(Received for publication, October 9, 1996, and in revised form, November 22, 1996)
From the Institut für Biologie III der Universität
Freiburg, Expression of the chicken lysozyme locus in
macrophages is regulated by at least six different positive and
negative cis-regulatory elements. Chromatin of the chicken lysozyme
locus is gradually reorganized during macrophage differentiation,
indicating that each cis-regulatory element is activated at a different
developmental stage. Irrespective of their differential developmental
activation, individual cis-regulatory regions are capable of driving
transcription of the lysozyme gene in mature macrophages of transgenic
mice. In order to examine the role of different cis-regulatory regions in lysozyme locus activation, we analyzed the time course of
transcriptional up-regulation of deletion mutants of the lysozyme locus
in a new in vitro differentiation system based on enriched
primary macrophage precursor cells from the bone marrow of transgenic
mice. We show that constructs carrying cis-regulatory elements which
are structurally reorganized early in development are also
transcriptionally active at an early stage. A construct in which the
early enhancer has been deleted shows a delay in transcriptional
activation. The presence or absence of a negative regulatory element
has no influence on the time course of transcriptional activation of
the lysozyme locus.
Most gene loci examined so far are regulated by a variety of
different cis-regulatory elements distributed over many kilobases of
DNA. The presence of a DNase I-hypersensitive site
(DHS)1 in chromatin is the result of the
assembly of transcription factor complexes; hence, a DHS in most cases
indicates the presence of an active cis-regulatory element. Many gene
loci exhibit changes in DHS patterns according to the developmental
stage, indicating that along with cellular differentiation, individual
cis-regulatory elements of a given gene locus display different
activity patterns. It has been argued that a reorganization of
chromatin at early stages of cellular differentiation may represent a
priming step required for the assembly of an active transcription
machinery at later stages of development (1-4). However, the
hierarchical relationship between the structural reorganization of
cis-regulatory elements and their actual ability to stimulate mRNA
synthesis is still unclear (1, 2, 5-7). The dissection of the role of
different cis-regulatory elements in the developmental control of gene
locus activation requires their individual analysis in an experimental
system where cell differentiation can be followed, thus enabling to
link a stage-specific chromatin structure with the transcriptional
activity of the gene.
We have been studying the chicken lysozyme locus as a marker for
macrophage differentiation. Cis-regulatory elements regulating gene
expression are located in the 5 We showed that the chromatin structure displayed by the lysozyme locus
in the various chicken cell types is faithfully reformed in lysozyme
expressing and non-expressing cells of transgenic mice. This holds true
for the DHS For each
experiment four mice were killed by cervical dislocation. Bone marrow
cells were flushed from femurs and tibias with phosphate-buffered
saline (PBS). Cells were collected, washed once in PBS, and maintained
on ice throughout the staining procedure. The cell suspension was
depleted of red blood cells by hypotonic lysis with an ammonium
chloride potassium buffer (Red Cell Lysis Buffer,
Sigma). To remove debris, the suspension was
centrifuged through a cushion of fetal calf serum (FCS; Life
Technologies, Inc.). The remaining cells were washed once and
resuspended in PBS supplemented with 3% FCS. After the suspension was
passed through a cell strainer (Falcon/Becton Dickinson), 3.5 × 107 cells in a volume of 200 µl were used for staining.
To suppress nonspecific binding, cells were first incubated for 5 min
with 15 µl of rat serum. Afterwards cells were incubated for 15 min with 16 µl of ER-MP20-FITC (BMA), washed with PBS, 3% FCS and subsequently incubated (5 min) with 16 µl of biotinylated ER-MP12 (BMA), followed by an addition of 16 µl of streptavidin-Red670 and
another incubation of 15 min. After two washes, the cells were
resuspended in PBS, 3% FCS to a final concentration of 2 × 106 cells/ml. Stained cells were analyzed and sorted on a
FACStarplus (Becton Dickinson). The fluorescence intensity
of individual cells was measured as relative fluorescence units. The
purity of the sorted cell populations was determined by flow cytometry and exceeded 95%. In order to characterize cells differentiated in vitro by antibody staining, cells were stained with
ER-MP12 and ER-MP20 as described above. In addition, cells were stained with R-phycoerythrin-coupled mAbs (Pharmingen) against B220
(RA3-6B2), Mac-1 (M1/70), F4/80 and IgM (isotype control). Stained
cells were analyzed by flow cytometry.
The medium used for the culture of sorted cells was
IMDM (Life Technologies, Inc.) supplemented with 10% FCS, 5%
conditioned medium containing IL-3, 10% L-cell conditioned medium
containing M-CSF, 2 mM glutamine, 100 units/ml penicillin,
100 units/ml streptomycin (Life Technologies, Inc.), and 1.5 × 10 Total RNA was
isolated from the adherent and non-adherent cells using 0.5 ml of
RNAzolTM B (Biotecx Laboratories, Inc.) according to the
manufacturer's instructions. cDNA of isolated total RNAs from the
different samples was prepared using random hexamers as primers and
Moloney murine leukemia virus reverse transcriptase (Life Technologies,
Inc.) in a reaction volume of 20 µl under conditions recommended by the manufacturer. Per reaction 2 units of ribonuclease inhibitor (Life
Technologies, Inc.) was added. cDNA was subsequently heated to
70 °C for 5 min to inactivate reverse transcriptase.
