
assess YAC integrity is shown. This YAC contains an intact -locus
with 39 kb of DNA upstream of the 5HS5 of the LCR and 23 kb of
DNA downstream of 3HS1. The HS sites of the LCR and the
3HS1 are displayed as arrows. The location of the
I-Ppo-I sites are shown as straight lines next to the vector
sequences (open boxes). YAC-vector sequences: TRP1 and LYS2, YAC
selectable markers for tryptophane and lysine prototrophy,
respectively; ARS1, yeast autonomous replicating sequence; CEN1,
centromere; PGK-Neo, selectable marker for G418 resistance. (B)
Structural analysis of the MEL(Ppo-155 -YAC) clones 15 and
17; I-Ppo-I digestion, PFGE, and capillary transfer were
performed as described in Materials and Methods. The blot was cut into
strips and each strip was hybridized with one of the three probes
indicated by the dotted lines between the YAC diagram and the
autoradiograph (ie, HS5, A-globin, and DF-10). The
strips were realigned to reconstruct the original membrane, and a
hybridization profile for each I-PpoI fragment was obtained.
Fragments with a positive hybridization signal in all three lanes were
considered as intact. Notice that clone 15 has two fragmented copies,
whereas clone 17 has two intact copies at 140 and 280 kb and a third
that extends from A to the 3 end of the globin
locus. (C) Graphic representation of the -YACs in the MEL(-YAC)
clones according to their hybridization profile. Solid lines represent
intact globin loci, whereas the dotted lines indicate lack of
hybridization and fragmented -YAC copies.
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Transfer of the Ppo-155 -YAC in MEL cells.
The Ppo-155 -YAC was transferred into MEL cells by
lipofection. Two variants of the 585 MEL cell line were used, one
semiadherent (585A) and one growing in suspension. Cells were plated in
culture medium containing 700 µg/mL of G418 and visible colonies were counted between days 10 and 12. The observed distribution of
G418-resistant colonies was very close to a Poisson distribution, with
an m value of 0.7 (where m is the number G418-resistant
cells present in every well). The transfection efficiency for the
semiadherent MEL cells was 1:104 cells and 1:2 × 104 for experiments performed with 5 and 10 µg of
lipofectin, respectively. Transfection efficiency of MEL cells growing
in suspension was 1:7 × 104 cells for either 5 or 10 µg of lipofectin.
Structural analysis of the transfected Ppo-155 -YACs.
For structural analysis, 3 × 107 cells from 20 randomly selected G418-resistant MEL clones were embedded in agarose
and the plugs were digested with I-PpoI and subjected to PFGE
as described in Materials and Methods. I-PpoI is a rare cutting
enzyme; for example, it cuts only once in S
cerevisiae chromosome 12. Therefore, if YAC copies are
rearranged upon integration into the mouse genome losing one or both
I-PpoI sites, it is highly unlikely that the new I-PpoI
fragment will be 144 kb in size. Thus, the presence of the 144-kb
fragment indicates that the -locus is intact. Fragments smaller or
larger than 144 kb have deletions of one or both I-PpoI sites
and possibly additional variable length deletions of sequences in the
-globin locus. Whether these aberrant PFGE fragments contain all or
part of the -globin locus can be determined with the method of
structural analysis that we used.
The initial screen of the 20 clones identified 8 clones with at least
one I-PpoI fragment of approximately 140 kb in size that
hybridized with the 0.9-kb -globin gene probe (data not shown).
Additional bands were present in 7 of these clones, and a complete
structural analysis of all the clones was undertaken to precisely map
the integrated -YAC fragments. The DNA was hybridized either with 12 probes spanning the entire -globin locus or with 3 probes located at
the 5 end, middle, and 3 end of the locus (5HS5,
A, and DF-10, respectively; Fig 1A). The latter method
has been tested in Ppo-155 -YAC transgenic mice and has been
shown to depict as accurately the extent of the -globin locus as the
complete structural analysis with the 12 probes.30
The hybridization pattern of two MEL-YAC clones (15 and 17) with the 3 probes is shown in the autoradiograph of Fig 1B; the results from the
other clones are depicted diagrammaticaly in the same figure. Fragments
hybridizing with all three probes were considered to contain an intact
locus and were included when per copy expression of the globin
genes was calculated. Clone 15 demonstrates why the analysis with the 3 probes is necessary for delineating the size of the -YAC fragments;
this clone at the initial PFGE screen was considered to contain an
intact fragment. However, as shown in Fig 1B, it clearly has two
incomplete fragments; one extends from 5HS5 to A
and the other from A to 3 end of the locus. This
was verified by hybridization of a PFGE blot with all 12 probes (data
not shown). Clone 17 has an intact fragment of approximately 140 kb and
another fragment of about 280 kb that has lost one or both
I-PpoI sites but contains an intact -globin locus.
