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Human Molecular Genetics Advance Access originally published online on September 9, 2005
Human Molecular Genetics 2005 14(20):3047-3056; doi:10.1093/hmg/ddi337
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Juxtaposition of the HPFH2 enhancer is not sufficient to reactivate the {gamma}-globin gene in adult erythropoiesis

Ping Xiang, Hemei Han, Grainne Barkess, Ivan Olave, Xiangdong Fang, Wenxuan Yin, George Stamatoyannopoulos and Qiliang Li*

Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA

* To whom correspondence should be addressed at: Medical Genetics, University of Washington, PO Box 357720, Seattle, WA 98195, USA. Tel: +1 2066164526; Fax: +1 2066164527; Email: li111640{at}u.washington.edu

Received April 30, 2005; Revised August 3, 2005; Accepted September 6, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have suggested that juxtaposition of a downstream enhancer to the fetal {gamma}-globin gene results in reactivation of the {gamma}-gene in adult erythrocytes of individuals with hereditary persistence of fetal hemoglobin (HPFH). To test the hypothesis in a much stricter basis, we produced ß locus YAC transgenic mice carrying an exact ß locus replicate of a deletional HPFH mutation, HPFH 2. Although the {gamma}-globin gene was expressed in the HPFH 2/ß locus YAC (HPFH2/YAC) transgenic mice in the early stage of development, it was completely silenced in the adult mice. The failure of {gamma}-gene reactivation by the juxtaposed HPFH2 enhancer contradicts the results of previous studies. We speculate that the discrepant results reflect differences in the distance between the locus of region (LCR) and the {gamma}-globin gene characteristic of the plasmid, cosmid or YAC constructs used for production of transgenic mice. The difference in the phenotype of the HPFH2/YAC transgenic mice and the humans with HPFH2 mutation suggests that in addition to juxtaposition of HPFH enhancers, the upstream region that is absent in the ß-YAC construct might be involved in {gamma}-gene reactivation in HPFH individuals. The DNase I hypersensitive sites of the LCR were well formed and the chromatin histones were acetylated. A moderate level of pol II binding was detected in the LCR, despite the fact that no transcription occurred in the globin-genes of the adult HPFH2/YAC transgenic mice. The results suggest that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human ß-globin cluster is situated on chromosome 11, and it contains five functional genes, {epsilon}, G{gamma}, A{gamma}, {delta} and ß, which are arranged in accordance with the developmental timing of their expression during ontogeny. The {gamma}-globin gene is normally expressed in the fetal stage of development and it is switched off in adult life. However, expression of the {gamma}-gene is not completely suppressed. A trace amount of {gamma}-globin chain can be detected in adult blood in healthy individuals; it accounts for about 1% of total globin chains and it is restricted to few erythrocytes, called F cells. {gamma}-gene expression can be stimulated in adult individuals by physiological or pharmacological manipulations producing acute erythropoietic stress, such as acute anemia or administration of cytotoxic drugs, such as 5-azacytidine and hydroxyurea (reviewed in 1Go). Histone deacetylase inhibitors, such as butyrate or other short-chain fatty acids, have the same effect (reviewed in 2Go). Naturally occurring mutations that delete various length of the region 3' to the {gamma}-gene (from a few kb to more than 100 kb DNA) result in increase of {gamma}-gene expression (reviewed in 1Go–4Go). On the basis of hematological findings in heterozygotes, these deletion mutations are classified into two groups, hereditary persistence of fetal hemoglobin (HPFH) and deletional {delta}ß-thalassemia. Another group of mutations, the non-deletional HPFHs are characterized by elevation of either the G{gamma} or the A{gamma}-globin chains of fetal hemoglobin in normal adult individuals, which carry mutations in either the G{gamma} or the A{gamma}-gene promoters (reviewed in 1Go).

Individuals heterozygous for deletional HPFH produce 14–30% hemoglobin (Hb) F and are clinically and hematologically normal; those with deletional {delta}ß-thalassemias produce 1–25% Hb F and have mild anemia and abnormalities of red cell morphology. The shared feature of deletional HPFH and thalassemia is that these deletions are able to enhance {gamma}-gene expression suggesting that there is a common mechanism whereby the {gamma}-gene can be reactivated by the deletions. On the other hand, deletions with similar length and breakpoints display different phenotypes, and completely different deletions result in similar phenotypes. These observations suggest that the mechanism of {gamma}-gene reactivation by these downstream deletions may be complex.