To ensure the use of comparable amounts
of RNA and cDNA for the analysis of the different samples, the
relative expression level of the housekeeping gene HPRT was used as a
standard for calibration. Only in the linear range of PCR reactions is
it possible to quantitate the intensity of PCR signals. The linear
range of PCR reactions is defined as the range in which the use of
double cDNA amounts correlates with a duplication of the signal
intensity. To define the linear range, serial 1:2 dilutions of the
different cDNA samples were used to perform a PCR with primers
specific for HPRT. For further analysis of the expression level of the endogenous mouse lysozyme and the different constructs of the transgenic chicken lysozyme, only those cDNA concentrations were used which gave rise both to signal intensities for HPRT within the
linear range as well as comparable intensities among the different samples. The corresponding sample used for HPRT calibration was further
diluted 1:2, 1:10, and 1:50 and used to perform a PCR with primers
specific for mouse lysozyme. Due to the different expression levels of
chicken lysozyme transgenes in the different mouse lines and in the
various differentiation stages, it was necessary to carry out PCR
reactions with a different range of dilutions for each mouse line.
Except for mouse line dXK.2, 1 µl of the following serial dilutions
from the determined dilution of every sample which gave rise to a
linear HPRT-PCR signal were used to analyze the different mouse lines:
mouse line XS.0b: 1:2, 1:10, 1:50; mouse line XSdSS.28: 1:2, 1:4, 1:8.
For mouse line dXK.2, 4, 2, and 1 µl were used. Primers used were as
follows: HPRT, 5 The different cis-regulatory elements
of the lysozyme gene are structurally activated at different macrophage
differentiation stages as indicated by a changing pattern of DHS in the
5
In order to follow transcriptional activation of wild type and mutant
lysozyme loci, we set up an in vitro differentiation system
based on enriched myeloid precursor cells as outlined in Fig.
2. It has been shown that developing macrophage
precursors can be characterized by a differential expression of the
ER-MP12 and the ER-MP20 surface antigens (28, 29). The most immature macrophage colony-stimulating factor (M-CSF) responsive precursors express a high level of the ER-MP12 and no ER-MP20 antigen (Fig. 2). During macrophage maturation, ER-MP12 expression is gradually switched off, whereas expression of ER-MP20 is switched on. Bone marrow
monocytes express a high level of the ER-MP20 antigen and have lost the
ER-MP12 antigen (30). We isolated
ER-MP12hi/20
Macrophages developing from ER-MP12hi
populations in vitro have up to now not been characterized
in detail. The ER-MP12hi population consists mainly of
small blast-like cells as described previously (30, data not shown).
From day 3 of culture onwards, cells progressively acquire an adherent
phenotype (see also Fig. 3). By day 6 most cells are
adherent and from morphology and surface marker expression (Fig. 5)
resemble monocytes. The ER-MP20hi population resembles
monocytic cells, which adhere quickly within 12 h of culture (see
Fig. 3). During progressive differentiation cells of both populations
acquire the characteristic features of macrophages, like irregular
shapes and granules.
Immunophenotypic characterization of
differentiating cells. Cell surface marker characterization of
ER-MP12hi/20
In order to further characterize cells developing in our culture
system, we analyzed their proliferative capacity. In each respective
differentiation culture we started with the same number of cells;
hence, the comparison of the total amount of HPRT mRNA present in
our RNA preparations from the different time points allowed an accurate
description of relative cell numbers. In the same way, we could measure
the proportion of adherent versus non-adherent cells. The
result of this experiment is depicted in Fig. 3, which shows the
relative increase in cell numbers and the changing proportion of
adherent versus non-adherent cells in the different cell
populations. An initial lag phase is observed for the
ER-MP12hi culture which is due to the presence of
contaminating non-myeloid cells dying in the first 2 days of in
vitro differentiation (30). Thereafter, both cell populations show
the same proliferative capacity. The proportion of adherent cells in
the ER-MP20hi population is an order of magnitude higher
compared with the ER-MP12hi population. Mature
ER-MP20hi cells cease to proliferate toward the end of the
culturing period. In contrast, the ER-MP12hi population
contains a significant number of proliferating non-adherent precursor
cells which successively mature and adhere. This indicates that the
non-adherent fraction of the ER-MP12hi cell population
contains the desired precursor cell types to use as a starting
population for the analysis of the time course of transcriptional
activation of different transgene constructs.
