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The region underlying this new peak of
pression is unremarkable, containing 3.7 kb
poorly conserved, predominantly noncoding
quence, although the tail of the peak exten
into the yz-globin gene. This region conta
17 SNPs, 10 of which have been previou
characterized in nonthalassemic individua
We therefore analyzed the segregation of
remaining seven SNPs and, as controls,
additional SNPs from nonrepetitive regio
of the a cluster (Fig. 1), within affected fa
ilies. In addition, we performed genetic linka
studies in 15 nonthalassemic Melanesian in
viduals (aa/aa), 22 with a thalassemia t
Eaa/(aa)T^, and 5 with HbH disease E(aa)T(aa
Six of the seven SNPs underlying the n
peak of transcription were found on both t
normal aa and abnormal (aa)T chromosom
Only the C allele of SNP 195 (C or T, locat
at coordinate 149709) segregated with thal
semia in the affected families and show
complete association with the (aa)T haploty
(table S2). This allele was not found in a se
arate analysis of 131 nonthalassemic, Melanes
individuals. SNP 195 changes the sequen
Fig. 1. Overview of thea-globin cluster andidentification of a rSNP.The genes located in thetelomeric region of chro-mosome 16 are num-bered as in (1), and theglobin genes are la-beled. The VNTR (3hypervariable region) isshown as a red zigzagline. A deletion (15)removing the regioncontaining the rSNP isshown as a black line.Below this, all DNAse1hypersensitive sites (DHSs)and erythroid-specificsites (eDHSs) are shown(3, 10, 16). MCS-P/R sum-marizes all evolutionar-ily conserved promoterand regulatory sequen-ces across this region(2). Probes used to pro-
file ChIP products areshown in pink, and re-peats are shown ingreen. Below this, allsequence differencesbetween the (aa)T andwild-type aa chromosome are shown. New Diffs refers to newly identifiedsequence differences that are not known to be polymorphic SNPs. SNPsanalyzed in genetic linkage studies described in this paper are shown inpurple. The rSNP described here is shown as a black diamond in All Diffsand New Diffs. A dashed vertical line runs from these diamonds throughthe array data. Below, the patterns of gene expression recorded on acustom-tiled Affymetrix array spanning this telomeric region in primaryerythroid cells from (A) a normal individual (aa/aa) and (B) patient L withthe (aa)T/(aa)T genotype are shown. The peak of z-globin expression in the
(aa)T
chromosome results from cross-hybridization to the highly expressed
abnormal transcripts across the homologous yz gene. (C) Estimates of differences in RNA expression between normal and abnormal chromsomes, based on independent quantitative PCR (QPCR), are shown bel(on a logarithmic scale). (D) Representation of how one or more of tconserved regulatory elements (contained within the region spannedthe horizontal black bar) normally interact with the a-globin promot[aa] and how they are proposed to interact less effectively (dashed linin the abnormal (aa)T chromosome. The direction of transcription of tglobin genes and the new promoter, created by the C allele of SNP 19
are indicated by the arrows.
20k 40k 60k 80k 100k 120k 140k 160k 180k 200k
1 2
4
56
73 163.1
DHSs
eDHSs
MCS-P/R
ChIP Probes
Repeats
All Diff.s
New Diff.s
16p13.3
Deletion
VNTR
R1 R2 R3 R4
100
1000
A
B
D 21
0
QPCR
0.01
1100
10000
()T
C
D
Fig. 2. In situ RNA analysis demonstrating reduced primary a-globin transcripts in patient L.Nascent a-globin (red) and b-globin (green) transcripts in intermediate erythroblasts from a normalcontrol and from patient L [with the (aa)T/(aa)T genotype] are shown. (Left) Representative nucleishow b-globin transcripts in both patient and control, but a-globin transcripts are present only inthe normal control. (Right) The proportion of nuclei containing none, one, or two signals wererecorded from the analysis of 100 cells.
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8/15/2019 Regulatory SNP causes new promoter 2006
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5-TAATAA-3 (T allele) to 5-TGATAA-3
(C allele), potentially creating a new binding site
for the key erythroid transcription factor GATA-1.
Conventional in vitro electromobility gel shift
assays and supershifts, using an antibody to
GATA-1, demonstrated that this SNP creates a
potential GATA-1 binding site (fig. S3). A
chromatin immunoprecipitation (ChIP) profile
using quantitative real-time PCR across the a-
globin cluster (coordinates 53195 to 185030)
showed that in addition to binding the known
regulatory elements, GATA-1also binds at the C
allele of SNP 195 in vivo (Fig. 3). The C allele
also nucleates the binding of a pentameric
erythroid complex including the transcription
factors SCL, E2A, LMO2, and Ldb-1 (Fig. 3),
which are frequently found with GATA-1 at
erythroid regulatory elements (10, 11). ChIP
profiles using antibodies that recognize modi-
fied histones EH4Ac, H3Ac, and H3K4me2
(Fig. 3 and fig. S4)^ demonstrated that binding
of GATA-1 at the C allele is associated with a
new peak of active chromatin in the a-globin
cluster. Finally, we showed that the C allele,
unlike the T allele, binds RNA polymerase II
(Fig. 3).
