• Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression.

The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the β-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δβ-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult β-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of β-hemoglobinopathies.

1.
Wienert
B
,
Martyn
GE
,
Funnell
APW
,
Quinlan
KGR
,
Crossley
M
.
Wake-up sleepy gene: reactivating fetal globin for β-hemoglobinopathies
.
Trends Genet.
2018
;
34
(
12
):
927
-
940
.
2.
Martyn
GE
,
Wienert
B
,
Kurita
R
,
Nakamura
Y
,
Quinlan
KGR
,
Crossley
M
.
A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site
.
Blood.
2019
;
133
(
8
):
852
-
856
.
3.
Martyn
GE
,
Wienert
B
,
Yang
L
, et al
.
Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding
.
Nat Genet.
2018
;
50
(
4
):
498
-
503
.
4.
Wienert
B
,
Martyn
GE
,
Kurita
R
,
Nakamura
Y
,
Quinlan
KGR
,
Crossley
M
.
KLF1 drives the expression of fetal hemoglobin in British HPFH
.
Blood.
2017
;
130
(
6
):
803
-
807
.
5.
Wienert
B
,
Funnell
AP
,
Norton
LJ
, et al
.
Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin
.
Nat Commun.
2015
;
6
(
1
):
7085
.
6.
Cancio
MI
,
Aygun
B
,
Chui
DHK
, et al
.
The clinical severity of hemoglobin S/Black (A γδβ)0 -thalassemia
.
Pediatr Blood Cancer.
2017
;
64
(
11
):.
7.
Wang
X
,
Xu
JZ
,
Conrey
A
, et al
.
Whole genome sequence-based haplotypes reveal a single origin of the 1393 bp HBB deletion
.
J Med Genet.
2020
;
57
(
8
):
567
-
570
.
8.
Thein
SL
.
The molecular basis of β-thalassemia
.
Cold Spring Harb Perspect Med.
2013
;
3
(
5
):
a011700
.
9.
Sankaran
VG
,
Xu
J
,
Byron
R
, et al
.
A functional element necessary for fetal hemoglobin silencing
.
N Engl J Med.
2011
;
365
(
9
):
807
-
814
.
10.
Fornari
TA
,
Lanaro
C
,
Albuquerque
DM
,
Ferreira
R
,
Costa
FF
.
Featured Article: Modulation of fetal hemoglobin in hereditary persistence of fetal hemoglobin deletion type-2, compared to Sicilian δβ-thalassemia, by BCL11A and SOX6-targeting microRNAs
.
Exp Biol Med (Maywood).
2017
;
242
(
3
):
267
-
274
.
11.
Katsantoni
EZ
,
Langeveld
A
,
Wai
AW
, et al
.
Persistent gamma-globin expression in adult transgenic mice is mediated by HPFH-2, HPFH-3, and HPFH-6 breakpoint sequences
.
Blood.
2003
;
102
(
9
):
3412
-
3419
.
12.
Gaensler
KM
,
Zhang
Z
,
Lin
C
,
Yang
S
,
Hardt
K
,
Flebbe-Rehwaldt
L
.
Sequences in the (A)gamma-delta intergenic region are not required for stage-specific regulation of the human beta-globin gene locus
.
Proc Natl Acad Sci USA.
2003
;
100
(
6
):
3374
-
3379
.
13.
Antoniani
C
,
Meneghini
V
,
Lattanzi
A
, et al
.
Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus
.
Blood.
2018
;
131
(
17
):
1960
-
1973
.
14.
Xiang
P
,
Han
H
,
Barkess
G
, et al
.
Juxtaposition of the HPFH2 enhancer is not sufficient to reactivate the gamma-globin gene in adult erythropoiesis
.
Hum Mol Genet.
2005
;
14
(
20
):
3047
-
3056
.
15.
Papanikolaou
E
,
Georgomanoli
M
,
Stamateris
E
, et al
.
The new self-inactivating lentiviral vector for thalassemia gene therapy combining two HPFH activating elements corrects human thalassemic hematopoietic stem cells
.
Hum Gene Ther.
2012
;
23
(
1
):
15
-
31
.
16.
Chung
JE
,
Magis
W
,
Vu
J
, et al
.
CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells
.