Construct XS carries the full set
of cis-regulatory elements of the lysozyme locus and is expressed at a
high level in macrophages of transgenic mice and independent of the
chromosomal position of the transgene (26). Macrophage precursor cells
from the bone marrow of transgenic mouse line XS.0b were isolated as
described above and subjected to in vitro differentiation,
and RNA prepared from the various cell populations was analyzed by
RT-PCR. Since the ER-MP20hi cell population is composed
almost entirely of adherent monocytes, only the adherent cell fraction
was examined. Fig. 4 shows the results of this
experiment. One example for such an RT-PCR analysis is depicted in Fig.
4A. The time course of transcriptional activation of
transgene and endogenous lysozyme gene was determined by quantifying PCR signals derived from the linear part of the amplification reaction
(Fig. 4B) for each time point.
Expression analysis for chicken lysozyme and mouse lysozyme expression
in the ER-MP12hi fraction at day 0 revealed a very low
level of expression of both genes. Expression of the chicken lysozyme
transgene in the differentiating non-adherent fraction linearly
increases from day 0.5 of in vitro differentiation onwards,
whereas expression of the endogenous mouse lysozyme gene increases
exponentially. The adherent ER-MP12hi population shows a
different time course of expression. Initially, both transgene and
mouse lysozyme mRNA levels increase during differentiation culture.
Surprisingly, progressive differentiation in culture leads to a
decrease of transgene but not of mouse lysozyme mRNA levels. A
similar phenomenon is observed with the differentiated ER-MP20hi cell population, where transgene expression is
initially high (probably as a result of activation via Fc receptors)
and sharply declines after a couple of days in culture. However, in
both populations LPS treatment leads to a reactivation of transgene
expression. For comparison, we measured the chicken lysozyme expression
level in thioglycolate elicited, relatively mature peritoneal
macrophages of the same mouse line. The mRNA level in these cells
that had been cultured for 1 day was comparable to the highest level
found in in vitro differentiated cells but did not decrease
during prolonged cell culture (data not shown). Only the more mature
ER-MP20hi population but not the non-adherent or the
adherent ER-MP12hi cell population responds to LPS
treatment at early time points of differentiation. Expression of the
endogenous mouse lysozyme gene was not induced by LPS treatment, as
shown before (27, 31).
In order to determine the differentiation
stage in which the chicken lysozyme transgene is activated in mouse
cells, we analyzed the surface marker expression of differentiating
cells. We stained cells at various time points of in vitro
differentiation with antibodies directed against the ER-MP12 and
ER-MP20 antigens, as well as against CD11b (Mac-1) (33) and F4/80 (34),
the latter being surface markers characteristic for mature macrophages. A variant of the common leukocyte-specific antigen CD45R is expressed predominantly in B-cells and a subset of macrophages (35) and is
recognized by the B220 antibody. An antibody against mouse IgM served
as isotype-matched control (36). The results of these analyses are
shown in Fig. 5. As predicted, in vitro
differentiation of the ER-MP12hi population leads to a
successive down-regulation of the expression of the ER-MP12 antigen and
to an up-regulation of the ER-MP20 antigen (30). The cells reach the
double positive stage around days 2-3 of differentiation, about 8% of
all cells display this phenotype at day 3 (data not shown).
Subsequently, the cells express more and more epitopes characteristic
for mature macrophages. Initially the expression of the B220 antigen is
high, indicative of the presence of B-cell precursors in this
population (37). Stimulated development of macrophages leads to a
decrease in the number of B220 positive cells. The
ER-MP20hi population is characterized by a high level of
expression of antigens specific for mature macrophages. Taken together,
our analysis shows that chicken and mouse lysozyme gene expression is
up-regulated from day 0.5 of differentiation onwards, where the cells
have not yet reached the ER-MP12/ER-MP20 double positive stage and
express only a low level of mature macrophage surface markers.
We analyzed the
time course of transcriptional activation of two different deletion
mutants of the lysozyme locus during in vitro
differentiation (26). Precursor cells of mouse lines XSdSS.28 and dXK.2
(Fig. 1) were isolated, and RNA from in vitro differentiated cells was analyzed by RT-PCR as described for mouse line XS.0b. Fig. 6,
A and B, displays the results
obtained for mouse line XSdSS.28, which carries a construct lacking the
late enhancer region. The kinetics of transcriptional activation of
this construct is undistinguishable from the construct carrying all
cis-regulatory elements (Fig. 4). Here also we observe a continuous
increase of expression in immature precursors followed by a decrease in more mature cell populations. The kinetics of transcriptional activation of construct dXK which lacks the
In our in vitro differentiation
experiments we analyzed two populations of macrophage precursor cells
representing two different maturation stages. Previous studies have
shown that the ER-MP12hi/20 Our three-color surface marker analyses of differentiating
ER-MP12hi/20 Transgene expression decreased at later macrophage differentiation
stages. This phenomenon was observed for all constructs tested.
