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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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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

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5

* * * *

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