PLoS One.
2019
;
14
(
1
):
e0208237
.
17.
Manca
L
,
Masala
B
.
Disorders of the synthesis of human fetal hemoglobin
.
IUBMB Life.
2008
;
60
(
2
):
94
-
111
.
18.
Choi
OR
,
Engel
JD
.
Developmental regulation of beta-globin gene switching
.
Cell.
1988
;
55
(
1
):
17
-
26
.
19.
Deng
W
,
Rupon
JW
,
Krivega
I
, et al
.
Reactivation of developmentally silenced globin genes by forced chromatin looping
.
Cell.
2014
;
158
(
4
):
849
-
860
.
20.
Kurita
R
,
Suda
N
,
Sudo
K
, et al
.
Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells
.
PLoS One.
2013
;
8
(
3
):
e59890
.
21.
Doerfler
PA
,
Feng
R
,
Li
Y
, et al
.
Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin
.
Nat Genet.
2021
;
53
(
8
):
1177
-
1186
.
22.
Ran
FA
,
Hsu
PD
,
Wright
J
,
Agarwala
V
,
Scott
DA
,
Zhang
F
.
Genome engineering using the CRISPR-Cas9 system
.
Nat Protoc.
2013
;
8
(
11
):
2281
-
2308
.
23.
Xu
P
,
Scott
DC
,
Xu
B
, et al
.
FBXO11-mediated proteolysis of BAHD1 relieves PRC2-dependent transcriptional repression in erythropoiesis
.
Blood.
2021
;
137
(
2
):
155
-
167
.
24.
Huang
P
,
Keller
CA
,
Giardine
B
, et al
.
Comparative analysis of three-dimensional chromosomal architecture identifies a novel fetal hemoglobin regulatory element
.
Genes Dev.
2017
;
31
(
16
):
1704
-
1713
.
25.
Prakobkaew
N
,
Fucharoen
S
,
Fuchareon
G
,
Siriratmanawong
N
.
Phenotypic expression of Hb F in common high Hb F determinants in Thailand: roles of α-thalassemia, 5′ δ-globin BCL11A binding region and 3′ β-globin enhancer
.
Eur J Haematol.
2014
;
92
(
1
):
73
-
79
.
26.
Ojwang
PJ
,
Nakatsuji
T
,
Gardiner
MB
,
Reese
AL
,
Gilman
JG
,
Huisman
TH
.
Gene deletion as the molecular basis for the Kenya-G gamma-HPFH condition
.
Hemoglobin.
1983
;
7
(
2
):
115
-
123
.
27.
Mayuranathan
T
,
Rayabaram
J
,
Das
R
, et al
.
Identification of rare and novel deletions that cause (δβ)0-thalassaemia and hereditary persistence of foetal haemoglobin in Indian population
.
Eur J Haematol.
2014
;
92
(
6
):
514
-
520
.
28.
Pissard
S
,
Raclin
V
,
Lacan
P
, et al
.
Characterization of three new deletions in the β-globin gene cluster during a screening survey in two French urban areas
.
Clin Chim Acta.
2013
;
415
:
35
-
40
.
29.
Nadkarni
A
,
Wadia
M
,
Gorakshakar
A
,
Kiyama
R
,
Colah
RB
,
Mohanty
D
.
Molecular characterization of delta beta-thalassemia and hereditary persistence of fetal hemoglobin in the Indian population
.
Hemoglobin.
2008
;
32
(
5
):
425
-
433
.
30.
Kutlar
A
,
Gardiner
MB
,
Headlee
MG
, et al
.
Heterogeneity in the molecular basis of three types of hereditary persistence of fetal hemoglobin and the relative synthesis of the G gamma and A gamma types of gamma chain
.
Biochem Genet.
1984
;
22
(
1-2
):
21
-
35
.
31.
Saglio
G
,
Camaschella
C
,
Serra
A
, et al
.
Italian type of deletional hereditary persistence of fetal hemoglobin
.
Blood.
1986
;
68
(
3
):
646
-
651
.
32.
Camaschella
C
,
Serra
A
,
Gottardi
E
, et al
.
A new hereditary persistence of fetal hemoglobin deletion has the breakpoint within the 3′ beta-globin gene enhancer
.
Blood.