The remaining clones have at least one intact -YAC fragment; the
structure of the integrated -globin loci is shown diagrammatically in Fig 1C. Clone 1 has three intact -globin loci 200, 150, and 90 kb
in size; clone 3 has two intact loci at 280 and 140 kb; clone 7 has
three intact loci at 280, 140, and 100 kb; clone 12 has three intact
loci at 180, 140, and 100 kb; clone 18 has three intact loci at 240, 220, and 200 kb; and clone 20 has one intact locus at 140 kb.
Expression of the globin genes of the Ppo-155 -YAC is
strongly influenced by the position of integration into the MEL cell
genome.
The 7 clones that contained intact -globin loci by structural
studies were used for analysis of human -like and murine -globin mRNA by RNase protection at several intervals between 20 and 120 days
of continuous culture. The results are shown in
Fig 2. In Fig 2A, the mRNA is hybridized
with a -globin probe. In Fig 2B, the mRNA is hybridized with the
- and A globin probes. Two clones synthesized only
-globin mRNA (3 and18) during the 4 months of culture and 2 clones
synthesized and mRNA (12 and 20), whereas 1 clone (17)
synthesized , , and mRNA. Clone 1 failed to express any human
mRNA, and clone 7 had minimal - and -globin expression at levels
less than 2% of murine (data not shown).
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| Fig 2.
Expression of the human globin genes in
MEL(Ppo-155 -YAC) clones. Total RNA was isolated from MEL
cells at the days shown above the autoradiogram and was subjected to
RNase protection assay as described in Materials and Methods. In (A),
the RNA was hybridized with the human -globin probe. In (B), the RNA
was hybridized with both - and -globin probes. The location of
the protected fragments is shown next to the autoradiogram. Notice the
striking variation between human globin mRNA levels between clones.
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Table 1 shows the levels of total (ie, + + ) human globin mRNA for each clone as the percentage of
mouse- (corrected for copy number) at different culture days; due to
different growth rates among clones, not all of them were available for
analysis on the particular days. The results are shown as a diagram in Fig 3. The striking dispersion of the human
mRNA values is characteristic. Thus, the mRNA levels of the 4 clones
available for analysis on day 22 range from 11.5% to 437% of
mouse- (38-fold variation), on day 60 from 0% to 115%, on day 75 from less than 2% to 193.5% (97-fold), on day 90 from 2% to 178%
(89-fold), and on day 120 from 2.5% to 139% (55.6-fold). These
results indicated that the expression of the Ppo-155 -YAC
strongly depends on the position of integration of the -YAC into the
MEL cell genome.
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| Fig 3.
Position-dependent expression of human globin genes after
direct transfer of the YAC into MEL cells. Total human globin mRNA
levels (ie, + + ) of the MEL (-YAC) clones are
expressed as the percentage of the endogenous mouse -gene corrected
for copy number. Each column represents results obtained by a single
clone identified with the number below the column. Clone 1 had 0% and
clone 7 less than 2% of mouse mRNA and are shown as flat lines.
Results from 6 culture days are shown. Notice the striking variation in
human globin gene expression between clones. Such findings indicate
that the expression of the globin genes is strongly influenced by the
position of integration of the -YAC into the mouse genome.
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Developmental expression of the human globin genes in the
MEL(Ppo-155 -YAC) cells is random.
As mentioned earlier, clones 3 and 18 synthesize only -globin mRNA,
whereas clones 12, 17, and 20 synthesize more than one globin mRNA. The
changes in human globin gene expression during the 120 days in culture
for the latter 3 clones are shown in Fig 4.