However, it was not observed with thioglycolate-elicited peritoneal
macrophages kept in culture for up to 2 weeks, indicating that the
lysozyme transgene is expressed at a high level in in vivo
differentiated cells. The uniformly high expression level of the
endogenous mouse lysozyme gene as well as the reactivation of a high
level of transgene expression by LPS treatment argues against a general
decrease of cell viability in our culture system. It rather indicates
that the cells reach a quiescent differentiation stage which does not
support transgene expression without a further signal. In
vivo, monocytes migrate into various tissues and differentiate into various mature macrophage types. In most cases such cells also
cease expression of the endogenous mouse lysozyme gene. We could show
that expression of the chicken lysozyme transgene, in contrast to the
mouse lysozyme gene, is a faithful marker for macrophage activation
(27, 31). Our experiments indicate that transgene expression is
up-regulated until the cells reach the monocyte stage where they
normally would leave the bone marrow is then down-regulated and is only
reactivated after further macrophage differentiation.
Thioglycolate-elicited macrophages obviously have received an
additional differentiation signal required to maintain a high transgene
expression level, which in addition can be further enhanced by
LPS treatment (31).
The various enhancer elements on the lysozyme locus
assemble transcription factors and thus become DNase I-hypersensitive at different stages of macrophage development. Our studies of the time
course of activation, as well as earlier chromatin structure analyses,
point to a different role of various cis-regulatory elements with
respect to the coordinated activation of the lysozyme locus during
development. In early macrophage precursor cells the early enhancers,
the promoter, and the silencer element are active, whereas the late
Each of these scenarios could be experimentally distinguished. If the
two latter models were true, mutants of the lysozyme locus carrying
deletions of early or late enhancer regions should have shown
characteristic differences in the time course of developmental activation as compared with a locus carrying all cis-regulatory elements. Such differences should also have been observed if the presence of the Our results demonstrate that the absence of the silencer element does
not alter the time course of transcriptional up-regulation, implying
that this element does not repress the action of the early enhancers.
Although we have no evidence to suggest this from the present analysis,
we prefer a model in which the Taken together our data indicate that initial locus activation is
performed by the interaction of the early enhancers with the promoter.
Maximal transcriptional activity, which is necessary during a bacterial
attack (simulated by LPS treatment), is achieved by the inactivation of
the negative regulatory element and the simultaneous activation of the
We are particularly indebted to M. van Bruijn
and P. Leenen, Erasmus University of Rotterdam, for generous advice and
the availability of information prior to publication. We thank Dr. Nicole Faust and Dr. P. J. Nielsen, MPI für Immunbiologie,
Freiburg, for critically reading the manuscript and Gudrun Krüger
for expert technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-half of the gene locus. Transfection
analysis revealed three enhancers located 6.1, 3.9, and 2.7 kb upstream
of the transcriptional start site, a hormone responsive element at
1.9 kb, and a complex promoter (8-14).2
A negative regulatory element is located at
2.4 kb (
2.4-kb silencer), which has been implicated in the repression of the lysozyme
locus in lysozyme non-expressing tissues (11, 15, 16). The activity of
each of these cis-regulatory elements is marked by the presence of DHS
in chromatin (17-20). In turn, reporter gene constructs carrying
particular cis-regulatory elements are only active when transfected
into cell types displaying a DHS at the position of the same element
(13, 14, 19). The analysis of lysozyme chromatin structure in
retrovirally transformed chicken cell lines resembling multipotent
myeloid progenitor cells (21) and various macrophage maturation stages
revealed that different sets of cis-regulatory elements are
structurally reorganized at different developmental stages and thus are
differentially active (20, 22). Multipotent progenitor cells do not
transcribe the lysozyme gene and display the chromatin structure of the
inactive locus. At the myeloblast stage, DHSs are formed at the
6.1-
and the
3.9-kb (early) enhancers as well as at the promoter form. The
silencer element is still active, as indicated by the presence of a
DHS, whereas the (late) enhancer at
2.7 kb displays no DHS and thus
is inactive at this differentiation stage. A low level of lysozyme
mRNA can be measured. At later stages of differentiation, a switch
in chromatin structure in the region between
2.7 and
2.4 kb is
observed. The DHS at the
2.4-kb element disappears, until it is lost
upon terminal differentiation and a new DHS at the
2.7-kb enhancer
element is formed. Along with chromatin reorganization, transcription
of the gene is up-regulated from a very low level in myeloblast-like
cells to an almost 100-fold higher level in activated macrophage-like
cells. These experiments correlate a high transcriptional level of the
lysozyme gene with conditions where all enhancers are active and where
the silencer element has been inactivated.