1990
;
75
(
4
):
1000
-
1005
.
33.
Henthorn
PS
,
Smithies
O
,
Mager
DL
.
Molecular analysis of deletions in the human beta-globin gene cluster: deletion junctions and locations of breakpoints
.
Genomics.
1990
;
6
(
2
):
226
-
237
.
34.
Anagnou
NP
,
Papayannopoulou
T
,
Nienhuis
AW
,
Stamatoyannopoulos
G
.
Molecular characterization of a novel form of (A gamma delta beta)zero-thalassemia deletion with a 3′ breakpoint close to those of HPFH-3 and HPFH-4: insights for a common regulatory mechanism
.
Nucleic Acids Res.
1988
;
16
(
13
):
6057
-
6066
.
35.
George
E
,
Faridah
K
,
Trent
RJ
,
Padanilam
BJ
,
Huang
HJ
,
Huisman
TH
.
Homozygosity for a new type of G gamma (A gamma delta beta)zero-thalassemia in a Malaysian male
.
Hemoglobin.
1986
;
10
(
4
):
353
-
363
.
36.
Lacerra
G
,
Prezioso
R
,
Musollino
G
,
Piluso
G
,
Mastrullo
L
,
De Angioletti
M
.
Identification and molecular characterization of a novel 55-kb deletion recurrent in southern Italy: the Italian (G) γ((A) γδβ)°-thalassemia
.
Eur J Haematol.
2013
;
90
(
3
):
214
-
219
.
37.
He
S
,
Wei
Y
,
Lin
L
, et al
.
The prevalence and molecular characterization of (δβ)0- thalassemia and hereditary persistence of fetal hemoglobin in the Chinese Zhuang population
.
J Clin Lab Anal.
2018
;
32
(
3
):
e22304
.
38.
Yamashiro
Y
,
Hattori
Y
,
Okayama
N
, et al
.
A novel (g)gamma(a)gamma(deltabeta)O-thalassemia with a 27 kb deletion
.
Hemoglobin.
2005
;
29
(
3
):
197
-
208
.
39.
Shiokawa
S
,
Yamada
H
,
Takihara
Y
, et al
.
Molecular analysis of Japanese delta beta-thalassemia
.
Blood.
1988
;
72
(
5
):
1771
-
1776
.
40.
Svasti
S
,
Paksua
S
,
Nuchprayoon
I
,
Winichagoon
P
,
Fucharoen
S
.
Characterization of a novel deletion causing (deltabeta)0-thalassemia in a Thai family
.
Am J Hematol.
2007
;
82
(
2
):
155
-
161
.
41.
Vives-Corrons
JL
,
Pujades
MA
,
Miguel-García
A
,
Miguel-Sosa
A
,
Cambiazzo
S
.
Rapid detection of Spanish (delta beta)zero-thalassemia deletion by polymerase chain reaction
.
Blood.
1992
;
80
(
6
):
1582
-
1585
.
42.
Anagnou
NP
,
Papayannopoulou
T
,
Stamatoyannopoulos
G
,
Nienhuis
AW
.
Structurally diverse molecular deletions in the beta-globin gene cluster exhibit an identical phenotype on interaction with the beta S-gene
.
Blood.
1985
;
65
(
5
):
1245
-
1251
.
43.
Trent
RJ
,
Svirklys
L
,
Jones
P
.
Thai (delta beta)0-thalassemia and its interaction with gamma-thalassemia
.
Hemoglobin.
1988
;
12
(
2
):
101
-
114
.
44.
Ottolenghi
S
,
Giglioni
B
,
Taramelli
R
, et al
.
Molecular comparison of delta beta-thalassemia and hereditary persistence of fetal hemoglobin DNAs: evidence of a regulatory area?
Proc Natl Acad Sci USA.
1982
;
79
(
7
):
2347
-
2351
.
45.
Zhang
JW
,
Stamatoyannopoulos
G
,
Anagnou
NP
.
Laotian (delta beta) (0)-thalassemia: molecular characterization of a novel deletion associated with increased production of fetal hemoglobin
.
Blood.
1988
;
72
(
3
):
983
-
988
.
46.
Palena
A
,
Blau
A
,
Stamatoyannopoulos
G
,
Anagnou
NP
.
