©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Organization of the -Globin Promoter and Possible Role of Nuclear Factor I in an -Globin-inducible and in a Noninducible Cell Line (*)

(Received for publication, April 18, 1995; and in revised form, June 15, 1995)

Theo Rein Reinhold Förster Anja Krause Ernst-L. Winnacker Haralabos Zorbas (§)

From the Institut für Biochemie der Ludwig-Maximilians-Universität München, Würmtalstrasse 221, D-81375 München, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nuclear factor I (NFI) was suggested to be involved in the expression of the human alpha-globin gene. Two established cell lines, which express alpha-globin differentially, were therefore compared for differences in binding of NFI at the alpha-globin promoter in vivo. HeLa cells, in which alpha-globin is repressed, show a high density promoter occupation with several proteins associated with structurally distorted DNA. Cell line K562, which is inducible for alpha-globin, surprisingly was found to be heterogeneous consisting mainly of cells (95%) unable to express alpha-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 alpha-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 alpha-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 alpha-globin transcription in HeLa cells. However, NFI is apparently not involved in establishing the latent transcriptional state of the majority of K562 cells.


INTRODUCTION

Expression of alpha-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 alpha-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 alpha-globin gene contains no GATA-1 site but instead basal transcription elements, a possible SP1/alpha-IRP site(8) , and a binding site for nuclear factor I (NFI; (9) ). (^1)

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(6))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(p) 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 alpha-globin gene expression. NFI has been, in fact, implicated in the multistep process of transcriptional activation of the human alpha-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 alpha-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 alpha-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 alpha-globin promoter in the chromosomal context could be correlated with a particular transcriptional state of the alpha-globin gene. This would clarify a possible importance of this site in vivo and provide clues for an implication of NFI in alpha-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 alpha-globin promoter in vivo. Our data are also compatible with the hypothesis that NFI species found in HeLa cells could act as repressors of alpha-globin transcription.


MATERIALS AND METHODS

RNA Isolation and Slot Blots

K562 cells were purchased from ATCC (CCL 243); HeLa cells were a laboratory stock. Logarithmically growing K562 or HeLa cells suspended in RPMI 1640 medium supplemented with 10% fetal calf serum were harvested by centrifugation (1000 rpm, at 4 °C), washed in phosphate-buffered saline, and RNA was isolated according to a standard protocol(36, 37) . Hemin induction of K562 cells was done with a final concentration of 50 µg/ml for the times indicated in Fig. 1A. Slot blots were performed on a Schleicher & Schuell apparatus (SRC 07210 Minifold II). 30 µg of total RNA in 100 µl of TE were added to 300 µl of 6.15 M formaldehyde, 10 SSC, denatured for 15 min at 65 °C, and transferred on a nitrocellulose filter. The filter was baked at 80 °C for 2 h. Specific probes for the alpha-globin mRNA were oligonucleotides C and E of the first primer set also used in the ligation-mediated PCR. Glyceraldehyde-3-phosphate dehydrogenase specific probes were oligonucleotides 5`-CCAGTGAGCTTCCCGTTCAGCTC-3` and 5`-CCACCACCCTGTTGCTGTAGCC-3`. They were radioactively labeled at the 5` ends as described(9) . The sarcosyl technique was used for hybridization(38) .


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 alpha-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 alpha-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.''



Protein Extracts

Purification of baculovirus-expressed NFI (amino acids 1-257) from infected Spodoptera frugiperda (Sf9) cells is described in (9) . HeLa and K562 whole nuclear extracts were prepared as described (39) with the exception that 1 mM phenylmethylsulfonyl fluoride was added to all solutions. Protein concentration was determined by a standard method (40) and was between 2.5 and 4 µg/µl. Whole cell extracts from uninfected Sf9 cells were kindly provided by M. Stanglmaier.

Electrophoretic Mobility Shift Assay and Antibody Supershifting

Oligonucleotides were synthesized by G. Arnold (Laboratory for Molecular Biology-Genzentrum, Martinsried). Oligonucleotide ``alpha-G wt'' contains the NFI site in the context of the alpha-globin promoter(9) . Oligonucleotide L1/2 with a higher affinity NFI site has been described in (41) . Oligonucleotide ``alpha-G mut.'' in which the NFI site has been inactivated is the same as oligonucleotide ``k'' in (9) . Purification and radioactive labeling of the oligonucleotides and the conditions for protein-DNA incubation are described(9) . The amount of the labeled double-stranded oligonucleotide alpha-G wt was usually 5 fmol, whole nuclear extracts were 2-4 µl; cold competitors were added before the binding reaction in 100-fold molar excess. In supershift experiments, the binding reaction was on ice, then 1 µl of nonimmune or anti-NFI-antiserum (described in (42) ) was added, and incubation was continued for 15 min. Native polyacrylamide gel electrophoresis (acrylamide:bisacrylamide = 30:0.8) and autoradiography were as in (43) .