and the nucleosomal phasing pattern as well as for the
reorganization of the
2.4/
2.7-kb region after terminal macrophage
differentiation (23, 24), demonstrating that the same chromatin
rearrangements take place in both species. Our experiments in
transgenic mice also showed that for position-independent expression of
the lysozyme locus in mature macrophages, the presence of all
cis-regulatory elements is necessary (25, 26). However, experiments
with deletion mutants of the lysozyme locus in transgenic mice
demonstrated that each enhancer region, despite their unique temporal
regulation, is capable of driving macrophage-specific expression in
mature macrophages (26). The question now remained of how the different cis-regulatory elements cooperate during earlier stages of cell differentiation. To this end, we have analyzed the time course of
transcriptional activation of wild type and mutant lysozyme locus
constructs during in vitro differentiation of myeloid
precursor cells isolated from the bone marrow of transgenic mice. We
show that constructs carrying a deletion of the late enhancer region including the silencer element are transcriptionally activated at the
same developmental stage as the wild type locus carrying all
cis-regulatory elements. The time course of activation of both
constructs is indistinguishable, indicating a coincidence of structural
reorganization of the early enhancers and the onset of mRNA
synthesis. A construct in which the early enhancer has been deleted
shows a delay in transcriptional activation. Our results suggest that
the presence of the
2.4-kb silencer element on the wild type locus
has no influence of the developmental onset of lysozyme locus
activation, which is solely determined by the activity of the early
enhancers.
Immunofluorescence Labeling and Cell Sorting
4 M monothioglycerol. Conditioned media
were prepared as described (27). After sorting the cell populations
ER-MP12hi/ER-MP20
and
ER-MP12
/ER-MP20hi were distributed into
24-well plates (Greiner) depending on the number of kinetic time points
and were cultured in a humidified environment with 5% CO2
in air at a temperature of 37 °C. Depending on the sorting
efficiency 1.5-3 × 104
ER-MP12hi/20
and 8-10 × 104 ER-MP12/20hi cells were plated per well.
When harvesting the cells in intervals of either 12 or 24 h over a
period of 6 days, the adherent cell fractions were separated from the
non-adherent fractions. When indicated, cells were stimulated with 5 µg/ml LPS for 12 h. In order to obtain cells for antibody
staining, cells were plated onto hydrophobic Petriperm cell culture
dishes (Heraeus). Test experiments indicated that no difference in gene
expression was found as compared with normal tissue culture dishes
(data not shown).
-CACAGGACTAGAACACCTGC-3
; 5
-
GCTGGTGAAAAGGACCTCT-3
; mouse lysozyme (m-lys),
5
-ACCCAGCCTCCAGTCACCAT-3
, 5
-CAGTGCTTTGGTCTCCACGG-3
; chicken
lysozyme (c-lys), 5
-GATCGTCAGCGATGGAAACGGC-3
,
5
-CTCACAGCCGGCAGCCTCTGAT-3
. HPRT-PCRs were performed with 1.25 mM MgCl2 and 18 pmol of each HPRT primer. PCRs
for mouse lysozyme used 1.25 mM MgCl2 and 15 pmol of each m-lys primer, whereas chicken lysozyme PCRs were performed
with 1.0 mM MgCl2 and 15 pmol of each c-lys
primer. To every PCR reaction 1 µl of the corresponding cDNA
dilution was added. PCR reactions were done in a total volume of 30 µl and with the use of 1.5 units of Taq polymerase (Life
Technologies, Inc.). PCRs were carried out in a Trio-Thermoblock
(Biometra) using a regimen of 94 °C for 40 s, 55 °C (HPRT)
or 62 °C (m-lys and c-lys) for 40 s, and 72 °C for 1 min.
After 35 cycles, the PCR was finished with a terminal elongation step
of 72 °C for 10 min. 15 µl of each sample was loaded on a 6%
polyacrylamide gel electrophoresis. Gels were stained with ethidium
bromide and photographed with a Cybertech-Videoprinter (Mitsubishi)
under 245 nm of UV light. Bands on digitized videoprints were
quantified with the Image 1.35 program on a Macintosh computer. Signal
intensities for mouse and chicken lysozyme were normalized to the HPRT
signal, and the absolute normalized expression level was calculated
from these values by multiplication with the dilution factors.
Experimental Strategy
-regulatory region of the lysozyme locus in various myeloid chicken
cell lines (20) (Fig. 1A). The highest
transcriptional level is observed in cell lines representing activated
macrophages that display DNase I hypersensitivity at the position of
all enhancer elements and where the DHS at the silencer element at
2.4 kb has completely disappeared. In order to evaluate the
contribution of different cis-regulatory region to the developmental
control of lysozyme expression, we analyzed the time course of
transcriptional activation in the three different mouse lines indicated
in Fig. 1, B and C (26). Mouse line XS.0b carries
a construct with the full set of cis-regulatory elements, whereas the
transgenes in mouse lines dXK.2 and XSdSS.28 carry either a deletion of
the early enhancer region around
6.1 kb or a deletion of the late
2.7/
2.4-kb enhancer region, respectively. The presence or absence
of domain bordering sequences has no influence on the expression of the
different constructs in macrophages, as shown earlier (26).
Fig. 1.
