Human Molecular Genetics

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Human Molecular Genetics, 2000, Vol. 9, No. 4 631-636
© 2000 Oxford University Press

ß-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice

Raouf Alami, John M. Greally¹, Keiji Tanimoto², Steven Hwang¹, Yong-Qing Feng, James D. Engel², Steven Fiering³ and Eric E. Bouhassira⁺

Division of Hematology, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA ¹Department of Genetics, Yale University School of Medicine, New Haven, CT, USA ²Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL and ³Microbiology Department, Dartmouth Medical School, Hanover, NH, USA

Received 12 November 1999; Revised and Accepted 7 January 2000.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human ß-globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter­dependent mechanisms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Expression of genes taken out of their natural context and introduced at ectopic chromosomal sites often differs from expression at the native locus either because the gene is silenced in a fraction of the cells (variegating position effects) or because the level of expression is lower in all cells (stable position effects). Position effects have been observed in many organisms including yeast, Drosophila and mouse (1–8), and are believed to be the consequence of differences in epigenetic organization of the transgene at different integration sites. Typically, larger transgenes are less subject to position effects than smaller ones, presumably because the inclusion of more native regulatory elements makes the transgene more likely to establish its normal epigenetic organization.

A great deal of historical evidence has shown that small constructs containing human ß-like globin gene and their proximal regulatory sequences integrated at ectopic loci in the mouse genome are expressed at levels that are generally very low and that depend greatly on the site of integration (9,10). In contrast, inclusion in the construct of the ß-globin locus control region (LCR) [a group of five DNase I hypersensitive sites (HSSs) located 15–25 kb upstream of the -globin gene (Fig. 1)] results in expression levels that are markedly increased, independent of the site of integration and dependent on the number of integrated copies (11). It is therefore well established that LCRs can substantially suppress position effects in general (12–14). These transgenic mouse experiments have led to the definition of an LCR as a cis-acting element required for transgene expression that is approximately proportional to the number of integrated copies in each line of transgenic mice. However, this operational definition does not address whether an LCR acts by overcoming stable position effects, overcoming variegating position effects or a combination of both. This distinction can only be addressed by experiments that assay expression in individual cells rather than the more common assays on bulk cell populations. Two recent experiments have examined the ability of mutated LCRs to conform to the above definition of an LCR. The studies concurred in their findings: both the ß-globin and the CD2 LCRs appeared to be fully suppressive of position effects unless the LCRs were mutated and the transgene was located in pericentromeric heterochromatin (15,16). These findings have added further defining char- acteristics to those previously attributed to LCRs.

View larger version (50K): [in this window] [in a new window]   Figure 1. Position effects in mice transgenic for human ß-globin YACs. (a) Expression analysis in mice transgenic for normal YACs; the YACs present in four transgenic lines (31-wt, 42-wt, 277-wt, 264-wt) are represented. The red arrows represent the five HSSs making up the LCR, the open boxes the five human ß-like globin genes. In all four lines the YACs were identical except for the presence of loxP sites (represented by black triangles) in opposite orientation flanking either the genes (31-wt and 42-wt) or the LCR (277-wt and 264-wt). The loxP sites were required for in vivo manipulation of the transgene (see text). Expression was analyzed at the single-cell level by FACS after staining of peripheral red blood cells with FITC-labeled anti-human ßA antibodies, and in cell pools by HPLC analysis of red blood cell lysates. The histograms illustrate the FACS results (x-axis, intensity of green fluorescence; y-axis, number of cells). The peak on the left represents red cells that do not express the human ß-globin transgene, the peak on the right red cells that do. The chromatograms illustrate the HPLC analysis (x-axis, time of elution; y-axis, OD at 220 nm). The results are summarized in the bar graph at the bottom. Yellow bars, percentage of human ß-globin chains relative to the mouse endogenous chains in the hemolysate; y-axis, percentage ß = [100 x ß/(ß + ßmin + ßmaj)]. Black bars, percentage ß per FACS-positive red cells; y-axis, percentage ß per fraction of FACS-positive cells. This study demonstrates that the 150 kb YACs are subject to variegating but not stable position effects (see text). (b) Chromosomal localization of the YAC transgenes: The integration sites of the YACs were determined by four-color FISH. The chromosomes were counterstained with DAPI (blue pseudocolor), the centromeres were identified with a mouse centromere-specific (minor satellite) probe detected with CY3 (red pseudocolor), the chromosomes were identified with chromosome-specific probes detected with CY3.5 (yellow pseudocolor) and the YAC transgenes with a BAC probe detected with FITC (green pseudocolor). In all four lines the transgenes are far from the centromere. (c) Expression analysis in mice transgenic for YACs with either an inverted LCR or inverted genes; the structure of the YACs after Cre-mediated in vivo manipulation is shown. Expression was analyzed as in (a). These experiments demonstrate that gene inversion eliminates the variegating position effects whereas LCR inversion creates a decrease in expression in line 264-inv. In line 277, LCR inversion potentiates the variegating position effect and likewise reduces the level of expression per cell.