Transgenic mice have been instrumental in the analysis of the mechanism of globin-gene switching during development. Transgenic mice carrying the human ß-locus in the context of a yeast artificial chromosome (ßYAC transgenic mice) display high level of {gamma}-gene expression in the yolk sac and early fetal erythropoiesis, but consequently switch to exclusive ß-globin gene expression (reviewed in 1Go). ßYAC or cosmid transgenic mice carrying {gamma}-gene promoter mutations known to produce a non-deletional HPFH phenotype in human, continue expressing the mutant {gamma}-gene in the adult stage of development, thus reproducing the phenotype of HPFH (5Go, 6Go and unpublished data). Such observations suggest that ß-globin locus YAC transgenic mice provide the proper experimental model for investigation of the mechanism underlying the continuation of fetal hemoglobin synthesis in individual with deletional HPFH and {delta}ß thalassemia mutations.

To search for a common mechanism responsible for elevated expression of the {gamma}-gene both in HPFH and {delta}ß-thalassemia, we planned to establish several HPFH and thalassemia models in ß-locus YAC transgenic mice. We initiated this study by producing transgenic mice carrying a 213 kb ßYAC which faithfully reproduced the structure of the ß-locus of the human HPFH2 mutation. In this form of HPFH, the deletion results in juxtaposition of an enhancer, which is situated downstream of the HPFH2 breakpoint, to the human A{gamma}-globin gene. It has been hypothesized that this ‘imported’ enhancer element results in activation of the {gamma}-gene in the adult stage. We found that ß-locus YAC transgenic mice carrying the HPFH2 mutation failed to activate the globin-gene in adult erythroid cells. The DNase I hypersensitive sites (HSs) of the ß-globin locus control region (LCR) of the HPFH2 transgene were normally formed, chromatin histones were acetylated and pol II was recruited to the region. Histones on the {gamma}-gene promoter were only slightly acetylated, or not at all, and no pol II was recruited to the {gamma}-promoter. Our results suggest that in the context of a ß-locus YAC, juxtaposition of the HPFH2 enhancer to the {gamma}-globin gene is not sufficient to reactivate {gamma}-gene expression; we postulate that upstream sequences missing in the ßYAC construct may be involved in {gamma}-gene reactivation in HPFH individuals. Our study also suggests that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Introduction of the HPFH2 deletion into the ßYAC construct
Figure 1 outlines the process of introduction of the ~83.5 kb HPFH2 deletion into a ~213 kb ßYAC construct by homologous recombination in yeast (7Go). The YIP deletion vector contained a 1.2 kb 5' fragment and a 2.0 kb 3' fragment surrounding the HPFH2 breakpoint. Between the 5' and 3' fragments an URA selection gene that was flanked by the loxP sequence was inserted. The YIP deletion vector was transfected into the ßYAC yeast (Fig. 1A). URA-positive clones were screened by Southern hybridization. Two clones containing the 83.5 kb HPFH2 deletion (Fig. 1B) were processed for removal of the URA gene. A cre-expression plasmid was transfected into yeast bearing the HPFH2/YAC/URA and selected on 5-FOA plate. The YAC structure of positive clones was examined by Southern hybridization using the probes spanning the locus, and five clones had the desired structure (Fig. 1C). The HPFH2/YAC had the expected size 129.5 kb measured by pulse field agarose gel electrophoresis (Fig. 1D).



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  		Figure 1. Construction of the HPFH2/YAC. The first line (A) diagrams the 213 kb ßYAC. The short arrows show the HS sites within the LCR and the region 3' the ß-gene. Filled boxes represent the globin-genes that are consecutively activated in order from 5' to 3' direction during development. The ~83.5 kb sequences that are deleted in the HPFH2 are bracketed by two long arrows. The HPFH2 enhancer that is juxtaposed to the {gamma}-gene is shown as an oval downstream to the breakpoint. The HPFH2 deletion was executed by transforming the ßYAC yeast with a YIP construct, which contained the 1.2 kb and 2.0 kb fragments homologous to the 5' and 3' sequences of the breakpoint, respectively. An URA gene flanked with the LoxP sites was placed between the two fragments and severed as selection mark after homologous recombination. The second line (B) represents the intermediate product, in which the ~83.5 kb sequences that are deleted in the HPFH2, were replaced with the URA selection gene. After transformed with a Cre-DNA recombinase expressing plasmid and selected against 5-FOA, the URA selection gene was removed and a LoxP was left behind (C). The panel in the figure (D) Shows the HPFH2/YAC has a corrected size (~129.5 kb) evaluated on pulse field gel electrophoresis.

 
Production of transgenic mice and structural analysis of the transgene
The 129.5 kb HPFH2/YAC was isolated from the yeast chromosomes on a 1% agarose gel by pulse field gel electrophoresis. The purified YAC DNA was used for transgenic production and 11 founders were found to be positive. Six transgenic lines were established and subjected to detailed structural analyses as previously described (8Go). In brief, single cell suspensions of transgenic mouse liver were embedded in agarose plugs, they were subjected to restriction with SfiI and the SfiI fragments were fractionated on an agarose gel by pulse field gel electrophoresis; Southern blots were cut into stripes and each stripe was hybridized with one of several probes spanning in the locus. All six transgenic lines contained intact HPFH2/YAC copies. Figure 2A shows four lines (designated lines A–D), which contain one 59 kb band that was expected for SfiI restriction. Other two lines contained extra band(s) in addition to the 59 kb SfiI fragment. These two lines were excluded in the further analysis.