Genomic Sequencing and in Vivo Footprinting

Conditions for base-specific modification in vitro and piperidine cleavage were as described(44) . For in vivo footprinting with dimethyl sulfate (DMS), K562 or HeLa cells were grown as described above. alpha-Globin induction of the K562 cells was with 50 µg/ml hemin for 24 h. Treatment with alpha-amanitin was at a final concentration of 10 µg/ml for 1 h. The cells were washed with an isotonic phosphate buffer and incubated at 3 10^7 cells per ml in RPMI containing 0.2% DMS (Merck). The reaction was at room temperature for 2 min and was stopped by adding to the cells 40 volumes of cold phosphate buffer with 2% beta-mercaptoethanol and subsequently by removing the medium by centrifugation. Cells remain viable after this treatment as controlled by trypan blue exclusion. The DNA was extracted by a standard protocol and cleaved at modified residues with piperidine. To visualize the DNA sequence, the ligation-mediated PCR method was used essentially as described(45) . For the extension step with the radioactive primer E, four PCR cycles were performed. The primer set used for analyzing the sense-strand was: primer C, 5`-CAGGAGACAGCACCATGGTGGGTTC-3`; primer D, 5`-GGTGGGTTCTCTCTGAGTCTGTGGG-3`; and primer E, 5`-AGTCTGTGGGGACCAGAAGAGTGCC-3`. The in vivo footprinting experiments were repeated several times; the results are reproducible in the sense that the patterns are exactly the same whenever the same in vivo methylated DNA batch was analyzed and similar, but never contradictory, in the variation of band intensities, when a different DNA batch or a different set of primers was used.

Flow Cytometry

Intracellular expression levels of alpha-globin and NFI were determined by the use of specific antibodies and flow cytometry (FACS) as described previously(46) . Briefly, K562 cells and HeLa cells were washed twice in phosphate-buffered saline, fixed with 2.5% paraformaldehyde (10 min, 4 °C), and permeabilized for 4 min at room temperature with 0.0025% digitonin (Sigma) in order to allow intracellular antibody binding. Cells were adjusted to 2 10^6 per ml in staining buffer (phosphate-buffered saline, 4% fetal calf serum, 5 mM EDTA, 0.1% NaN(3)) and were incubated for 20 min at 12 °C with a rabbit anti-alpha-globin immune serum (Sigma; final dilution: 1:3000) or with protein A-purified rabbit anti-NFI polyclonal antibodies (100 µg/ml)(42) . After 2 washes in staining buffer, cells were incubated with F(ab`)(2) fluorescein isothiocyanate-conjugated anti-rabbit IgG and IgM antibodies (1:160, Dianova, Hamburg, FRG). Cells were washed twice, counterstained with propidium iodine (5 µg/ml in 4 mM sodium citrate, 0.1% Triton X-100, pH 7.0) and analyzed by flow cytometry (Becton Dickinson, Heidelberg, FRG).


RESULTS

The alpha-Globin-inducible Cell Line, K562, Is Heterogeneous with Respect to the alpha-Globin Gene Expression

In order to define a system for the study of alpha-globin expression, we used K562 cells, a commonly used erythroleukemic cell line, for the investigation of expression of the globin genes (cf. for example, (47) ). K562 cells can be stimulated to actively transcribe the alpha-globin gene after hemin induction for different periods of time (Fig. 1A). There is a clear difference in the amount of alpha-globin mRNA in K562 cells compared to the non-alpha-globin expressing HeLa control cells (at least 12.5-fold more alpha-globin mRNA at 72 h). However, in the K562 cell population, alpha-globin mRNA already displays a high uninduced level (0 h) and is induced by a comparatively very low factor of only about 2.5-fold after 72 h. We wondered whether this reflects a uniformly low level of induction of all or most of the K562 cells, or rather a heterogeneous composition of this cell line, with some cells expressing high levels of alpha-globin and others expressing low levels or no alpha-globin. We therefore determined alpha-globin expression in K562 cells by FACS analysis using specific anti-alpha-globin antibodies; we indeed detected different subpopulations (Fig. 1B). In the uninduced state, the great majority of cells essentially does not express alpha-globin, whereas a few cells show comparatively high expression; the latter may be the reason for alpha-globin mRNA being already detectable before induction (see Fig. 1A). After hemin stimulation, the fraction of cells expressing alpha-globin increases, but does not exceed 5% of the overall population. This means that a small number of cells are actually responsible for virtually all of the alpha-globin expression in this cell line. As expected, no HeLa cell expresses alpha-globin detectably (Fig. 1B), which parallels the RNA analysis (see above). Accepting that protein levels directly mirror ongoing alpha-globin mRNA synthesis, these results mean that most of the K562 cells do not actively transcribe the alpha-globin gene, i.e. their state of alpha-globin expression is equivalent to that of HeLa cells.