 
To determine whether the LCR is equally dominant over all types of position effect, globin gene expression was analyzed in mice transgenic for a 150 kb yeast artificial chromosome (YAC) containing the human ß-like globin genes and the LCR in their normal configuration (17). In order to detect heterocellular position effects, the proportion of erythrocytes expressing these transgenes was measured by flow cytometry after staining with fluoroscein isothiocyanate (FITC)-labeled anti-human adult ß-globin monoclonal antibodies (18). To detect pancellular position effects, the level of expression of the ß-globin gene was quantified relative to the endogenous mouse ß-type globin genes in cell pools using an HPLC assay of ß-globin chains (19). This revealed that single-copy YAC transgenes containing an unmodified human ß-globin locus are subject to variegating but not stable position effects at three of the four integration sites tested. In vivo manipulations of the transgene indicated that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhance- ment. DNase I HSS studies suggest that variegated position effects but not reduction in the level of expression within each expressing cell are associated with lack of LCR formation.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ß-globin YAC transgenes including a normal LCR are often subject to variegating position effects
Four lines of mice (20) carrying a 150 kb human ß-globin YAC transgene (21) were analyzed. Each of these lines had previously been shown to carry a unique, intact copy of the YAC using both quantitative and qualitative methods that relied on detection by Southern blotting of either the arms of the YAC (end-fragment assay) or of 100 and 10 kb SfiI fragments encompassing the entire ß-globin locus (20). Flow cytometric analysis of human ß-globin expression was performed on peripheral blood from three or more 3-month-old mice from each of the lines (31-wt, 42-wt, 277-wt and 264-wt). This revealed that 95.6 (± 0.5), 89.0 (± 3.0), 41.5 (± 1.5) and 99.5 (± 0.4)%, respectively, of the red cells expressed the transgene (Fig. 1a). Thus, at three of the four integration sites, the 150 kb YAC was silenced in a fraction of the erythroid precursor or progenitor cells. Analysis of at least three 10-day-old mice from each line by the same method showed the same proportions of expressing cells (data not shown), indicating that the pattern of silencing did not vary with age. Student’s t-tests revealed statistically significant differences (P < 0.001) between the proportion of expressing cells in the variegating lines compared with the non-variegating 264-wt line.

Variegating position effects are not caused by pericentromeric integration
To ascertain whether the position effects that we observed were associated with pericentromeric integration, we performed fluorescence in situ hybridization (FISH) on metaphase chromosome preparations from these transgenic mice. We used a human ß-globin BAC probe, a mouse centromere-specific (minor satellite) probe, a mouse L1 banding probe (22), mouse chromosome-specific probes (23) and DAPI counterstaining. These experiments showed that none of the transgenes had integrated close to a centromere (Fig. 1b). These combined results demonstrate that the complete ß-globin locus, including the unmodified LCR, can be subject to variegating position effects even when integrated far from the particularly repressive chromatin at the centromere (15,16). Since three of the four YACs tested exhibited position effect variegation, these repressive sites must be quite frequent.

The level of expression of the transgene in expressing cells is uniform at all integration sites
Quantification of human and mouse globin chains by HPLC showed variability of transgene expression levels between lines (Fig. 1a). However, after normalization to the proportion of expressing cells, the ratios of human ß chains relative to the mouse ß-globin minor and major chains (which is an approximation of the level of human ß-globin expression within each expressing cell) were statistically the same at all four integration sites and ranged from 15.2 ± 1.5 to 17.1 ± 2.5 (Fig. 1a, bottom). A comparable result was obtained by normalizing the previously reported transgene mRNA levels (20) to the proportion of expressing cells. We therefore conclude that within the context of a large transgene that includes the entire locus, the ß-globin promoter is not subject to detectable stable position effects.

