Expression of a Gene Located 300 kb from the α-Cluster Is Controlled by the α-Globin Regulatory Elements.

To determine whether expression of eNME4 in erythroid cells is regulated by the α-globin MCS-R elements, we analyzed expression of eNME4 mRNA transcripts in red blood cells obtained from rare individuals, each with a different deletion of the MCS-Rs on one allele (ΔMCS-R/αα) (9) (see Fig. 1A). We found that while the expression of control erythroid-specific genes [EPOR and β-globin (HBB)] was not significantly affected by these deletions, eNME4 was significantly reduced (n = 7, P = 0.01) to ≈50% of its normal level of expression (Fig. 2A). This suggested that enhanced expression of the NME4 erythroid-specific transcript, like α-globin, depends on the α-globin MCS-R elements located ≈300 kb upstream of this gene.

Open in a separate window

Fig. 2.

Allele-specific effect on the expression of eNME4 by various deletions of the α-globin cluster. (A) Expression of the three erythroid-specific genes in the terminal 500 kb of chromosome 16p, and two erythroid-specific control genes, EPOR and HBB. For each gene, the expression in control samples is set to 100%, and each group of deletions is calculated relative to controls. Student's t test P values are calculated for each gene for each group of samples compared to controls; *, P < 0.05; **, P < 0.005. Controls, n = 15; ΔMCS-R/αα, n = 7; -α/αα, n = 14; --/αα, n = 22. (B) The proportion of the G allele of NME4 contributing to total transcription, as determined by pyrosequencing (see Tables S4 and S5 for details). All samples are heterozygous (A/G) for SNP rs14293 (controls, n = 12; ΔMCS-R/αα, n = 7; --/αα, n = 11) except for an A/A and a G/G control (indicated by *). Samples for which phase of the α-globin locus deletion and the SNP allele could be determined are shown in color; deletion in phase with the A allele are shown in red; deletion in phase with the G allele are shown in green; samples where phase could not be determined are shown in black. The P value is calculated by an f test for differences in variation.

Given the relatively large distance between NME4 and the α-globin regulatory elements, it seemed possible that the effect on expression might be mediated in cis or in trans [as suggested for other long-range interactions (reviewed in ref. 10)]. To establish the effect of the MCS-R deletions on each copy of NME4, we analyzed allele-specific expression in red blood cells, using a highly polymorphic, synonymous transcribed SNP in exon 4 of NME4 (A/G, rs14293). This SNP is contained in both the full-length transcript and the truncated erythroid-specific transcript (Fig. 1C). With one exception, six individuals with MCS-R deletions, who were informative for this SNP, displayed allele-specific expression of NME4 (Fig. 2B). Using somatic cell hybrids containing a single copy of human chromosome 16 derived from these individuals, we determined which SNP was in phase with the deletion and found that in all cases examined, deletion of the α-globin MCS-R occurred in cis to the eNME4 allele that was under-expressed. The reduced level of expression specifically from one allele in cis with the MCS-R deletion demonstrates that the erythroid-specific enhanced expression of NME4/eNME4 is regulated in cis by the α-globin major regulatory element (MCS-R2).

Identification of Long-Range Interactions Across the Terminal Region of Chromosome 16.

We hypothesized that this functional interaction between the MCS-Rs and NME4/eNME4 was mediated via a physical interaction. We have previously used chromosome conformation capture (3C) to demonstrate physical interactions between the α-globin genes and the MCS-Rs both in mouse (11) and human (12). Recently, we and others (13, 14), have developed this methodology into a modified circular 3C method (4C), which, after cross linking and ligation of chromosome loops, uses an inverse PCR protocol to detect all physical interactions with a genomic fragment of interest. Our assay has been modified to give extremely high sensitivity and is analyzed using a microarray-platform (see Methods). By using a DpnII fragment containing MCS-R2 (the major α-globin regulatory element) as the anchor fragment, we performed 4C analysis on nonerythroid and erythroid cells (Fig. 3A).

Open in a separate window

Fig. 3.