A, map of the 5-regulatory region of
the lysozyme locus (upper panel) with the coding region with
intron and exons indicated as a gray box and black
boxes, respectively, the transcriptional start site depicted as a
horizontal arrow, and the DNase I-hypersensitive sites
(DHS) as vertical arrows. The lower
panel schematically displays the developmental changes in the DHS
pattern (vertical arrows) and the transcriptional activity
of the lysozyme locus in various retrovirally transformed chicken cell
lines representing different stages in macrophage differentiation.
B, map of the complete chicken lysozyme locus with DHS and
the three different constructs analyzed in transgenic mice. The
dotted and the striped box on the maps indicate
the position of deletions of cis-regulatory regions. E,
enhancer; S, silencer; P, promoter;
HRE, hormone responsive element. C, the three
different transgenic mouse lines analyzed in this study, their
transgene copy numbers, and the relative expression levels of the
different chicken lysozyme transgenes in macrophages.
[View Larger Version of this Image (26K GIF file)]
cells (ER-MP12hi
population) by fluorescence activated cell sorting in order to follow
gene expression during early macrophage differentiation. To compare the
mRNA levels in monocytes differentiated in vitro with
the level found in bone marrow monocytes, we also analyzed ER-MP12
/20hi cells (ER-MP20hi
population). The isolated cell populations were cultivated under macrophage growth promoting conditions in the presence of M-CSF and
interleukin-3 (IL-3) for a period of 6 days. Bacterial
lipopolysaccharide (LPS) is known to promote macrophage activation and
has been shown to stimulate expression of the chicken lysozyme gene in
chicken and mouse macrophages (10, 20, 27, 31). To examine at which
stage of differentiation the cells acquire LPS responsiveness, cultures
were treated with LPS for 12 h, either immediately after isolation
or after 5 days of culture. Along with progressive macrophage maturation the cells acquire an adherent phenotype. Since we were interested in studying gene activation starting with a precursor population as immature as possible, we separated the cells into more
mature adherent and immature non-adherent fractions. Cells were
harvested at 12- or 24-h intervals as indicated in Fig. 2, and total
RNA was prepared which was subjected to RT-PCR analysis for the
expression of the following genes: HPRT as an internal calibration
standard, the endogenous macrophage-specific mouse lysozyme M gene as a
control marker for a successful and reproducible in vitro
differentiation, and the chicken lysozyme transgene. In order to define
the developmental stage at which transgene activation takes place, we
immunostained cells at various time points of differentiation with
labeled antibodies directed against markers indicative of different
macrophage maturation stages (32).
Fig. 2.
Experimental strategy for the examination of
transgene activation in developing macrophages. FACS,
fluorescence-activated cell sorting.
[View Larger Version of this Image (24K GIF file)]
Fig. 3.
A, proliferative capacity of
differentiating cells of the ER-MP12hi/20
(left panel) and the ER-MP12
/20hi
(right panel) cell population. Relative cell numbers
were determined by calculating absolute HPRT mRNA levels for the
adherent and the non-adherent cell fraction. Signal intensities as
measured by RT-PCR were multiplied with the dilution factor and added
to determine the proliferative capacity of the total cell population. Dotted circles, cells at day 6 of differentiation (6+)
stimulated with LPS for 12 h. B, ratio adherent
versus non-adherent cells in the differentiating
ER-MP12hi/20
(left panel) and the
ER-MP12
/20hi (right panel) cell
population. Absolute HPRT mRNA levels for the adherent and the
non-adherent fraction were determined as described in A.
Dotted squares and circles, cells at day 6 of differentiation (6+) stimulated with LPS for 12 h.
[View Larger Version of this Image (22K GIF file)]
Fig. 5.
(A) and
ER-MP20hi/12
cells (B) during
their in vitro differentiation in the presence of IL-3 and
M-CSF and unseparated bone marrow (BM, upper panel, A). All samples were stained with biotinylated (+RED
670) ER-MP12, ER-MP20-FITC and the corresponding
R-phycoerythrin-labeled third mAb specific for either of the indicated
surface markers. A mAb against mouse IgM was used as an isotype-matched
control (IgG 2b) for the macrophage-specific mAbs Mac-1 and F4/80. For
analysis of the third mAb at day 0 live gates were set on either
ER-MP12hi/20
(A) or
ER-MP20hi/12
(B). Histograms in
A were generated from 100,000 (bone marrow), about 1000 (day
0), 500 (day 1), 1000 (day 3), and 2000 (day 6) events; histograms
in B were generated from about 5000 (day 0) and 500 (day 6)
events.
[View Larger Version of this Image (49K GIF file)]
Fig. 4.