Clone 20 expressed only -globin initially, but after 60 days in
culture, small levels of -globin mRNA appeared. In clone 12, similar
levels of - and -globin mRNA were present from 30 to 120 days in
culture so that the / + mRNA ratio remained constant. Initially, in clone 17, there was predominantly - and some
-globin mRNA; as the culture time advanced, and mRNA
decreased, and by day 60 the expression pattern had switched to
-globin only. Thus, several patterns of globin gene expression were
observed: an adult pattern from the onset of observation (clones 3 and
18), an initial and later partial activation pattern (clone
20), a nonswitching pattern of globin gene expression (clone 12), and a
pattern typical of the to switch (clone 17). These results suggest that the globin genes of the -YAC are activated randomly when the -YAC is directly transferred into the MEL cells.
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| Fig 4.
Developmental regulation of the globin genes in the
MEL(-YAC) clones. Human -, -, and -globin gene expression
levels are plotted as a percentage of total human mRNA output from the
-locus. Results are shown from three MEL(-YAC) clones that
express more than one mRNA species. ( ) -Globin mRNA; ( )
-globin mRNA; ( ) -globin mRNA. Notice the highly inconsistent
pattern. In clone 12 there is no change in or gene expression.
Clone 17 shows a typical  switch.
Clone 20 displays a delayed gene expression.
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Transfer of the Ppo-155 -YAC in L-cells.
The results obtained in the MEL-YAC cells were surprising, because the
155-kb -YAC contains an intact LCR. Several previous studies in
transgenic mice have clearly shown that an intact LCR protects the
globin gene from effects of position of integration.1,12,33 Therefore, the manifestation of strong position effects in the presence
of an intact LCR was unexpected. Also, previous studies in which a
248-kb -YAC was transfected into L-cells and subsequently into MEL
cells by LxMEL cell fusion have shown that the globin genes of the
-YAC were correctly developmentally regulated.22 There
are two explanations for the difference between the results of the
previous and the present study: (1) the Ppo-155 -YAC may be
missing sequences that are present in the 248-kb -YAC and are
essential for protecting the -globin locus from position effects; or
(2) the transfer of the YAC into L-cells first may have affected the
locus in a manner that allowed normal function of the LCR when the
248-kb -YAC was subsequently transferred in the MEL cells by fusion.
To test the latter hypothesis, the Ppo-155 -YAC was
transfected into L-cells by lipofection. After 12 days in culture, 14 G418-resistant clones were randomly picked and expanded under further
selection. The clones were screened for the presence of -YAC DNA by
conventional Southern analysis. Three clones were identified (LY2, 5, and 7) that carried the correct size fragments from the -locus (data
not shown). The structural integrity of the transfected -YACs was
analyzed by I-PpoI digestion and PFGE; all 3 clones carried at
least one intact copy of the YAC (data not shown).
Fetal/embryonic pattern of globin gene expression of the
Ppo-155 -YAC transferred into L-cells.
Previous studies have shown that human globin gene transcripts are
present in L-cells transfected with the 248-kb -YAC. The pattern of
-YAC gene expression was predominantly fetal-embryonic, with about
80% -globin mRNA, 15% , and 5% .22 There was no transcription of the endogenous murine globin genes.
In the present study, the 14 L(-YAC) clones were screened for globin
gene expression by RNase protection. Globin mRNA was present only in
the 3 clones that had an I-Ppo-I fragment containing the entire
-globin locus (LY2, 5, and 7). Globin gene expression patterns
varied between clones. Clone 2 synthesized exclusively mRNA; in
clone 5, 85% of the transcripts were and 15% , and clone 7 had
72% , 23% , and 5% mRNA. These results provide further
evidence that the genes of the -YAC are activated when transferred
into the nonerythroid environment of the L-cells. In addition, the
fetal pattern of expression of the Ppo-155 -YAC in this
study is similar to the expression pattern of the 248-kb -YAC.22
Position-independent human globin expression in L(-YAC)x MEL cell
hybrids.
The three L-cell clones containing structurally normal -YACs
were fused with MEL-585, and L(-YAC)xMEL cell hybrids were obtained
by selecting cells growing in suspension and in the presence of
G418. Parental cells were eliminated because of their G418 sensitivity
(MEL cells) or adherent growth (L-cells).