The General alpha-Globin Promoter Configuration in Vivo Differs between K562 and HeLa Cells

Since HeLa cells could not be induced to express alpha-globin (data not shown), the condition, in which promoter elements of the alpha-globin gene in these cells are, may reflect the dormant, or fully repressed state. On the other hand, the few K562 cells, which are transcribing alpha-globin and can even be stimulated (e.g. by hemin; this work), define the active state of the alpha-globin gene. We wondered whether the alpha-globin promoter, in the great majority of K562 cells which are silent, is also in a repressed state as in HeLa cells. To compare the promoter structure of alpha-globin in the two cell lines, we performed in vivo dimethyl sulfate (DMS) footprinting analysis. Typical results are shown in Fig. 2, A and B; all data are summarized in Fig. 3. (^2)To properly interpret the results, we consider band intensities to be altered in vivo only when they are flanked by any two guanosines, the intensity of which is not altered compared to the in vitro signals.


Figure 2: In vivo footprinting (A and B) and genomic sequencing (C) of the alpha-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, alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-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.

Occupation of the NFI Site in the alpha-Globin Promoter in Vivo Differs between K562 and HeLa Cells

The performed in vivo footprint analysis provides the opportunity to examine protein interactions at the NFI site of the alpha-globin promoter in the chromosomal context of the two cell lines (cf. introduction). In vivo footprints with K562 cells revealed no stable protein occupation of the NFI site (Fig. 2, lane 5), in spite of the fact that the DNA region does not seem to be particularly inaccessible due to tight protein packaging (cf. instead the corresponding region in HeLa cells; previous section). Since clear protection footprints are obtained only if a sufficiently high portion of the DNA site in question is stably occupied, one reason for the lack of NFI footprints in K562 cells could be a low rate or a merely transient protein binding to its site. alpha-Amanitin has been used to visualize binding of RNA polymerase II in vivo by trapping the enzyme at the promoter(49) . In an attempt to enhance a hypothetical insufficient factor binding at the alpha-globin promoter by the same rationale, we therefore performed in vivo footprints after treatment of induced K562 cells with alpha-amanitin. Again, no occupation of the NFI site was detectable (data not shown). Thus, by the methods used, the NFI site within the alpha-globin promoter does not become bound in K562 cells.

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(6))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''). (^3)Therefore, it is not possible to diagnose unambiguously whether protection of the NFI site of the alpha-globin promoter in HeLa cells is due to NFI binding. Nevertheless, it is clear that the NFI site present at the alpha-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.

All CpG Dinucleotides in the alpha-Globin Promoter in Both Cell Lines Are Unmethylated

Binding of several eukaryotic proteins to DNA is known to depend on the methylation state of cytosines. Most proteins are inhibited from binding when cytosines are methylated (at so called CpG or HTF islands, (50) ), but some require methylated cytosines in order to bind(51, 52) . Therefore, the distinct footprinting pattern of the alpha-globin promoter could be, at least in part, due to cell line specific methylation of this region of the genomic DNA. In particular, differential CpG methylation might have been a cause for lack of binding of NFI to the NFI site in K562 cells. The HTF islands of the alpha1- and alpha2-globin gene loci have been investigated in several cell types and tissues(53, 54, 55) . In fact, they have been shown to be unmethylated in K562 cells(55) , whereas the same loci appear heavily methylated in HeLa cells(53, 54) . However, in HeLa cells also, unmethylated sites are apparent at the promoter region, just in front of the alpha-globin genes(53, 54) . These investigations which are based on the use of methylation-sensitive restriction enzymes only detect part of the CpG dinucleotides. Therefore, to investigate a possible effect of DNA methylation on the protein binding at this region, we performed direct genomic sequencing of the alpha-globin promoter of both cell lines. We found that all cytosines of K562 DNA, and hence all 12 CpG pairs from -90 to -1, are not methylated (Fig. 2C, lane 7). (Again, these results mirror the methylation state of the majority of the K562 population; cf. above.) The same cytosine residues are also not methylated in HeLa cells (Fig. 2C, lane 8). Thus, both non-alpha-globin-expressing cell populations have equivalent cytosine methylation patterns. From these results we conclude, that, firstly, cytosine methylation cannot be the reason for differential protein associations of the alpha-globin promoter of the two cell lines. Secondly, methylation-free CpG islands may be necessary but not sufficient for activation of the alpha-globin promoter. Thirdly, these observations exclude inhibition by methylation as a possible reason for lack of NFI binding to the alpha-globin promoter in K562 cells.