Developmental activation of construct XS
carrying the full set of cis-regulatory elements. A,
expression of the chicken lysozyme transgene (c-lys), the
endogenous mouse lysozyme gene (m-lys), and the HPRT gene in
the non-adherent (ER-MP12hi na) and the adherent
(ER-MP12hi a) fraction of the
ER-MP12hi/20 cell population as well as from
the adherent fraction (ER-MP20hi a) of the
ER-MP12
/20hi cell population. Selected RT-PCR
experiments using high cDNA concentrations are shown which
emphasize early stages of differentiation where transgene and mouse
lysozyme expression levels are low. Note, therefore, that the PCR
signals at later differentiation stages are outside the linear range of
the amplification reaction and are shown only for reasons of
completeness. B, quantification of PCR signals. Three
different cDNA concentrations were used for the PCR reaction to
ensure that amplification was in the linear range. Bands were
densitometrically scanned as described under "Experimental
Procedures," the resulting signals specific for c-lys (upper
panel) and m-lys (lower panel) were normalized for RNA
variation against the HPRT signal and plotted against culture duration.
Curves were calculated with a Kaleidagraph program on a Macintosh
computer. The plot depicts mean values (where possible) of two
independently performed but overlapping kinetics. Cells were harvested
either every 12 h over a period of 3 days or harvested every
24 h over a period of 6 days. Open circles and
squares, cells of the freshly sorted cell population that
had not been separated by selective adherence. Dotted
circles and squares, cells stimulated with LPS for
12 h.
[View Larger Version of this Image (43K GIF file)]
6.1-kb enhancer region was different (Fig. 6, C and D). Expression in
freshly isolated ER-MP12hi cells at day 0 was high,
probably as a result of contaminating more mature cells, which were
lost due to their adherence after 1 day in the differentiation medium.
Note that these contaminating cells also have already acquired LPS
responsiveness (Fig. 6C, ER-MP12hi a).
Expression in precursor cells did not increase until day 2.5 of
differentiation and thereafter was up-regulated with a kinetics similar
to that observed for the other constructs. As with the other
constructs, expression in the ER-MP20hi cell population was
high in the beginning but decreased toward terminal differentiation of
the cells (Fig. 6, C and D, ER-MP20hi
a). Expression in the adherent ER-MP12hi population was
barely up-regulated. Expression from the XSdSS
and most likely also
from the dXK construct was refractory to LPS stimulation at early
differentiation stages but could be induced at later stages as it has
been shown before (31). This holds true for all cell populations
analyzed. The time course of activation of expression of the endogenous
mouse lysozyme M gene in all in vitro differentiation
cultures was basically indistinguishable, indicating that the time
course of transgene activation is a function of the various constructs
analyzed and is not due to differences in the differentiation kinetics
of the cells.
Fig. 6.
Developmental activation of deletion
constructs. A and B, developmental activation of
construct XS.dSS28 carrying a deletion of the late enhancer region.
A, expression of the chicken lysozyme transgene
(c-lys), the endogenous mouse lysozyme gene (m-lys), and the HPRT gene in the non-adherent
(ER-MP12hi na) and the adherent (ER-MP12hi a)
fraction of the ER-MP12hi/20 cell population
as well as from the adherent fraction (ER-MP20hi a) of the
ER-MP12
/20hi cell population. B,
quantification of PCR signals. C and D,
developmental activation of construct dXK carrying a deletion of the
early enhancer region including the silencer element. C,
expression of the chicken lysozyme transgene (c-lys), the
endogenous mouse lysozyme gene (m-lys), and the HPRT gene in
the non-adherent (ER-MP12hi na) and the adherent
(ER-MP12hi a) fraction of the
ER-MP12hi/20
cell population as well as from
the adherent fraction (ER-MP20hi a) of the
ER-MP12
/20hi cell population. D,
quantification of PCR signals. For further details see legend of Fig.
4.
[View Larger Version of this Image (36K GIF file)]
The Wild Type Chicken Lysozyme Locus Is Transcriptionally Activated
in Mouse Macrophages at the Same Developmental Stage as in Chicken
Macrophages
population
contains about 50% morphologically undifferentiated blast cells, and
the other half represents identifiable blasts cells of the myeloid, the
lymphoid, and the erythroid lineage (30, 38). 12% of the total cell
population represents a mixture of granulocyte macrophage-colony
forming cells (GM-CFCs) and macrophage-colony forming cells
(M-CFCs/monoblasts) with a high proliferative potential (30).3 The heterogeneous composition of
this population explains the apparent lag phase in proliferation, since
precursor cells of other hematopoietic lineages are unable to
proliferate under our culture conditions. However, since we observe no
reduction of relative cell numbers, we assume that myeloid precursors
start proliferating immediately after plating. The
ER-MP12
/20hi population contains
predominantly monocytic cells (74%) with a few immature blasts cells
(Ref. 30 and this study), which both immediately start to proliferate.