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  		Figure 2. Structural analysis of the HPFH2/YAC transgene in four mouse lines. (A) Analysis of intactness of the HPFH2/YAC transgene in the mouse genome. Agarose-embedded mouse genomic DNA was digested with SfiI and separated by pulse field gel electrophoresis. Southern blot was cut into strips and each strip was hybridized with a probe corresponding to the thin lines linking the construct diagram on top and the reconstituted Southern blots below. (B) Copy number measurement. Mouse genomic DNA (10 µg) was digested with HindIII and electrophoresized on an agarose gel. Southern blots were hybridized with probes of HS3, the {epsilon} and {gamma}-genes, respectively. Ten micrograms of EcoRI-restricted human genomic DNA (Promega) served as the copy number standard (the H lanes on panels). (C) Confirmation of configuration of the HPFH2 deletion in the transgene. The line diagram shows that the correct configuration in the HPFH2/YAC transgene should create an 8 kb NheI fragment when tested with a probe 3' to the {gamma}-gene (open box). The Southern blot below the diagram shows hybridization results of the NheI restricted mouse genomic DNA from the four lines with the wild and HPFH2/YAC DNA as control (the two left lanes).

 
The copy numbers of the HPFH2/YAC transgene in the four lines were determined by Southern blot hybridization. DNA prepared from carcasses of transgenic embryos was digested with HindIII and the blots were separately hybridized with three probes (HS3, {epsilon} and {gamma}-genes, Fig. 2B). All hybridization bands had the expected sizes (1.9 kb for HS3, 8.1 kb for {epsilon}-gene, 6.8 kb and 3.3 kb for G{gamma} and A{gamma}-genes), indicating that there were no internal deletions in these fragments. Copy numbers were calculated using human genomic DNA (digested with EcoRI) as the standard. Calculations from blots hybridized with three different probes yielded the same copy number for a given line, suggesting that no extra small pieces of YAC DNA were present. The copy numbers were 2, 2, 3 and 1 for line A, B, C and D, respectively.

Previous studies have suggested that the enhancer 3' to the HPFH2 breakpoint was responsible for {gamma}-gene reactivation in HPFH2 individuals. Southern hybridization was used to confirm the intactness of the HPFH2 enhancer sequence and its juxtaposition to the {gamma}-gene in the four HPFH2/YAC transgenic lines. We found that the physical maps of the HPFH2 enhancer region in the wild-type YAC and the HPFH2/YAC construct were identical (data not shown), suggesting that the enhancer was intact in the deletion construct. To document the juxtaposition of the HPFH2 enhancer to the {gamma}-gene, genomic DNA from the four transgenic lines was digested with NheI and hybridized with a probe 3' to the A{gamma}-gene. As shown in Figure 2C, the probe was able to detect a 24 kb fragment in the wild-type ß-globin locus. The HPFH2 deletion removed the first adjacent NheI site and brought the next NheI site closer to the {gamma}-gene, generating a new 8 kb fragment. Figure 2C shows that the new 8 kb NheI fragment was detected in the four HPFH2/YAC lines, indicating that the juxtaposed HPFH2 enhancer is present in the predicted position.

The {gamma}-gene is silenced in the adult HPFH2/YAC mice
Expression of the globin-genes in HPFH2/YAC mice was measured by RNase protection assays in day 12 blood and yolk sac, which represented embryonic erythropoiesis, in day 12 fetal liver and day 14 blood and fetal liver, which represented fetal erythropoiesis and in adult blood. As shown in Figure 3, the {epsilon} and {gamma}-globin genes were highly expressed in embryonic erythropoiesis in all four HPFH2/YAC lines. In fetal erythropoiesis, only a slight amount of {gamma} mRNA could be detected and {epsilon} mRNA was absent. No {gamma}-gene expression could be detected in the adult blood of the HPFH2 transgenic mice. When we increased the RNA samples by 10-fold in RNA protection assay (from 50 to 500 ng of total RNA), a weak band of the protected {gamma} mRNA could be seen on the overexposed autoradiograph. Under the same condition, a {gamma} band with a similar intensity was also seen in the wild-type ßYAC control. The amount of {gamma} mRNA was estimated at about 0.1% of mouse {alpha} mRNA in both wild-type and HPFH2/ßYAC mice; this level could be an overestimate because the labeled {alpha} RNA probe was saturated in this assay condition. In addition, no {gamma}-globin chain could be detected in adult peripheral blood of the HPFH2/YAC transgenic mice by fluorescent staining with anti-{gamma} chain monoclonal antibody (data not shown). These results suggest that the HPFH2 deletion is unable to reactivate {gamma}-gene expression in adult erythropoiesis of the HPFH2/YAC transgenic mice.



