(Received for publication, April 18, 1995; and in revised form, June 15, 1995)
From the
Nuclear factor I (NFI) was suggested to be involved in the
expression of the human -globin gene. Two established cell lines,
which express
-globin differentially, were therefore compared for
differences in binding of NFI at the
-globin promoter in
vivo. HeLa cells, in which
-globin is repressed, show a high
density promoter occupation with several proteins associated with
structurally distorted DNA. Cell line K562, which is inducible for
-globin, surprisingly was found to be heterogeneous consisting
mainly of cells (
95%) unable to express
-globin. However, the
promoter of the nonexpressing K562 cells was clearly different from
that of HeLa cells, being occupied only at basal transcriptional
elements. Therefore, the
-globin gene in these K562 cells may not
be truly repressed, but in an intermediate state between repression and
active transcription. The NFI site of the
-globin promoter
appeared occupied in HeLa but free of proteins in K562 cells. All cells
of both cell lines produce NFI, but the composition and DNA binding
affinity of NFI species differ significantly between the two cell
lines. Therefore, distinct forms of NFI may repress
-globin
transcription in HeLa cells. However, NFI is apparently not involved in
establishing the latent transcriptional state of the majority of K562
cells.
Expression of -globin is regulated in vivo by the
interplay of the locus control region at -40 kilobases and
diverse promoter elements(1, 2) . Activation of a
particular gene in the
-globin cluster is supposed to be achieved
by interaction of factors binding to the locus control region and
factors binding to promoter and enhancer elements, thereby keeping the
chromatin free of histones(3) . However, this histone-free
state which also correlates with DNase I hypersensitivity (4) bestows upon the globin genes only transcriptional
competence. Additional events or factors that bind at the regulatory
elements are required for a particular gene to be actively transcribed.
In the erythroid lineage, the major specific transcription factor for
globin gene expression is GATA-1(5, 6, 7) .
However, the promoter of the human
-globin gene contains no GATA-1
site but instead basal transcription elements, a possible SP1/
-IRP
site(8) , and a binding site for nuclear factor I (NFI; (9) ). (
)
NFI was originally isolated from HeLa
cells as a host protein required for the efficient replication of
adenovirus 2/5 DNA in vitro and in
vivo(10, 11) . NFI specifically recognizes the
DNA consensus sequence
5`-TGG(N)GCCAA-3`(12, 13, 14) .
NFI binding sites are found in many viral and cellular promoters and
enhancers (see (3, 4, 5, 6) and
7-12 in (9) ) suggesting a role of NFI as transcriptional
regulator. Most of these genes display tissue specificity in their
expression(15, 16, 17, 18, 19, 20, 21, 22) .
However, NFI is a ubiquitous factor, and it is not known whether it can
influence transcription in a tissue-specific manner. NFI may be
involved in transcription as a ubiquitous factor with specificity
provided through association with other, cell-specific factors.
Precedents for this mode include, for example, the association of the
ubiquitous Jun and Fos with the lymphoid-specific NF-AT
factor(23) . Alternatively, NFI may act as a
cell-specific transcriptional regulator in spite of its ubiquitous
expression. The latter view is supported by the observation of the
presence of different forms of NFI in different cell types (24, 25, 26) . These forms can arise by
expression of different NFI genes (24, 27) , by
differential splicing(28) , by diverse covalent
post-translational modifications(29, 30) , or by
heterodimerization (31) .
In this context it is of interest
whether NFI could contribute tissue specificity in -globin gene
expression. NFI has been, in fact, implicated in the multistep process
of transcriptional activation of the human
-globin gene by in
vitro(32) and in vivo transient assays with
reporter plasmids(28, 33) . These assays revealed a
weak but clear stimulation of
-globin transcription following
binding of NFI to the promoter sequence. Originally it was thought that
stimulation occurs by binding to the general positive cis-acting CCAAT genetic element of this
promoter(34) . It was this assumption which led to the
definition of NFI as ``CTF'' (=CCAAT-box transcription
factor) implying a role for NFI as a general transcription
factor(32) . However, we demonstrated that specific and fairly
strong binding of NFI actually occurs at an adjacent previously
unrecognized NFI site within the
-globin promoter(9) .