Our experiments clearly demonstrate that under our culture conditions
both sorted cell populations proliferate and simultaneously
differentiate. The ER-MP12hi/20
population
reaches the differentiation state of the
ER-MP12
/20hi population around day 6 as
judged from mouse lysozyme and surface marker expression levels as well
as from the ratio of adherent to non-adherent cells.
cells indicate that they transiently mature
into cells expressing both the ER-MP12 and the ER-MP20 antigen around
days 2-3 of in vitro differentiation. A cell population
isolated from the bone marrow expressing this surface marker
combination has been shown to consist of some M-CFCs and promonocytes
(30). The increase in chicken lysozyme expression begins at day 0.5 of
differentiation whereby half-maximal expression is reached by day 3. The onset of transgene expression occurs therefore most likely at the
GM-CFC stage (which is analogous to the myeloblast stage in the chicken system), indicating a concordance in developmental regulation between
the two species (20). Since also the same developmentally controlled
chromatin rearrangements occur in both species, we were able to draw
relevant conclusions from the analysis of wild type and mutant gene
loci in transgenic mice.
2.7-kb enhancer is inactive (20, 22, 23). Hence, several scenarios
could be envisaged. Either the early (
6.1-kb and
3.9-kb) enhancers
and the promoter drive lysozyme transcription at a low frequency
irrespective of the presence of the silencer at
2.4 kb or the
functional silencer element competes with the enhancers and inhibits
transcription at early differentiation stages. This would allow the
gene to be transcribed only after the developmentally controlled
inactivation of the silencer element and the simultaneous activation of
the late
2.7-kb enhancer. The third possibility would be a
developmentally controlled reorganization of the early enhancers by
transcription factors synthesized later in differentiation which in
turn would be necessary for their interaction with the basal
transcription machinery. The two latter models would imply that the
structural reorganization of the early enhancers is uncoupled from
their ability to drive transcription.
2.4-kb silencer element had any influence on the
onset of lysozyme expression at early differentiation stages, i.e. if it would repress expression of the lysozyme locus in
early precursors. However, the time course of transcriptional
activation of the chicken lysozyme transgene, with or without the
silencer element, is identical. Both transgenes up-regulate
transcription at day 0.5 of in vitro differentiation and
decrease expression at later differentiation stages with similar
kinetics. This is in contrast to the construct carrying a deletion of
the
6.1-kb enhancer region. Here, in concordance to the chromatin
studies, we observe a 2-day delay in the onset of transcriptional
activation. At day 2.5 to 3 the cells reach the ER-MP12/ER-MP20 double
positive (promonocytic) stage. At this stage, in analogy to the chicken system, the
2.7-kb enhancer is reorganized and thus activated. Our
results also indicate that the presence of the early
3.9-kb enhancer
alone is not sufficient to activate transcription in early
progenitors.
2.4-kb element is involved in
repressing the macrophage-specific
2.7-kb enhancer element at early
developmental stages of myeloid differentiation. The same element might
also be responsible for the repression of the
2.7-kb enhancer in the
chicken oviduct, where the gene is expressed under steroid hormone
control. Support for this idea comes from experiments that demonstrate
the presence of a DHS at the
2.4-kb element in the chicken oviduct,
where the
2.7-kb enhancer is not hypersensitive and thus not active
(18). Previous chromatin analyses have demonstrated that the silencer
element and the immediately juxtaposed enhancer element are each
organized in a positioned nucleosome and may form an integrated
cis-regulatory element. The spacing of binding sites is such that they
may face the same side on each nucleosome, thus bringing them in close contact (23). Chromatin rearrangements at the
2.4-kb and at the
2.7-kb elements are strictly parallel, and the appearance of MNase
and DNase I-hypersensitive sites at the enhancer correlates with the
disappearance of such sites at the negative regulatory element,
indicating that factor binding at both elements is mutually exclusive.
2.7-kb enhancer. In addition, the activity of all enhancers as well
as the promoter can be modulated by LPS and other macrophage activating
agents (10; 31; 39),4 indicating that the
expression status of the chicken lysozyme locus, as an endogenous gene
in chicken and as a transgene in the mouse, is strongly dependent on
the physiological status of a macrophage cell.
*
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (to C. B.) and by the Max Planck Society.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-761-203-2761; Fax: 49-761-203-2745; E-mail:
bonifer{at}sun2.ruf.uni-freiburg.de.
1
The abbreviations used are: DHS, DNase
I-hypersensitive site; mAb, monoclonal antibody; LPS, bacterial
lipopolysaccharide; HPRT, hypoxanthine phosphoribosyltransferase; kb,
kilobase pair(s); PBS, phosphate-buffered saline; FCS, fetal calf
serum; RT-PCR, reverse transcription-polymerase chain reaction; M-CSF,
macrophage colony-stimulating factor; IL-3, interleukin-3; c-lys,
chicken lysozyme; m-lys, mouse lysozyme; GM-CFCs, granulocyte
macrophage-colony forming cells; M-CFC, macrophage-colony forming
cells.
2
M. C. Huber, G. Krüger, and C. Bonifer,
unpublished observations.
3
M. de Bruijn and P. Leenen, personal
communication.
4
N. Faust, C. Bonifer, and A. E. Sippel,
manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.