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  		Figure 3. Globin-gene expression measured by RNase protection assays. Four lines (A, B, C, and D) carrying this transgene were established. Total RNA was prepared and RNase protection assay was performed using RNA probes for human {varepsilon}- and {gamma}-globin, which were marked by Hu {varepsilon} and Hu {gamma}. Mouse {alpha} and {zeta} mRNAs serve as internal control, which were marked by Mo {alpha} and Mo {zeta} on the left side of the panel. At least three transgenic individuals were used for RNA measurement, but only one set of samples of each line is presented in this figure. Bl, Blood; YS, yolk sac; FL, fetal liver.

 
{gamma}-gene expression is reduced in embryonic and fetal erythroid cells
Table 1 shows copy number-corrected levels of {gamma}-gene expression in the four HPFH2/YAC lines. Mean levels of {gamma} mRNA in the four lines were 18.7% of murine {alpha} mRNA per copy in d12 blood and 27.1% in d12 yolk sac (Table 1). These levels were ~70% of those in corresponding samples of the wild-type control (27.0 and 37.1%, respectively). Thus, in embryonic erythropoiesis, {gamma}-gene expression was decreased by ~30% in the HPFH2/YAC transgenic mice. The decrease was more prominent in fetal erythropoiesis. In d12 fetal liver, the mean level of {gamma}-gene expression in the four HPFH2/YAC lines was 3.9% of murine {alpha}mRNA (Table 1). The corresponding level in the wild-type ßYAC mice was 16.7%. In d14 blood and fetal liver, {gamma} mRNA was reduced to 3.5 and 1.3% in HPFH2/YAC mice, while the {gamma}-gene was expressed at 10.7 and 6.4% of mouse {alpha}-mRNA in the wild-type control. Thus, in fetal erythropoiesis {gamma}-gene expression in the HPFH2/YAC mice was reduced to 20–30% of that in the wild-type ßYAC mice.


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  		Table 1. Expression of the human {gamma}-gene in HPFH2 YAC mice
 
The DNase I HSs of the LCR are formed in the HPFH2/YAC mice
In HPFH2/YAC mice the ß-gene is absent and both the {epsilon} and {gamma}-genes are silenced in the adult stage of development (Fig. 4). We asked whether the HS sites of the LCR were formed in erythroid cells when no globin-genes were expressed in the locus. Nuclei were prepared from phenylhydrazine-treated adult spleen of a HPFH2/YAC transgenic mouse (line A, two copies of the transgene). Nuclei of the drug-treated spleen of a wild-type ßYAC mouse (three copies of the transgene) served as the positive control. After DNase I partial digestion, DNA was restricted with either BamHI or MfeI and then blotted. The results of HS assays demonstrated that the HS sites were formed in the adult erythroblasts of the HPFH2/YAC mice exactly as the wild-type control (Fig. 4A and B). As mentioned before, {gamma}-gene expression was undetectable in adult erythropoiesis, these results suggest that HS formation in the LCR region does not depend on an active gene transcription of the downstream genes.



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  		Figure 4. DNase I hypersensitivity of the LCR and the HPFH2 enhancer of HPFH2/YAC transgenic mice. Nuclei from adult spleen of transgenic mice were treated with DNase I and the partial digested DNA was restricted with BamHI (A and C) or MfeI (B). Sizes of the subbands are indicated alongside the blots.

 
Figure 4C shows Southern hybridization analysis of HS formation in the downstream region of the HPFH2 breakpoint. In this region, HS site was formed in the 3D enhancer (9Go), which was located next to the HPFH2 enhancer and was also juxtaposed to the {gamma}-gene. A faint band with expected size (2.0 kb) corresponding to the HS site could be seen in the sample of HPFH2/YAC transgenic mice (Fig. 4C, right). The Southern blot used in this experiment was the same as in the analysis of HSs 1–3 (Fig. 4A). Although the subbands of HSs 1–3 in Figure 4A were intensive, intensity of the 3D subband was weak. However, the intensity of the 3D enhancer in HPFH2/YAC transgenic mice was comparable to that in K562 cells (Fig. 4C, left), suggesting that DNase I sensitivity at the position of the 3D ‘enhancer’ is not prominent.