Expression of the a-globin genes normally
occurs late in erythropoiesis after what appears
to be a well-defined order of transcription fac-
tor binding to the upstream regulatory elements
(MCS1 to MCS4), followed by recruitment of
the pre-initiation complex and RNA polymer-
ase II. These events are thought to result in the
formation of a DNA/protein complex including
one or more of the regulatory elements and
the a-globin promoter(s) (10). We and others
have shown that the insertion of active
heterologous promoters (such as PGK Neo)
in some regions of the a-globin cluster can
disrupt a-globin expression, probably as a
result of preferential interaction of the heter-
ologous promoter with the upstream ele-
ments, out-competing the endogenous
globin promoters (1214). SNP 195 create
new promoterlike element between t
upstream regulatory elements and their co
nate promoters. This element, when activat
causes significant down-regulation of the a
a2, and a1 genes that lie downstream (F
1D), thereby causing a thalassemia.
These findings not only demonstrate
additional mechanism causing human gene
disease but also illustrate two important poiwhen searching for SNPs that may influen
gene expression (9). First, to distinguish fun
tional from nonfunctional SNPs, it has be
suggested that searches should be concentra
in areas of the genome likely to contain c
regulatory elements (8) (such as multispec
conserved elements). The gain-of-function re
ulatory SNP (rSNP) identified here, located i
region of the a-globin cluster that we kn
may be deleted with no discernible effect on
globin expression (Fig. 1) (15), demonstra
that SNPs in such areas should not be dismiss
as of no potential importance. Second, the u
of densely tiled arrays for analysis of transcrtion and ChIP profiles provides a rapid a
efficient in vivo strategy to distinguish n
functional from functional rSNPs that m
underlie the altered patterns of express
responsible for a wide range of human gene
diseases.
References and Notes1. J. Flint et al., Nat. Genet. 15, 252 (1997).2. J. R. Hughes et al., Proc. Natl. Acad. Sci. U.S.A. 102
9830 (2005).3. D. R. Higgs et al., Genes Dev. 4, 1588 (1990).4. M. H. Steinberg et al., Eds., Disorders of Hemoglobin
(Cambridge Univ. Press, Cambridge, 2001).5. D. R. Higgs et al., Blood 73, 1081 (1989).6. A. P. Jarman, R. D. Nicholls, D. J. Weatherall, J. B. Cle
D. R. Higgs, EMBO J. 5, 1857 (1986).7. R. Sachidanandam et al., Nature 409, 928 (2001).8. J. C. Knight, J. Mol. Med. 83, 97 (2005).
9. T. Pastinen, T. J. Hudson, Science 306, 647 (2004).10. E. Anguita et al., EMBO J. 23, 2841 (2004).11. I. A. Wadman et al., EMBO J. 16, 3145 (1997).12. C. Esperet et al., J. Biol. Chem. 275, 25831 (2000).
13. A. Leder, C. Daugherty, B. Whitney, P. Leder, Blood 9
1275 (1997).14. E. Anguita et al., Blood 100, 3450 (2002).15. P. Winichagoon et al., Nucleic Acids Res. 10, 5853
(1982).16. P. Vyas et al., Cell 69, 781 (1992).
17. Materials and methods are available as supportingmaterial on Science Online.
18. M.D.G. is a Ph.D. student in Pharmacology and
Experimental and Clinical Therapy at the University oTurin, Italy. We thank J. Sloane Stanley, J. Sharpe,J. Green, J. Brown, N. Ventress, K. Clark, and the OxfComputational Biology Research Group for technicalsupport. V.V. is supported by the Thailand Research Fu
Supporting Online Materialwww.sciencemag.org/cgi/content/full/312/5777/1215/DC1Materials and MethodsFigs. S1 to S4
Tables S1 and S2References
21 February 2006; accepted 25 April 2006
10.1126/science.1126431
Fig. 3. Chromatin immunoprecip-itation demonstrating the acquisi-tion of a new transcription factorbinding site (arrowed). The newbinding site is located at coordinate149709. Names of transcriptionfactors and chromatin modificationsare shown at left. Chromatin immu-noprecipitation was performed aspreviously described (10) using prim-ers and antibodies described in thesupporting online material (17). Thedegree of enrichment in a normalindividual (black columns) and in anindividual with the (aa)T/(aa)T geno-type (white columns) is shown on they axis, and coordinates of theregions sampled by QPCR are shownon the x axis. Asterisks indicatewhere insufficient primary cells wereavailable for analysis.
2D 1c16orf35 LUC7LDIST
0
0.2
0.4
0.6
0.8
1
0
0.4
0.8
1.2
1.6
2
0
1
2
3
0
2
4
6
8
10
1031
42
532
40
14
777
1
149
779
150
916
15
278
7
1629
08
163
130
163
55
8
16
587
7
18
499
2
HS
-40
HS
-3
3
GATA1
SCL
Ac-H3
Pol II
11
002
5
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