4C analysis from MCS-R2 identifies a physical interaction with NME4 in --/αα erythroid material. (A) 4C material hybridized to a tiled microarray. The dashed line represents the fixed fragment of MCS-R2. Shaded boxes show α-globin locus and NME4; nonerythroid, EBV-transformed B-lymphocyte cell line; erythroid, two-phase culture system for generation of erythroid cells. All tracks are representative of two biological replicates. Zoomed section shows signal from Lower. Actual enrichment of 4C-amplified material relative to genomic DNA (based on real-time PCR; QPCR) is shown for two amplicons (389776 and 396875). The y axis is a log scale. Arrows represent transcription of NME4 and eNME4. Primer sequences can be found in Table S6. (B) Pyrosequencing tracks from an --/αα individual informative for SNP rs14293 at NME4. (Upper) Genomic DNA; (Lower) 4C-amplified DNA. Peaks used for calculations are shaded; peak 4 corresponds to the G allele, peak 8 corresponds to the A allele; dispensation order of nucleotides is shown on the x axis; E, enzyme; S, substrate. Further information can be found in Tables S4 and S5.

In nonerythroid cells there are no interactions between MCS-R2 and genes in the α-globin locus (see Fig. 3A Top). In erythroid cells, where MCS-R2 becomes active and the α-globin genes are highly transcribed, there is a strong interaction between MCS-R2 and the α globin genes (see Fig. 3A Middle, erythroid controls 1 and 2). In these cells, although there is not a consistent interaction with NME4, we have observed rare interactions represented by small peaks of enrichment (for example, erythroid control 2) in approximately one in three 4C experiments (n = 9). Therefore, even though the functional data and some 4C data suggest an interaction does occur between MCS-R2 and NME4 in a normal, intact chromosome, it seems that when the α-globin genes are present, their interaction with the MCS-Rs may out-compete the much weaker and occasional interactions with NME4 (see below). Such infrequent interactions (MCS-R2/NME4) may often lie below the level of detection using this assay.

Deletion of the α-Globin Genes Increases Expression of NME4 via Competition for the Shared Regulatory Element (MCS-R2).

The level of expression of eNME4 in erythroid cells is regulated by the α-globin MCS-Rs, and provisional evidence suggests that NME4 and MCS-R2 may physically interact, albeit rarely. Therefore, it seemed likely that NME4, even though it lies hundreds of kilobases from the α-globin cluster, may compete for the activity of the remote regulatory elements (MCS-R1 to -R4). To test this hypothesis, we examined the expression of eNME4 in the red cells of patients who have inherited chromosomes with either one (-α) or no (--) α genes rather than the normal duplicated pair of genes (αα). We observed that deletion of a single α-globin gene (-α) resulted in a small increase (factor of two) in expression of eNME4 (see Fig. 2A). However, deletions removing both α-globin genes (--) resulted in a dramatic increase (8-fold) in eNME4 expression when compared to normal controls (n = 22, P = 3.18 × 10⁻⁹) (see Fig. 2A). This group consisted of individuals each carrying mutations on one of three different chromosomes (--^SEA, n = 18; --^FIL, n = 3; --^MED, n = 1).

As described before, we also determined whether up-regulation of eNME4 was caused by an increase in expression from one or both alleles. We found highly skewed patterns of NME4 expression in all informative individuals carrying these deletions (see Fig. 2B). Again, by generating somatic cell hybrids where material was available, we were able to link the nucleotide at the NME4 SNP to the deletion, and found that the up-regulated allele was always on the chromosome from which the α-globin gene deletion had occurred. This confirms that not only is the expression of eNME4 under the influence of the MCS-Rs lying ≈300 kb away, but the level of expression dramatically increases as the number of competing α-globin promoters in cis is reduced.

Cell Types.

Erythroid cells were obtained using a two-phase culture system as previously described (25). Red blood cells were purified from whole blood (according to ref. 26), for expression analysis. hES cells were obtained from the H1 HES cell line, grown according to manufacturer's instructions. Nonerythroid cells were either primary T lymphocytes (ChIP, Northern analysis), or EBV-transformed B-lymphocyte cell lines (expression analysis, 4C analysis).

Expression Analysis.

For all cell types, RNA was extracted with Tri reagent as per manufacturer's instructions (Sigma). For Northern blots, 20 μg of total RNA were assayed, using the NorthernMax-Gly kit as per manufacturer's instructions (Ambion). For real-time expression analysis, RNA was DNaseI treated (Ambion) and cDNA was generated with SuperScript III (Invitrogen) as per manufacturer's instructions. Real-time PCR assays were obtained from either Applied Biosystem's Assay-On-Demand resource, or designed with Primer Express software. Both +RT and −RT templates were analyzed to detect genomic contamination. Expression in hES and erythroid cells was calculated relative to a control sequence in the 18S ribosomal RNA gene (Eurogentec RT-CKFT-18S). Expression in red blood cells was calculated relative to CD71, to correct for stage of erythropoiesis. For the latter, the mean of expression of each gene in the control samples is set to 100%, and expression in the deletion patient samples is expressed relative to this mean for each gene analyzed. For details of assays, real time primers and probes, and Northern probes see Tables S1–S3.