Furthermore, in vivo analysis with reporter plasmids suffers
from copy number effects and does not account for the influence of
chromatin which is known to play an important role in gene
transcription via the presence of specific histones, nucleosomes, and
higher order structures, such as the 30-nm-diameter chromatin filament,
locus boundary elements, and the nuclear matrix or
scaffold(3) . Chromatin structure is particularly important for
NFI binding and function; for example, Lee and Archer (35) demonstrated recently that NFI can bind and activate the
murine mammary tumor virus promoter from transiently transfected,
``naked'' plasmid templates, whereas the chromatin version of
the same sequence in the same cell is refractory to NFI action. For
these reasons, we wondered whether occupation of the NFI site of the
-globin promoter in the chromosomal context could be
correlated with a particular transcriptional state of the
-globin
gene. This would clarify a possible importance of this site in vivo and provide clues for an implication of NFI in
-globin gene
expression and regulation in situ. To start approaching this
question, we compared the in vivo footprints of an inducible
(K562) and a noninducible (HeLa) cell line. Our present results suggest
that the transcriptional state of K562 cells does not correlate with
NFI binding to the
-globin promoter in vivo. Our data are
also compatible with the hypothesis that NFI species found in HeLa
cells could act as repressors of
-globin transcription.
Figure 1:
A, slot blot
analysis of RNA of HeLa and K562 cells. An autoradiograph of a
representative experiment is shown. The values on the top of
the bars indicate relative amount of expression of
-globin mRNA. This amount was arbitrarily set 1 for the K562 cells
prior to induction with hemin (0 h). Values were normalized by using
the glyceraldehyde-3-phosphate dehydrogenase signals as a standard. B, FACS analysis of
-globin in K562, K562 hemin-induced,
and HeLa cells. A nonimmune serum served as a control. The broken
line serves as a comparison of differences in fluorescence. For
details see ``Materials and
Methods.''
Figure 2:
In vivo footprinting (A and B) and genomic sequencing (C) of the
-globin promoter. Genomic DNA from HeLa or K562 cells was modified in vitro or in vivo as indicated, and specific
regions were detected with the ligation-mediated PCR as described under
``Materials and Methods.'' Amplification products were
analyzed by sequencing gel electrophoresis and autoradiography. The
localization of the NFI, CCAAT,
-IRP, and ATA sites is indicated. Lines with open dots denote decreases, arrows denote
increases, and rectangles are examples for bands with equal
intensity of in vivo and in vitro modified DNA. Lane 5 is a longer exposure of lane 4, which was
performed to visualize the signals of the top region of the gel. The dotted lines indicate regions of lanes 4 and 5, which were not evaluated.
Figure 3:
Summary of the in vivo footprinting data of the -globin promoter in K562 and HeLa
cells. Symbols are as in Fig. 2. For comparison, a summary of
NFI footprints is also displayed, which was obtained by methylation
interference analysis of this region in
vitro(9) .
The in vivo footprinting patterns obtained with both cell lines is
clearly different from that of the protein-free DNA methylated in
vitro. This indicates that, in vivo, several proteins
occupy the -globin promoter in both cell lines (Fig. 2, lanes 1 and 4 or 5 versus lanes 2 and 3). However, the in vivo footprinting pattern is
distinct for each cell line (Fig. 2, lane 1 versus lane 4 or 5): HeLa cells generally display a high density
occupation of the promoter with proteins, revealed by the DMS-protected
guanosines (denoted by lines with open dots in Fig. 2, lane 1, and in Fig. 3), whereas K562 cells show only a
minimal occupation of the promoter (Fig. 2, lane 4 or 5, and Fig. 3). Additionally, the promoter in HeLa
cells shows a particular region of about 25 nucleotides with many
DMS-hypersensitive purine residues (denoted by arrows in Fig. 2, lane 1, and in Fig. 3). Also, two
neighboring cytosine residues at two sites (within the
-IRP site
and 3` of it; compare Fig. 3) become methylated to some extent
in this region, presumably at the N-3 positions which can occur only
after strong distortion of the double strand state of the DNA (cf., for example, ``Discussion'' in (48) ,
and references therein). For these reasons, we suggest that the DMS
hypersensitivity unequivocally indicates a dramatic alteration of the
secondary structure at this region of the DNA in HeLa cells. In
contrast, unusual reactivity of the DNA bases was not observed in K562
cells. Interestingly, protein binding in K562 cells seems to happen
exclusively next to basal transcription elements, such as CCAAT-box,
ATA-box and cap-site, as opposed to HeLa cells, in which at least the
last two sites appear essentially protein-free (Fig. 2, lane
4 or 5 versus lane 1; see summary in Fig. 3). In
summary, despite the equivalence in the expression pattern, the in
vivo footprinting data indicate that K562 cells possess an
-globin promoter structure clearly different from HeLa cells. In
the latter, the promoter is packed tightly with proteins and the DNA
structure is pronouncedly distorted. In contrast, in K562 cells, the
promoter shows an ``open'' chromatin configuration with
proteins bound only at distinct sequence elements. Therefore, we
suggest that the
-globin gene in the analyzed K562 cells may not
be truly repressed, as in HeLa cells, but in an intermediate state
between repression and active transcription.