The chromatin histones are acetylated in the LCR of the HPFH2/YAC mice
To further delineate the chromatin structure, we measured histone H3 acetylation in the LCR by chromatin immunoprecipitation (ChIP) assay. As ChIP assays of experimental and control samples were performed in parallel, but in different tubes, it was necessary to evaluate the comparability of data tested from the two samples. Four mouse endogenous genes, whose expression presumably is not affected by the YAC transgenes, served as internal indications of the comparability. As shown in Figure 5A, ratios of histone H3 acetylation between the mouse ß-actin, aire, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ßmaj-genes were identical in HPFH2/YAC and wild-type ßYAC mice, indicating that the ChIP data of the two-type transgenic mice were comparable. Figure 5B presents levels of acetylated histone H3 in the LCR and the {gamma}-gene promoter in phenylhydrazine-treated adult splenic cells of transgenic mice. As expected, histone H3 was barely or not acetylated on the {gamma}-gene promoter in both HPFH and wild-type ßYAC transgenes, in agreement with the silenced status of the {gamma}-gene. In the LCR of the HPFH2/YAC, transgene acetylation of histone H3 of the HS sites seems to be comparable to that of the control. We conclude that chromatin histone H3 is acetylated at a moderately high level in the HPFH2/YAC mice.



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  		Figure 5. Histone acetylation and pol II recruitment in the LCR measured by ChIP assays. (A) Demonstration of comparability of the ChIP data between the HPFH and wild-type YAC mice. Histone acetylation levels on the promoters of four mouse endogenous genes (actin, aire, GAPDH and ßmaj), that should not be affected by the transgene, were measured in the samples of the wild and HPFH2/YAC mice. The ChIP data are expressed as fold difference by setting the acetylation level on the mouse aire gene as one unit. As shown in (A), levels of histone acetylation are similar in wild and HPFH2/YAC mice, indicating that the ChIP data are comparable between the two types of mice. (B) Measurement of acetylated histone H3 in the LCR and the {gamma}-gene promoter. (C) Measurement of pol II recruitment in the LCR and the {gamma}-gene promoter.

 
Pol II is recruited to the LCR in the HPFH2/YAC transgene
As no transcription occurred in the ß-globin locus in the HPFH2/YAC transgenic mice, we asked whether the LCR-mediated pol II recruitment was maintained. As shown in Figure 5C, no or little pol II was recruited on the {gamma}-gene promoter in both the HPFH2 and wild-type YAC transgenic mice. On the other hand, compared with the wild-type control, approximately half of pol II was recruited to HSs 2, 3 and 4 in the HPFH2/YAC transgenic mice. Although the recruitment was reduced, the level was still higher than that on the {gamma}-gene promoter, which was silenced in the adult erythropoiesis, suggesting that LCR is able to recruit pol II independent of gene transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Three different, but not mutually exclusive, hypotheses have been proposed for explaining {gamma}-gene activation by the HPFH deletions. The juxtaposition model suggests that the deletions juxtapose an enhancer neighboring the HPFH breakpoints to the vicinity of the {gamma}-globin gene (9Go–14Go). The silencer model proposes that the HPFH deletions remove sequences residing within the region between the {gamma} and {delta}-genes, which are necessary for the silencing of the genes in adult life (15Go,16Go). The chromatin subdomain model postulates that there are ‘fetal’ and ‘adult’ chromatin domains in the globin-gene cluster (17Go,18Go). Presumably, persistent gene expression of the {gamma}-globin could result from the loss of sequences delineating stage-specific functional chromatin subdomains in the locus.

The hypothesis that in deletional HPFHs, juxtaposition of the downstream enhancer to the {gamma}-gene results in reactivation of the fetal globin-genes in adult erythrocytes (12Go) has received broad support because the phenotype of {gamma}-gene expression in the adult has been partially reproduced in transgenic mice carrying cosmid ß-globin gene constructs with a juxtaposed HPFH enhancer (10Go,13Go). Arcasoy et al. (10Go) produced a construct carrying a 4.7 kb µLCR linked to a 13 kb fragment containing the G{gamma} and A{gamma}-gene, to which a 12 kb fragment containing the HPFH2 3' breakpoint was attached. These authors reported that the juxtaposition of the 12 kb of HPFH2 3' breakpoint DNA sequence to the {gamma}-gene increased {gamma}-gene expression in adult blood of transgenic mice from <0.4% in the control to 3.4–8.0% in the HPFH2 transgenic mice. In HPFH2 transgenic mice, the level of A{gamma}-gene expression was greater than that of G{gamma}-gene, while this ratio was reversed in the control mice. These results were interpreted to suggest that an ‘imported’ downstream enhancer was the cause of the {gamma}-gene reactivation in adult erythrocytes. Katsantoni et al. (13Go) reported a similar study using a cosmid construct containing 22 kb of the LCR sequence linked to the 5.6 kb A{gamma}-gene, to which a 0.7 kb sequence of the HPFH2 enhancer was attached. In the control transgenic mice, expression of the A{gamma}-gene ranged from 0 to 0.89% in adult blood, whereas {gamma}-gene expression ranged 0–19.2% when the HPFH2 enhancer was linked to the cosmid construct. After reduction of the transgenes to single copy by the LoxP/cre technique, {gamma}-gene expression was 0.24, 0.72 and 6% in three of HPFH2 transgenic lines, respectively, while it was 0, 0.1 and 0.14% in three lines of the control animals. These studies were interpreted to suggest that the HPFH2 enhancer is able to inconsistently increase {gamma}-gene expression in the context of the cosmid transgenes.