Gene inversion eliminates variegating position effects
The YAC transgenes in these four lines contain loxP sites in opposite orientations flanking either the LCR (lines 31-wt and 42-wt) or the ß-globin genes (lines 264-wt and 277-wt). Expression of Cre recombinase in fertilized oocytes from these lines produced mice with inversions of these DNA segments (20). As these modified and unmodified transgenes are integrated at the same site, the effects of manipulations can be dissociated from variability due to position effects. Specific effects of these transgene manipulations on the developmental regulation of the locus have previously been reported based on quantification of mRNA in cell pools (20). We report on the effect of these manipulations at the single-cell level.

Fluorescence-activated cell sorter (FACS) analysis of three or more of these mice showed that inversion of the ß-like globin genes (lines 31-inv and 42-inv) caused >99.5% of the red cells to express the transgene (Fig. 1c); inversion of the genes relative to the LCR therefore eliminates variegating position effects. HPLC analysis revealed that inversion of the genes did not significantly change expression levels per expressing cell (Fig. 1c).

LCR inversion decreases gene expression levels and can increase variegating position effects
Inversion of the LCR also revealed striking effects on single-cell expression patterns. In the original 264 line (264-wt), there was no evidence for variegating position effects. With inversion of the LCR (line 264-inv), there were still no variegating position effects but the level of ß-globin expression decreased from 16.1 (± 0.3) to 6.7 (± 0.2)% of the endogenous mouse ß-globin genes, a change of statistical significance (Student’s t-test, P < 0.01). The peaks observed after FACS analysis were reproducibly broader than those of the parental mice, suggesting that the amount of globin within expressing cells is more variable after LCR inversion (Fig. 1c). In line 277-inv, inversion of the LCR likewise led to a statistically significant decrease in the level of ß-globin expression per expressing cell from 15.2 (± 1.5) to <5.1 (± 0.2)%. In addition, the proportion of expressing cells was decreased from 41.5 to 14.0%. Inversion of the LCR therefore appears to attenuate its characteristically strong transcriptional enhancement and to accentuate pre-existing variegating position effects (Fig. 1c).

Variegating position effects are associated with loss of DNase I sensitivity at the LCR
We then analyzed DNase I HSS formation at the LCR in spleen erythroblast nuclei obtained from lines 264-wt, 264-inv, 277-wt and 277-inv. In the 264-wt and 264-inv lines, the HSSs of the LCR were clearly and equally formed (Fig. 2a and b). The presence of the HSSs therefore correlates with the proportion of expressing cells within the whole population (99.5% in both) rather than with the level of expression per positive cell (16.1 and 6.7% of endogenous mouse ß-globins, respectively). In line 277-wt, the HSSs were weaker than in line 264-wt, correlating with the lower proportion of cells (41.5%) expressing the transgene; in line 277-inv, in which even fewer cells express the transgene (14.0%), the HSSs were weaker still (Fig. 2c).

View larger version (45K): [in this window] [in a new window]   Figure 2. DNase I hypersensitive site mapping. (a) Schematic representation of the 10.4 kb EcoRI fragment of the LCR used to detect the HSSs. The location of HSSs 2, 3 and 4, the probe used to detect the fragments (hatched box) and the predicted fragments after DNase I digestion are shown. (b and c) The markers shown on the left depict the positions of HSSs within the human LCR (HS4, 2, 3 and 3/4) and within the mouse ßmaj globin gene promoter. (b) 264-wt and 264-inv transgenic mice; (c) 277-wt and 277-inv transgenic mice. The HSSs form equally in 264-wt and 264-inv but are weak in line 277-wt and even weaker in 277-inv.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Although the striking positive effects of the ß-globin LCR on gene expression have long been recognized, the influence of integration sites on transgenes containing intact LCRs is not well understood. Using our single-cell analytical approach, we have dissected the different effects that the site of integration can have on YAC transgenes that include all the regions believed to be important for regulating the locus. The variegation we document is reproducible, statistically significant and occurs at three of the four non-centromeric integration sites studied, indicating that non-centromeric regions causing variegation of large transgenes containing powerful local regulatory elements may be frequent in the mammalian genome.

The clear demonstration that the LCR does not always prevent variegating position effects, and the marked changes in levels of expression per cell associated with specific alterations of the LCR, suggest that at ectopic chromosomal sites, the principal effect of the LCR is to provide strong transcriptional enhancement rather than to dominantly overcome local epigenetic organization. This is consistent with the previous report in which deletion of the LCR from the endogenous mouse ß-globin locus did not affect chromatin accessibility to DNase I, and the ß-like globin genes continued to be expressed at low levels (24). The observation that inversion of the genes relative to the LCR increases the amount of ß-globin expression is consistent with prior data showing that relative proximity to the LCR greatly increases gene expression (25–27). We show that this is mediated by suppression of variegation in these transgenes, not by changes in the level of expression per cell.