In
contrast, binding of the NFI site is clearly evident in HeLa cells,
where the first two guanosines of the first half of the NFI site are
consistently found to be protected from in vivo methylation
(underlined in GGG(N)GCCAG; see Fig. 2, lane
1, and summary in Fig. 3). However, this protection pattern
deviates from DMS footprints of NFI made in vitro ( (9) and (14) ; see ``Discussion''). (
)Therefore, it is not possible to diagnose unambiguously
whether protection of the NFI site of the
-globin promoter in HeLa
cells is due to NFI binding. Nevertheless, it is clear that the NFI
site present at the
-globin promoter is utilized differentially in
each cell line, and this may point toward a distinct, hitherto
unrecognized genetic function of this site in the chromosomal context.
All conclusions in the last two sections are based on the analysis of the sense strand. In spite of the application of various experimental conditions for the PCR (use of formamide, deoxynucleotide analogues, dimethyl sulfoxide, different temperatures, and concentrations of compounds), we never obtained interpretable signals from the antisense strand. This is most probably due to the even higher GC content of the DNA upstream of the NFI site, which we believe is responsible for the attenuation of the signals beyond the displayed region also of the sense strand (not shown). Ambiguous annealing of the PCR primers to this region may impede the analysis of the antisense strand.
Figure 4: FACS analysis of NFI in K562, K562 hemin-induced and HeLa cells. A nonimmune serum served as a control. The broken line serves as a comparison of differences in fluorescence. For details see ``Materials and Methods.''
Figure 5:
A, binding and competition analysis of
NFIDNA complexes. 20 ng of purified recombinant NFI protein or 10
µg of whole nuclear extracts each of HeLa and K562 cells were
incubated with 5 fmol of radioactively labeled
-G wt oligonucleotide. Competition of complex formation was done with
the indicated oligonucleotides (100-fold molar excess over the labeled
probe) prior to the addition of proteins and analyzed by native gel
electrophoresis and autoradiography. B, supershift analysis of
NFI
DNA complexes by anti-NFI antiserum. Incubation and analysis
was performed as above. - (lanes 1, 4, 7, and 10) denotes no antiserum, 0 (lanes 2, 5, 8, and 11) denotes nonimmune serum, and
+ (lanes 3, 6, 9, and 12)
denotes anti-NFI antiserum added to the incubation reactions prior to
analysis.
The specificity of the polypeptides
in the shifted bands as NFI site-recognizing species and their relative
DNA binding affinity was checked by competition assays with 100-fold
molar excess of unlabeled DNA (Fig. 5A). Homologous
competition decreased the intensity of the control and of all shifted
bands of HeLa and of K562 cells (Fig. 5A, lanes
3, 7, and 11, respectively). Competitor L1/2 with a higher affinity NFI site (41) competes for binding
again with all polypeptides of the control and both cell lines (Fig. 5A, lanes 4, 8, and 12, respectively). In contrast, a mutant oligonucleotide
(designated -G mut.), in which the NFI site of
-G wt had been inactivated(9) , has no
significant competition effect on the interaction of the control, as
well as of any polypeptide of either cell line with the NFI binding
site (Fig. 5A, lanes 5, 9, and 13, respectively). Since in this assay all shifted bands from
both cell lines show essentially the same qualitative behavior as the
control NFI protein, we conclude that all bands represent NFI protein
species capable of DNA binding. However, the bands of the K562 cell
extracts appear to be more resistant to homologous competition than
HeLa-derived complexes (Fig. 5A, cf. lanes 11 and 12 versus lanes 7 and 8); this striking
behavior points toward a possible difference in the inherent DNA
binding affinities of the NFI species in the two cell types.