NFI Is Present and Evenly Distributed in All HeLa and K562 Cells

NFI was described as ubiquitous protein in mammalian cells (25 and references therein). Previous Northern blot analysis of HeLa and K562 cells has shown in particular that both cell lines contain at least two prominent NFI mRNAs with the same length (8.6 and 4.5 kilobases) and same relative ratio(28, 56) . However, those types of analysis are not able to trace NFI in individual cells. Therefore, we wondered whether the absence of an NFI footprint at the alpha-globin promoter of K562 cells could be attributed to a heterogeneous distribution of NFI proteins and thus to their absence from the majority of the cells, as was detected with alpha-globin (see above). We approached this question by FACS analysis using antibodies raised against recombinant, baculovirus-expressed NFI, consisting of amino acids 1-257(42) . The results in Fig. 4demonstrate that NFI is present and expressed in all K562 and HeLa cells. Therefore, mechanisms other than heterogeneous expression seem to be responsible for the lack of NFI binding in vivo at this site in K562 cells.


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.''



K562 and HeLa Cells Contain Different Forms of NFI

NFI is expressed by several mechanisms as a family of homologous polypeptides (cf. introduction) which may exert distinct functions in the cell(24, 25, 26) . Thus, differences in the protein binding pattern at the NFI site in vivo might be attributable to differences in the NFI populations. In order to assay different NFI forms between the two cell lines, we performed electrophoretic mobility shift assays of whole nuclear extracts of K562 and HeLa cells (Fig. 5A). Comparable protein amounts of these extracts were allowed to bind the target alpha-G wt DNA (provided in excess) which contains the NFI site in the context of the alpha-globin promoter. A recombinant NFI fragment with an apparent molecular mass of 35 kDa (cf. above and (9) ) encompassing the DNA binding domain was used in a control reaction (^4)(Fig. 5A, lane 2). Both cell extracts gave rise to proteinbulletDNA complexes (Fig. 5A, lanes 6 and 9). However, the two cell types differ in the number of the DNA binding species: HeLa cells contain several (6 or 7) distinct species that form diverse complexes with DNA (Fig. 5A, lane 6). This is in agreement with earlier reports by us (9) and others(24, 25, 32) , describing multiple DNA binding forms of NFI in HeLa cells. In contrast, extracts from K562 cells form only 2-3 distinct bands in this assay, a strong one and one or two rather faint bands, with lower and higher mobilities (Fig. 5A, lane 10). Moreover, no two distinct bands of the HeLa and of the K562 extract accurately comigrate with each other. We may infer, therefore, that the alpha-G wt-DNA binding activities in these two cell types also differ in molecular shape and/or charge. (^5)Differences in conformation, or subunit composition, were further substantiated by proteolytic clipping with trypsin (data not shown).


Figure 5: A, binding and competition analysis of NFIbulletDNA 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 alpha-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 NFIbulletDNA 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 alpha-G mut.), in which the NFI site of alpha-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 alpha-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 NFIbulletDNA complex running a short distance into the gel (Fig. 5B, lane 6). The complexes formed by HeLa and K562 nuclear extracts with alpha-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 NFIbulletDNA 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.


DISCUSSION

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 alpha-globin transcription(28, 32, 33) . However, the involvement of NFI in alpha-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 alpha-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 alpha-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 alpha-globin, is consistent with the relatively weak RNA synthesis and inducibility found here and by others(65, 66) , with the low level of alpha-globin proteins (67) and with the reduced DNase I hypersensitivity in the alpha-globin promoter compared to other genes in the alpha-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 alpha-globin detectably, they differ profoundly in their respective alpha-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 alpha-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 alpha-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 alpha-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 alpha-globin promoter of the majority of K562 cells may define a distinct stage of alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-globin-inducible population. However, degeneration to a heterogeneous population with respect to alpha-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 alpha-globin transcription in vivo, we may conclude that NFI is apparently not implicated in the preactivation phase of cellular alpha-globin expression detected here. In this instance, it is interesting to note that in an investigation with the alpha-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 alpha-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 alpha-globin reproducibly show a clear occupation of the NFI site (contacts at the underlined Gs in GGG(N(6))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(6))GCCAA; (14) ).^3 When methylated in vitro, the underlined residues in GGG(N(6))GCCAG interfere with binding in the particular NFI site of the alpha-globin promoter (see Fig. 3and (9) ). To our knowledge, no other DMS footprint of NFI in vivo and no DMS protection footprints of the alpha-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 alpha-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 alpha-globin after transient transfection of reporter plasmids into HeLa cells, which normally keep the endogenous gene silent. In this study, alpha-globin (as opposed to beta-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 alpha-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 alpha-globin promoter, occupation in the repressed state indicates that this site might be a negative cis-acting element in the alpha-globin expression.