In this study, we demonstrate that juxtaposition of the HPFH2 enhancer to the {gamma}-gene in the context of a ß-locus YAC construct is not sufficient to reactivate {gamma}-gene expression in adult transgenic mice. Failure of {gamma}-gene reactivation was observed not only in a single copy line, but also in multi-copy lines. It is unlikely that in the context of a ßYAC construct, the human {gamma}-globin gene is permanently silenced in adult mouse erythroid cells. The {gamma}-globin gene promoter carrying a G->A mutation at -117 (Greek HPFH) was able to drive {gamma}-gene expression at high level in adult transgenic mice (5Go,6Go), suggesting that YAC transgene is a suitable system for the study of {gamma}-gene reactivation. The discrepancy between our results and those of the two previous transgenic studies could be attributed to differences of the constructs used in the three labs. For instance, the distance between the LCR and the {gamma}-gene promoter in the three types of constructs could be one of the factors causing the distinct behaviors of the {gamma}-gene. Many lines of evidence suggest that distance between the LCR enhancer and the downstream globin-gene promoters is a critical parameter for determining a developmental profile of globin-gene expression (19Go,20Go). For instance, the {gamma}-gene is expressed in the adult stage of development when the distance between the LCR and {gamma}-gene promoter is shorter than 2 kb, e.g. in plasmid-based constructs (21Go–23Go). When the distance is increased to ~10 kb, the {gamma}-gene is autonomously silenced in transgenic mice carrying a single copy of the transgene, but it is improperly expressed at various levels in multiple copy-transgenic mice (24Go). When the distance is >30 kb as in ß-locus YAC constructs, the {gamma}-gene is completely silenced even in the multi-copy transgenic mice (7Go,25Go,26Go). These observations suggest that the potential of {gamma}-gene reactivation in adult transgenic mice is progressively decreased with an increase in the distance between the LCR and the {gamma}-gene promoter. The distance between the A{gamma}-gene promoter and HS2 of the LCR was ~10 kb in the constructs used either by Arcasoy et al. (10Go) or Katsantoni et al. (13Go). It is likely that within this distance the {gamma}-gene can be the reactivated by the LCR. In fact, Katsantoni et al. (13Go) noticed that higher levels of {gamma}-gene expression were observed in the later fetal liver stage in the 27.6 kb LCRA{gamma} animals compared with transgenic mice carrying the 70 kb complete human ß-globin locus, and attributed this to the greater proximity of the {gamma}-gene to the LCR in the small cosmid construct. It is likely that when the {gamma}-gene is closely located to the LCR, placement of the HPFH2 enhancer next to the {gamma}-gene increases the probability that the LCR will interact with the {gamma} promoter, resulting in {gamma}-gene activation. It is of interest that {gamma}-gene expression was barely increased in transgenic mice by juxtaposition of the putative enhancers 3' to HPFH3 or HPFH6 in the context of cosmid constructs (13Go), suggesting that it is unlikely that enhancer juxtaposition is a general mechanism for {gamma}-gene reactivation in deletional HPFH.

The phenotype discrepancy between HPFH2 individuals who express high level of {gamma}-globin chain and the HPFH2/YAC transgenic mice that display no {gamma}-gene expression is very informative. The deletion in the HPFH2/YAC construct exactly mimics the natural mutation and the distance between the LCR and the {gamma} promoter is identical in the both cases. This discrepancy in phenotypes suggests that factor(s) other than the juxtaposed sequences are involved in {gamma}-gene reactivation in human who carry deletional HPFH mutations. The ß-locus YAC construct we used in this study contains ~40 kb sequence 5' to the {epsilon}-gene and ~30 kb sequence 3' to the HPFH2 break point. As implied in the mouse LCR knockout study, the far upstream region, which is absent in the ß-locus YAC construct, might be involved in chromatin opening of the ß-globin locus in situ (27Go). Using the chromatin conformation capture (3C) technique, it has been shown that a HS at 110 kb upstream of the {epsilon}-gene, which is absent in the HPFH2/YAC construct, was a component for the ‘hub’ structure in erythroid cells (28Go). It is possible that formation of the ‘hub’ might be a pre-requisite for re-establishment of interaction between the LCR and the {gamma}-gene driven by a natural promoter in the natural cluster configuration in adult erythroid cells. Our unpublished data showed that several alternatively spliced globin mRNA starting from the ~236 kb and ~77 kb upstream region occurred in human primary tissues. In addition, many erythroid-specific HS sites were identified within the region. It is possible that these HS sites and intergenic transcription might be involved in the anti-silencing mechanism of the {gamma}-globin gene in situ.