Conclusively, the structural differences in the NFI populations between
the two cell lines correlate with a different DNA binding mode of these
factors to the NFI site of the
-globin promoter.
It has been
reported that some cells contain a protein that binds exactly to the
NFI site but is not NFI(56) . NFI proteins share their greatest
sequence homology in the N-terminal DNA binding domain of the
polypeptide(57) . Therefore, in order to ascertain that the
complexes investigated here were due to NFI or NFI-like species, we
determined the immunological characteristics of the electrophoretic
mobility shift assay complexes by reaction with a polyclonal antiserum
directed against the recombinant NFI bearing the DNA binding domain (42) (Fig. 5B). This serum caused a strong
reduction of the DNA complex of the recombinant NFI (control) and
induced the formation of a supershifted NFIDNA complex running a
short distance into the gel (Fig. 5B, lane 6).
The complexes formed by HeLa and K562 nuclear extracts with
-G
wt all reacted similarly with the anti-NFI antiserum in that the
intensities of all bands were reduced to the same extent as that
observed with the recombinant NFI (Fig. 5B, lanes 9 and 12). Supershifted complexes with the nuclear extracts
were not apparent here; since all NFI
DNA complexes in the
extracts (Fig. 5B, lanes 7 and 10)
appear larger than the DNA complex of the recombinant NFI fragment (Fig. 5B, lane 4), we presume that the
additional increase in size caused by the antibody association
precluded entirely entering into the gel matrix. However, we consider
the impairment of the DNA binding by the anti-NFI antiserum to
sufficiently prove that the bound proteins were in fact NFI or, to a
large extent, NFI-like proteins.
NFI has been implicated in several aspects of DNA
transcription and replication. Functions of NFI include the potential
to act as transcriptional
activator(28, 32, 58) , repressor (16,
59-62, and (5) and (49) -52 and references
therein), antirepressor(63) , place-holder in chromatin
organization as a kind of ``antirepressor of
replication''(64) , and replication protein in viral
systems(12) . Several studies suggested that NFI also
stimulates -globin
transcription(28, 32, 33) . However, the
involvement of NFI in
-globin expression in situ has
never been tested. To define a differential state of transcription in
order to check NFI involvement, we first compared the
-globin
expression of two different human cell lines, K562 and HeLa, which are
known to be inducible or dormant for this gene, respectively. However
and to our surprise, the great majority of K562 cells, which is an
inducible cell line commonly used in globin gene research, was silent
for
-globin expression and could not be induced by hemin. To our
knowledge, the heterogeneity found in the K562 cell population is
demonstrated here for the first time. This heterogeneity, with only a
minor cell fraction actively expressing
-globin, is consistent
with the relatively weak RNA synthesis and inducibility found here and
by others(65, 66) , with the low level of
-globin
proteins (67) and with the reduced DNase I hypersensitivity in
the
-globin promoter compared to other genes in the
-globin
gene cluster(68) . In the latter case, the investigators
already mentioned heterogeneity in the K562 cell population as a
speculative explanation for their findings.