Figure 5 shows the results of RNase
protection after 54 and 72 days in culture. Notice that in hybrids 2 and 5, both and globin genes are transcribed at similar levels
so that the / + ratios are 0.68 and 0.67, respectively.
Hybrid 7 had only -globin mRNA on days 54 and 72. This was a
robustly growing hybrid and cells for RNA studies were available from
day 19 postfusion; at this time-point, a small amount of mRNA was
present (/ + ratio of 3.7%), raising the possibility that
this hybrid underwent a rapid to switch.
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| Fig 5.
Human globin gene expression of -YACs of LxMEL
hybrids. The -YACs were first transferred into L-cells by
lipofection and subsequently into MEL cells by cell fusion. Globin mRNA
expression was analyzed in L(Ppo-155 -YAC)xMEL hybrids at
various time points after hybrid formation. Hybrid 7 shows extinction
of expression of the human and murine globin genes. The visual
differences in expression levels in the autoradiogram were diminished
when the human mRNA levels were corrected for the copy number of the
integrated -YACs and the level of expression of the murine
globin gene. Quantitative data are shown in Fig 6.
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The total human globin mRNA (ie, + + ) synthesized at
culture days 54 and 72 is expressed in Fig
6 as the percentage of murine -globin mRNA. Notice that at day 54 postfusion, the levels of expression per copy of the YAC in the three
hybrids are very close to the levels of expression of the endogenous
gene. Human mRNA per copy ranged from 112.3% to 170.4% of murine ; ie, there was only a 1.5-fold variation in expression between the
three hybrids. By day 72, the levels of human mRNA decreased and ranged
from 37.6% to 92.5% of murine , ie, they displayed a 2.5-fold
variation. Thus, in sharp contrast to the position-dependent expression
pattern obtained after the direct transfer of the Ppo-155 -YAC into MEL cells, the genes of the chromosomally transferred YAC
were transcribed in a position-independent manner after transfer into
MEL cells.
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| Fig 6.
Position-independent expression of globin genes of the
YAC in L(-YAC)xMEL hybrids. Total human mRNA levels of
L(Ppo-155 -YAC)xMEL hybrids shown in Fig 5 were corrected
for the number of copies of the integrated human globin genes present
in the hybrids and expressed as the percentage of one copy of the
murine- gene. Results from culture days 54 and 72 postfusion are
shown. Notice the small degree of variation in human globin expression
between the three hybrids. Globin mRNA levels varied 1.5-fold on day 54 and 2.5-fold on day 72.
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Activation of the silent globin genes of a MEL(-YAC) clone after
fusion with L-cells.
Our results so far have shown that passage of the -YAC through the
L-cells resulted in position-independent expression in the LxMEL
hybrids. This was interpreted as evidence of the ability of the L-cells
to modify the incoming naked DNA in a way that resulted in proper
function of the LCR when the locus was chromosomally transferred into
MEL cells. We then asked whether L-cells could also influence the
expression of a -YAC that was first transferred into MEL cells; to
this effect, hygromycin-resistant L-cells were generated and fused with
three MEL(-YAC) clones. Hybrids were selected on the basis of dual
resistance to G418 and hygromycin. We analyzed hybrid pools that
maintained their ability for erythroid differentiation as evidenced by
exposure to HMBA. One hybrid, formed from the nearly silent
MEL(-YAC) clone 7, fulfilled the above-noted criteria and was
further analyzed. The expression levels for the and globin
genes increased 2.7- and 34-fold, respectively, in the hybrid cells
(Fig 7). The level of total human mRNA
output from the locus increased 13-fold (from 1.6% to 21% of murine
), indicating that a silenced -YAC can be reactivated upon
transfer into L-cells.
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| Fig 7.
Activation of human globin gene expression of a silent
MEL(-YAC) clone after transfer into L-cells. The nearly silent
MEL(-YAC) clone 7 (see Fig 3) was fused with HygroR L-cells. A
hybrid cell population was selected on the basis of its dual resistance
to G418 (from the MEL YAC cells) and hygromycin (from the L-cells).
This hybrid retained the potential for globin gene expression upon
induction of erythroid differentiation with hemin-HMBA. Total human
globin mRNA expression of t |
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Abstract
Introduction