We were puzzled by our finding that expression of the {gamma}-gene is reduced in fetal, and to a lesser extent, in embryonic erythropoiesis in transgenic mice carrying the HPFH2/YAC construct. It is unlikely that the LoxP site left in the construct suppresses globin-gene expression. The Cre/Lox system has been wildly used in gene and genome manipulations, and there are no reports showing that loxP sequence per se detrimentally affects gene expression. It was previously reported that a floxed hygromycin B resistance gene left between HS1 and the {epsilon}y-gene after homologous recombination substantially inhibit expression of the downstream ßmaj-gene (29Go). However, after deleting the selection gene, but leaving the loxP sequence behind, expression of the ßmaj-gene returned to the normal level, suggesting the loxP sequence itself has no negative effects on globin-gene expression. To date, no information is available regarding in vivo effects of the deletion mutation on {gamma}-gene expression in the fetal stage of development in HPFH2 individuals. When the entire sequences 3' to the {gamma}-gene were deleted in the context of a ßYAC construct, {gamma}-gene expression was retained at normal levels in embryonic and fetal erythrocytes (Li et al., unpublished data), and it is properly silenced in adult erythropoiesis (5Go). Comparing the HPFH2/YAC mice to the ßYAC mice carrying a deletion in the entire region 3' to the {gamma}-gene, the negative effect could be attributed to the HPFH enhancer and/or other downstream sequences.

The establishment of HPFH2/YAC transgenic mice provided us with an opportunity for studying properties of the LCR in the absence of any globin-gene transcription. Our results demonstrate that the HS of the LCR can be detected in the splenic erythroblasts of HPFH2/YAC mice, suggesting that the formation of DNase I HSs is independent of transcription of the globin-genes. Our observations are concordant with the results of previous studies reporting that an erythroid-specific HS site was formed by a 1.4 kb 5'HS4 fragment in three of three transgenic lines or by a 1.9 kb 5'HS2 fragment in nine of 10 transgenic lines (30Go). Reitman et al. (31Go) reported that a DNA fragment containing the chicken LCR consistently forms an HS site only when linked to a ß-globin gene; in the absence of the ß-globin gene only three of six animals formed an HS site. This result suggests that the chicken LCR does not form HS sites autonomously and that there may be differences in the mechanism in which the chicken and human LCR sequences function. In addition to normal formation of the HS sites, histone acetylation in the LCR of HPFH2 mice is retained at levels comparable to those of the wild-type ßlocus YAC mice. We conclude that the general chromatin conformation of the LCR is maintained in erythroid cells when no transcription occurs in the locus.

Recent studies have shown that the LCR is capable of recruiting pol II in MEL and K562 cells and the recruitment has a positive correlation with the binding of GATA1, but not with NF-E2 (32Go–34Go). Differentiation induction results in a small increase in the pol II recruitment. In studies to be reported elsewhere, we have shown that the deletion of the HS3 core in the context of the ßYAC abolishes pol II recruitment at the LCR in transgenic mice (35Go). These results provide strong evidence that pol II is bound to the LCR and are in agreement with the observations of the LCR-originating transcription (36Go,37Go). In HPFH2/YAC mice, pol II is recruited to the LCR, although the levels are moderately reduced on the HS3 and HS4 cores. Our results further suggest that the LCR possesses its own ability for pol II recruitment independent of the occurrence of active transcription of the globin-genes in the locus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DNase I was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). Rabbit polyclonal antibodies against histone H3 acetylated at lysines 9 and 14 (06-599) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Rabbit polyclonal antibodies against pol II (N-20, sc-899) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Syber green PCR kit were from Qiagen (Valencia, CA, USA).

Constructs
All constructs were generated using standard molecular cloning procedures. To introduce the 83.5 kb HPFH2 deletion (UCSC coordinates (hg15): Ch11, 5,223,022-5,139,442) into the yeast artificial chromosome (YAC) containing the human ß-globin locus, a deletion construct (designated YIP in Fig. 1) was made. The YIP/deletion construct contains 1.2 kb sequences (5,224,185-5,223,027) 5' to 2.0 kb sequences (5,139,443-5137,434) 3' to the HPFH2 breakpoint. An URA gene with flanking LoxP sites was inserted between the two fragments. The floxed URA plasmid was constructed by cloning the URA gene (as a SspI fragment released from pRS 406 (Stratagene, La Jolla, CA, USA) into a SmaI site between the two loxP sites in pBS246 (GibcoBRL, Grand Island, NY, USA).