Although both HeLa cells
and the majority of K562 cells do not express -globin detectably,
they differ profoundly in their respective
-globin promoter
configurations in vivo. In the inactive HeLa cells, it is
highly packed with various proteins. In contrast, in the majority of
K562 cells it is scarcely bound with proteins but shows distinct
occupation at basal transcription elements. What could be the cause for
the apparent DMS hypersensitivity of the HeLa promoter between the
-IRP and ATA-box? This hypersensitivity suggests distortion of DNA
conformation which may be due to a positioned nucleosome at this locus:
x-ray analysis showed that the DNA does not wind smoothly around a
nucleosome core, but is rather bent fairly sharply or kinked at several
locations(69) . These kinks are not in direct contact with
histones and display departure from good stacking spread over several
base pairs(69) . These features would permit purine
methylation, also enhanced methylation and methylation at functional
groups that are normally not accessible in dsDNA. Indeed, DMS
modification of nucleosomal DNA in vitro reveals no apparent
periodic modulation of reactivity corresponding to the twist of the DNA
and no sites of purine protection in either DNA groove but rather sites
of enhanced reactivity(70) ; these sites were inferred to be
located within and next to a sharp bend(69) . Thus, the
methylation pattern of this particular region of the
-globin
promoter in HeLa cells is entirely consistent with a kinked region of
the DNA wrapped around a nucleosome. The signals of decreased intensity
may be due to proteins other than nucleosomes, which may help to
establish the repressed state of HeLa. These characteristics are in
accordance with the transcriptional silence of this promoter in tightly
packed chromatin and may reflect the truly repressed state. In
contrast, the promoter in K562 cells very significantly lacks the DMS
hypersensitive region of that in HeLa cells, suggesting absence of
nucleosomes. This parallels earlier investigations which revealed DNase
I hypersensitivity at the human
-globin promoter of K562 cells (68) indicating an open, i.e. nucleosome-free and/or
loosely packed chromatin state. Our findings are also in agreement with
the hypothesis that the locus control region keeps the globin gene
region free of nucleosomes in erythroid cells(3) . Thus, the
-globin promoter of the majority of K562 cells may define a
distinct stage of
-globin expression in a cascade of activation
events, which is different from the repressed state of HeLa and of the
active state of the few expressing K562 cells. A latent transcriptional
state in the course of transcriptional activation, known as
``transcriptional competence'' from other globin (see
references cited in (4) ), but also non-globin genes (e.g. the heat shock protein 70 gene; (71) ), is associated with
increased in vivo sensitivity of chromatin to DNase I
(originating from binding of transcription factors; (4) ),
absence (72) or rephasing (73) of nucleosomes, and
relaxation of higher structural levels of chromatin, probably by
partial depletion of histone H1(74) . Therefore, from the data
quoted above and presented here, we suggest that the latent
-globin promoter in most K562 cells may be in the state of
transcriptional competence, or, alternatively, of an even earlier stage
of transition of an extinct promoter to one undergoing maximal gene
expression, because it cannot be induced.
We do not know the reason
why most of the cells cannot be induced by hemin to activated
-globin expression. Since K562 is an undifferentiated
erythroleukemia cell line(75) , maybe most of the cells do not
possess all required specific DNA binding activators for
-globin
gene induction. Alternatively, transcriptional
``coactivators,'' a hemin receptor, or a connecting link in a
signal transduction pathway other than a DNA binding transcription
factor may be missing, thus rendering the cells apparently
unresponsive. In the latter case, the chromatin configuration
of their
-globin promoter might be equivalent to that of the few
cells that can be induced. We are currently attempting to clone
individual K562 cells with the aim to generate a homogeneous
-globin-inducible population. However, degeneration to a
heterogeneous population with respect to
-globin expression may
occur as a stochastic process in cell culture.
The in vivo footprints reveal absence of NFI binding at the NFI site of the
promoter in K562 cells. Although it is not possible to assay from the
present data for a function of NFI in active -globin transcription in vivo, we may conclude that NFI is apparently not implicated
in the preactivation phase of cellular
-globin expression detected
here. In this instance, it is interesting to note that in an
investigation with the
-globin promoter as a model, one NFI member
(CTF-1) has been discussed as a factor needed for antirepression in
vitro(76, 77) , counteracting the binding and
action of histone H1, rather than for subsequent direct transcriptional
activation(63) . NFI was thus suggested to act at early but not
later stages of transcriptional activation. Our system provides a
platform on which this proposal may be tested in vivo.
However, our results argue against an involvement of NFI. Apparently,
NFI does not belong to the factors which prepare the
-globin
promoter for active transcription and, thus, does not contribute to
cell-specific expression at this stage in vivo.
In contrast
to K562 cells, HeLa cells which uniformly do not express -globin
reproducibly show a clear occupation of the NFI site (contacts at the
underlined Gs in GGG(N
)GCCAG). However, it is difficult to
diagnose whether this site is occupied in fact by NFI, because in
vitro the pattern of methylation protection of the consensus
sequence is different (underlined in TGG(N
)GCCAA; (14) ).