The effects of LCR inversion are complex. The reduced amount of human ß-globin in the red cells of the mice with inversion of the LCR could be caused either by a reduction in the rate of transcription during the entire erythroid differentiation period, or by premature silencing during terminal erythroid differentiation. In line 264, the DNase I HSSs formed equally whether the LCR was inverted or not. Since these studies were performed on pools of red cell precursors, this observation supports the hypothesis that reduced expression occurs throughout the erythroid differentiation and maturation period. However, the broader than normal peaks that we observed in the LCR inversion lines raises the possibility that premature silencing might also be occurring very late in differentiation.

In line 277, the intensity of the HSSs correlates with the proportion of expressing cells and therefore with transcription at the ß-globin promoter. Although we cannot exclude the possibility that the cause of the low intensity of HSSs in these lines is dynamic, transient or imperfect HSS formation in all cells, these results suggest that variegating position effects in these lines are associated with the complete absence of LCR formation in a fraction of the cells. As in line 264, the production of human globin chains in the expressing red cells of line 277 is lower after the LCR inversion. Therefore, LCR inversion can affect both the level and the probability of ß-globin gene expression. Combined with the demonstration that proximity to the LCR reduces position effect, this indicates that transcriptional enhancement and protection against variegating position effects are interdependent in a complex manner (28). This might explain the reports that in smaller constructs, the LCR appeared to have a specialized role in preventing position effects (11).

The unexpected susceptibility of the unaltered ß-globin locus to variegating position effects at most sites and the dramatic alterations in expression patterns in adult cells associated with simple modifications of the locus probably reflect the fact that the locus has evolved to function in its endogenous site and not as a transgene. Critical determinants of gene expression at ectopic chromosomal sites therefore remain to be discovered.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Flow cytometry
Detection of ß-globin in red cells was performed as previously described (18). Briefly, peripheral red blood cells were fixed by incubation in 4% formaldehyde/phosphate-buffered saline (PBS) for 1 h at room temperature followed by addition of 0.25 vol of 0.05% glutaraldehyde/PBS and gentle shaking for 2 min. The cells were then washed twice with PBS, blocked by shaking for 10 min at room temperature in 5% non-fat dry milk/PBS, rinsed in PBS, and permeabilized in a 0.01% Triton, 0.1% bovine serum albumin solution in PBS. Cells (100 000) were then stained by incubation with 3–5 µg of FITC-labeled anti-human ß-globin monoclonal antibodies and analyzed on a FACSCAN analyzer.

HPLC analyses were as previously described (19) using a linear acetonitrile gradient ranging from 35 to 55%.

DNase I sensitivity assays
These studies were performed as previously described (29). Nuclei were isolated from anemic spleen cells from 1-month-old mice and digested with increasing concentrations of DNase I. Genomic DNA was isolated, digested with EcoRI and separated by electrophoresis on a 1% agarose gel. After transfer to a nylon membrane, the blots were hybridized to a ³²P-labeled probe from the 3' flanking region of HS4 (Fig. 2b and c, top in each). After stripping the LCR probe, the blots were re-hybridized to a unique sequence probe that detects the mouse ß major globin gene promoter HSS as an internal control (Fig. 2b and c, bottom in each).

FISH analysis
Mouse splenocyte culture and FISH were performed as previously described (22). A bacterial artificial chromosome (kindly provided by Dr T.J. Ley, Washington University Medical School, St Louis, MO) spanning the human ß-globin locus was labeled with digoxigenin, the mouse minor satellite repeat with CY3 and either a mouse L1 probe (22) or chromosome-specific probes (23) were labeled with biotin. Detection of probes was with antidigoxigenin–FITC and avidin–CY3.5. Once DAPI and L1 banding had indicated the likely chromosome of integration of the transgene (data not shown), this was subsequently confirmed by probing with chromosome-specific clones. Digital grayscale images for each fluorochrome were collected and merged to generate a final pseudocolored image using Adobe Photoshop.

	ACKNOWLEDGEMENTS

 
This work was supported by grants NIH HL38655 and HL- 554350 (E.E.B.), NIH DK 02467 (J.M.G.), NIH HL24415 (J.D.E. and K.T.), and a Burroughs-Wellcome Career Development Award (S.F.). R.A. is supported by grant NIH HL07556.

	FOOTNOTES

 
⁺ To whom correspondence should be addressed. Tel: +1 718 430 2188; Fax: +1 718 824 3153; Email: bouhassi@aecom.yu.edu

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