Statistical Analysis.

Significance of differences in expression between control and deletion samples was calculated with a two-tailed Student's t test assuming nonequal variance. Significance in variation of allele-specific expression was calculated with an f test.

ChIP and ChIP-chip.

ChIP was performed as previously described (5). Briefly, for one immunoprecipitation, 1 × 10⁷ cultured primary human erythroblasts or T lymphocytes were cross-linked with 1% formaldehyde for 10 min. DNA was sheared by sonication to fragments under 500 base pairs. Antibodies used were H3Ac (06–599, Upstate), H4ac (06–866, Upstate), H3K4me2 (07–030, Upstate), H3K4me3 (ab8580, Abcam), CTCF (07–729, Upstate), GATA1 (sc1234, Santa Cruz), and SCL (gifted by C. Porcher). ChIP DNA was analyzed by real time PCR, calculated relative to input, and normalized to β-actin promoter. For details of primers and probes see Table S6. ChIP-chip was performed by hybridization to a custom α-globin tiled microarray as previously described (5), and enrichment was validated with real-time PCR.

Modified Circular Chromosome Conformation Capture.

For 4C, 1 × 10⁷ cells were fixed with 2% formaldehyde in medium for 10 min at room temperature with agitation. Following quenching with glycine, cells were lysed (10 mM Tris pH8.0, 10 mM NaCl, 0.2% Nonidet P-40, 1× proteinase inhibitor) and resuspended in 1× DpnII digestion buffer (NEB), 0.3% SDS, 2% Triton X-100, and 500U DpnII at 37 °C overnight with shaking. The enzyme was inactivated at 65 °C for 25 min with shaking, and the total volume resuspended in 7 ml 1× ligation buffer with 1% Triton X-100. Following incubation at 37 °C for 1 h, the samples were cooled on ice for 2 min and 240 units of high-concentration T4 DNA ligase (Fermentas) was added. Following incubation overnight at 16 °C, 300 μg of proteinase K was added and cross-links reversed at 65 °C overnight with rotation. The samples were treated with 15 μg RNase (Roche) at 37 °C for 30 min. Following phenol/chloroform extraction and ethanol precipitation, DNA was resuspended in 500 μl 1× ligation buffer and 60 units high-concentration T4 DNA ligase (Fermentas) and incubated for 2 h at 16 °C with shaking. Following phenol/chloroform extraction and ethanol precipitation, DNA was resuspended in 100 μl water, of which 10 μl was used as template in Advantage-GC PCR (Clontech) as per manufacturer's instructions. Primer sequences can be found in Table S6. The resultant amplified DNA was ethanol precipitated and resuspended in 20 μl water, of which 5 μl was hybridized to a customized α globin tiled microarray using sonicated genomic DNA as input, as previously described (5). Enrichment of NME4 in 4C material was quantitated by real-time PCR, and normalized to an unenriched amplicon.

Pyrosequencing.

The ratio of expression of allele-specific transcripts and 4C-amplified DNA of NME4 was ascertained by pyrosequencing. Primer and dispensation information is contained in Table S4 and S5. Peak height is directly proportional to the amount of nucleotide incorporated. Analysis was performed in duplicate and an average obtained.

We thank the clinicians and the members of the families studied for their participation, particularly Dr. C. L. Harteveld (Leiden University Medical Center, The Netherlands), Dr. D. Rund (Hadassah University Hospital, Israel), Dr. D. Filon (Hadassah Medical Center, Israel), Dr. H. Frischknecht (Institute for Medical & Molecular Diagnostics Ltd, Switzerland), Dr. S. L. Thein (King's College Hospital, United Kingdom) Dr. N. Gattermann (Heinrich-Heine University, Germany), Dr. J. Finlayson (QEII Medical Centre, Western Australia), and Dr. R. Hutch (Cork, Ireland). We thank Dr. C. Porcher for the kind gift of the SCL antibody, Dr. I. Dunham for assistance with microarrays, the Computational Biology Research Group, Oxford University, for bioinformatic support, and Prof. W. Wood for critical reading of the manuscript. This work was supported by the Medical Research Council, the Wellcome Trust and the National Institute for Health Research Biomedical Research Centre Program. K.M.L. was supported by an Oxford Nuffield Medical Fellowship, Oxford University.