When methylated in vitro, the
underlined residues in GGG(N
)GCCAG interfere with binding
in the particular NFI site of the
-globin promoter (see Fig. 3and (9) ). To our knowledge, no other DMS
footprint of NFI in vivo and no DMS protection footprints of
the
-globin NFI site in vitro have been ever reported
which could be compared directly with our results. However, the
following in vitro data point toward a possible involvement of
NFI. The first residue in the consensus is also protected by NFI (14, 43) and the third residue (G) is less affected by
methylation(9) ; also, the first G of the second half is
considered by some authors not to be necessary for interaction with NFI (e.g.(27) and (59) ). There are also
examples of other proteins that give similar but different footprint
patterns in vivo than in vitro(78) . For
these reasons, the in vivo pattern indeed may have been caused
by NFI protein species.
Binding of NFI, or one NFI isoform, to the
-globin promoter in HeLa cells would be consistent with a
repressor function in this system. This hypothesis is in agreement with
the results of Treisman et al.(79) , who reported a
(weak) expression of
-globin after transient transfection of
reporter plasmids into HeLa cells, which normally keep the endogenous
gene silent. In this study,
-globin (as opposed to
-globin)
expression was shown to be extremely sensitive to the copy number of
plasmids. This deregulated expression would be consistent with a
repressor function of NFI, since titrating out repressing NFI molecules
would allow escape synthesis of
-globin. NFI has been repeatedly
discussed as a repressor of transcription also in other systems (16,
59, 60, 62 and references cited in (5) and (49) -52). Particularly, from analyses of other globin
genes it has been speculated that displacement of positive factors by
NFI is a general globin gene regulatory mechanism (80 and references
therein; see also (22) ). Regardless of the identity of the
protein which interacts with the NFI site of the
-globin promoter,
occupation in the repressed state indicates that this site might be a
negative cis-acting element in the
-globin expression.
May we expect to see in vivo footprints of the -globin
NFI site at all? Making some plausible assumptions, (
)we
estimated that, theoretically, NFI in both cell lines should give
almost complete coverage of all sites, provided it were freely
diffusible and the DNA fully accessible. Therefore, we think that NFI
should have a real chance to bind to the
-globin promoter in the
nucleus yielding visible in vivo footprints. However, our
results suggest that the NFI site, at least in K562 cells, is not
bound. The presence of a factor in nuclear extracts contrasted by the
lack of in vivo binding to regulatory regions has been
reported for several genes, including the HLA-B7 transgene, the
endogenous H-2K
gene, the tyrosine aminotransferase gene,
MHC class II genes, and the muscle creatine kinase gene (83 and
references cited therein). In these examples, the reasons for
transcription factors being precluded from interaction with their
respective sites in the nucleus are not clear. Why should the NFI site
of the
-globin promoter not be recognized by NFI in vivo?
We can think of several reasons; for example, occupation of the site by
other factors or a specific orientation of the site toward a positioned
nucleosome (cf. (35) ), which renders it inaccesible
for NFI; lack of additional factors, which promote or stabilize NFI
binding to
-globin promoter; cytosine methylation inhibiting NFI
from binding(84, 85) ; elusive binding of NFI;
heterogeneous cellular distribution of NFI; active or passive
sequestration of NFI to particular regions of the nucleoplasm
(heterogeneous nuclear distribution; see (86) and (87) ); or modified binding characteristics of the
polypeptides. We could preclude heterogeneous cellular distribution of
NFI,
C methylation of the NFI site, and possibly elusive
binding of NFI as reasons for the lack of in vivo footprints
in K562 cells. Additionally, from the in vivo analysis we have
no indication of an inhibition of binding due to other factors or
nucleosomes. However, we do get preliminary evidence suggesting that
NFI polypeptides from the two cell lines, which differ in their
structure and composition, might also have different DNA binding
affinities. Binding experiments in vitro revealed that NFI
from K562 is apparently more insensitive toward homologous competition
than HeLa NFI (Fig. 5A). This unexpected binding
behavior may be explained by the assumption that large amounts of NFI
in K562 cells possess lower binding affinity compared to that of HeLa
cells, thus not forming visible protein
DNA complexes under the
applied conditions. Adding more DNA would then mobilize this
supplementary protein compensating apparently for homologous
competition. Studies are currently in progress to validate this
hypothesis.