Yeast transformation
Yeast transformation was performed using YEASTMARKER (Yeast Transformation System 2, CLONTECH) following the manufacturer's supplied protocol.

Production of transgenic mice
The HPFH2 ßYAC were purified by pulse field agarose gel electrophoresis (PFGE). The purified fragment was injected into fertilized eggs from B6/C3F1 donors, which were then transferred to pseudopregnant B6/D2F1 foster recipients. Founder candidates were identified by hybridizing Southern slot blots with a 32P labeled probes for human HS3. The positive candidates were further identified by PCR using primer sets spanning the ßYAC construct. The animals positive to all primers were bred with non-transgenic B6/D2F1 mates and F1 progeny were used for structure analysis.

Structure analysis
DNA from single-cell liver suspension of F1 progeny was embedded in 1% agarose gel and digested with SfiI as described previously (5Go). The digested DNAs were separated on a 1% (w/v) SeaKem Gold GTG agarose gel (BioWhittaker Molecular Applications, Rockland, ME, USA) in 0.5x TBE by PFGE, 200 V, 14 s switch, for 20 h at 14°C. The DNA was capillary-transferred overnight onto Zeta-probe positive-charged nylon membrane (Bio-Rad, Hercules, CA, USA) with 0.4 N NaOH. The membranes were cut into strips. Each strip was hybridized with a different radioactive labeled probe spanning the ß-globin locus from HS5 to the HPFH1 breakpoint. After hybridization, the strips were reassembled and subjected to autoradiography. The DNA fragments used as probes for the structural analyses were as follows: 2.6 kb HindIII HS5, 1.4 kb BamHI/SpeI HS4, 0.7 kb PstI HS3, 1.9 kb HindIII HS2, 0.7 kb BamHI {epsilon}-globin gene, 0.9 kb BamHI/EcoRI fragment of the G{gamma} or A{gamma}-globin gene, 0.7 kb SacI/SpeI fragment at the 3' end of HPFH2 breaking point and 0.8 kb SpeI/BsaI fragment at the 3' end of HPFH1 breaking point. The 0.6 kb probe located at the 5' end of HPFH2 deletion (5,225,915-5,225,288) was PCR amplified. The fragments were radio labeled using a Decaprime II random probe labeling kit following the manufacturer's instructions (Ambion, Austin, TX, USA).

RNase protection assay
Total RNA was prepared using guanidine thiocyanate-acid-phenol. Human globin transgene transcripts were analyzed by RNase protection as previously described (38Go). Total RNA was hybridized overnight at 48oC with 106 c.p.m. of each radiolabeled probe. After digestion with RNase A and T1, the protected fragments were separated on 6% polyacrylamide-8 M urea gels and autoradiography was performed without intensifying screens. Signal intensities were quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). Expression levels of {gamma}-globin were calculated as a percentage per copy of mouse {alpha}-like globin. To minimize experimental error, multiple measurements were performed on at least three individual animals and mean and standard deviation were calculated.

Dnase I hypersensitive assay
Single-cell spleen suspensions were prepared from phenylhydrazine-treated 10–12-week-old ß-YAC transgenic mice. Nuclei isolation, DNase I digestion, and southern blotting were done as described previously (39Go). The probes shown in Figure 4 were a 0.7 kb fragment (coordinate: 5,265,816-5,265,106) for detection of HSs 1, 2 and 3 and a 0.6 kb fragment (5,264,760-5,264,168) for detection of HSs 3, 4 and 5.

Chromatin immunoprecipitation assay (ChIP)
Single-cell spleen suspensions were prepared from 10–12-week-old ßYAC transgenic mice after 4 days of phenylhydrazine-induced hemolytic anemia. ChIP assays were performed as previously described with modifications (40Go). The mouse autoimmune regulator (mAire) gene, ß-actin gene, GAPDH-gene and the ßmaj-globin gene were selected as internal controls. Immunoprecipitations were performed at least three times on different days. Each DNA was diluted at three dilutions and the PCR readings of the three dilutions should be within the range of the standard DNA curve. PCR was performed on an Opticon 2 (MJ Research). All data were expressed as ratio of the PCR readings of a given primer set over the internal control and standard deviation (SD) was shown.


   ACKNOWLEDGEMENTS
 
We thank Xin Ye and Mary Stafford for skillful technical help. This study was supported by grants from the National Institutes of Health DK61805 and HL73439 to Q.L. and DK45365 to G.S.

Conflict of Interest statement. None declared.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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