May we expect to see in vivo footprints of the alpha-globin NFI site at all? Making some plausible assumptions, (^6)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 alpha-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^b 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 alpha-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 alpha-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 proteinbulletDNA 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.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant Zo 59/2-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 49-89-74017-450; Fax: 49-89-74017-448; zorbas{at}lmb.uni-muenchen.de.

(^1)
The abbreviations used are: NFI, nuclear factor I; DMS, dimethyl sulfate; PCR, polymerase chain reaction; FACS, fluorescence-activated cell sorter.

(^2)
The displayed in vivo DMS protection results with K562 cells were obtained after 24 h of hemin induction. However, equivalent results were obtained with uninduced cells (data not shown). This is not surprising, since the footprints of the K562 genomic DNA are representative for the bulk of the cells (about 95%) which do not express alpha-globin in either case.

(^3)
T. Rein, R. Förster, A. Krause, E.-L. Winnacker, and H. Zorbas, unpublished results.

(^4)
The recombinant NFI used in this study had been used previously to characterize the binding behavior of NFI to the NFI consensus sequence, as well as to this particular NFI site found in the alpha-globin promoter (9). It was expressed from a baculovirus-based vector in Sf9 cells; it consists of amino acids 1-257 (about 35 kDa) and is thus a truncated version of a full-length polypeptide (reported molecular masses of functional NFI species 52-66 kDa, see (32) ). This recombinant NFI encompasses the DNA binding and dimerization domain, which is highly homologous between different NFI species, and which confers all DNA binding characteristics. In in vitro binding experiments and footprints, it interacts with the NFI binding site in an indistinguishable manner when compared to full-length polypeptides (9).

(^5)
Significant proteolytic degradation of the extracts which would obscure the interpretation of the above results was checked and excluded by electrophoretic and Western blot analyses (data not shown).

(^6)
The number of binding-active NFI molecules per HeLa nucleus is 10^4 to 10^5 (own calculations based on quantitative electrophoretic mobility shift assay and footprinting experiments), and the number of all canonical NFI sites per diploid nucleus was estimated to be approximately 1 out of every 100 kilobases (81), i.e. in total about 10^4 per genome. This corresponds to 100 accessible sites, assuming that about 1% thereof resides in open chromatin (4). Allowing for mutations at the two positions of the NFI site found in the alpha-globin promoter reading 5`-GGG(N)(6)GCCAG-3`, which differ from the canonical sequence, there may be approximately 1600 NFI-like sites in the same nucleus, with which these NFI molecules may interact with comparable affinity. Thus, there is numerically sufficient NFI that in principle could interact with them to give extensive coverage and thus clear protections (protein to DNA ratio = 6:1 to 60:1). To which extent this happens depends on the NFI affinity to these sites and on the actual concentrations of the reaction partners. The affinity of NFI for the site in the alpha-globin promoter was previously determined quantitatively (41) and was found to be 2 10^8M, i.e. 50-fold weaker than that of the consensus. Thus, NFI interacts with lower affinity with the NFI site of the alpha-globin promoter than with the canonical NFI site. However, its affinity to the alpha-globin site approximates, for example, that of the trp repressor (82), a specifically DNA-binding protein. By using the above estimates and equilibrium binding constant, we obtain then for an average nucleus of 10 µl volume, a concentration of NFI-like sites of 27.2 nM and an NFI concentration of 0.17 µM, yielding 96.6% coverage of all sites, or an NFI concentration of 1.7 µM, yielding 99.7% coverage of all sites.


ACKNOWLEDGEMENTS

Oligonucleotides were synthesized by G. Arnold (Laboratory for Molecular Biology-Genzentrum, Martinsried). Whole cell extracts from uninfected Sf9 cells were kindly provided by M. Stanglmaier (Institute for Biochemistry, Martinsried). We are indebted to H. Ibelgaufts and M. Müller for critical reading of the manuscript and valuable comments and suggestions.


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