Eastern European (delta beta) zero-thalassemia: molecular characterization of a novel 9.1-kb deletion resulting in high levels of fetal hemoglobin in the adult
.
Blood.
1994
;
83
(
12
):
3738
-
3745
.
47.
Craig
JE
,
Kelly
SJ
,
Barnetson
R
,
Thein
SL
.
Molecular characterization of a novel 10.3 kb deletion causing beta-thalassaemia with unusually high Hb A2
.
Br J Haematol.
1992
;
82
(
4
):
735
-
744
.
48.
Gilman
JG
,
Huisman
TH
,
Abels
J
.
Dutch beta 0-thalassaemia: a 10 kilobase DNA deletion associated with significant gamma-chain production
.
Br J Haematol.
1984
;
56
(
2
):
339
-
348
.
49.
Popovich
BW
,
Rosenblatt
DS
,
Kendall
AG
,
Nishioka
Y
.
Molecular characterization of an atypical beta-thalassemia caused by a large deletion in the 5′ beta-globin gene region
.
Am J Hum Genet.
1986
;
39
(
6
):
797
-
810
.
50.
Dimovski
AJ
,
Efremov
DG
,
Jankovic
L
,
Plaseska
D
,
Juricic
D
,
Efremov
GD
.
A beta zero-thalassaemia due to a 1605 bp deletion of the 5′ beta-globin gene region
.
Br J Haematol.
1993
;
85
(
1
):
143
-
147
.
51.
Anand
R
,
Boehm
CD
,
Kazazian
HH
Jr
,
Vanin
EF
.
Molecular characterization of a beta zero-thalassemia resulting from a 1.4 kilobase deletion
.
Blood.
1988
;
72
(
2
):
636
-
641
.
52.
Thein
SL
.
Molecular basis of β thalassemia and potential therapeutic targets
.
Blood Cells Mol Dis.
2018
;
70
:
54
-
65
.
53.
Orkin
SH
,
Kolodner
R
,
Michelson
A
,
Husson
R
.
Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene
.
Proc Natl Acad Sci USA.
1980
;
77
(
6
):
3558
-
3562
.
54.
Orkin
SH
,
Old
JM
,
Weatherall
DJ
,
Nathan
DG
.
Partial deletion of beta-globin gene DNA in certain patients with beta 0-thalassemia
.
Proc Natl Acad Sci USA.
1979
;
76
(
5
):
2400
-
2404
.
55.
Shah
PS
,
Shah
ND
,
Ray
HSP
, et al
.
Mutation analysis of β-thalassemia in East-Western Indian population: a recent molecular approach
.
Appl Clin Genet.
2017
;
10
:
27
-
35
.
56.
Martyn
GE
,
Quinlan
KGR
,
Crossley
M
.
The regulation of human globin promoters by CCAAT box elements and the recruitment of NF-Y
.
Biochim Biophys Acta Gene Regul Mech.
2017
;
1860
(
5
):
525
-
536
.
57.
Traxler
EA
,
Yao
Y
,
Wang
YD
, et al
.
A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition
.
Nat Med.
2016
;
22
(
9
):
987
-
990
.
58.
Métais
JY
,
Doerfler
PA
,
Mayuranathan
T
, et al
.
Genome editing of HBG1 and HBG2 to induce fetal hemoglobin
.
Blood Adv.
2019
;
3
(
21
):
3379
-
3392
.
59.
Huisman
TH
.
Levels of Hb A2 in heterozygotes and homozygotes for beta-thalassemia mutations: influence of mutations in the CACCC and ATAAA motifs of the beta-globin gene promoter
.
Acta Haematol.
1997
;
98
(
4
):
187
-
194
.
60.
Maragoudaki
E
,
Kanavakis
E
,
Traeger-Synodinos
J
, et al
.
Molecular, haematological and clinical studies of the -101 C −−> T substitution of the beta-globin gene promoter in 25 beta-thalassaemia intermedia patients and 45 heterozygotes
.
Br J Haematol.
1999
;
107
(
4
):
699
-
706
.
61.
Badens
C
,
Jassim
N
,
Martini
N
,
Mattei
JF
,
Elion
J
,
Lena-Russo
D
.
Characterization of a new polymorphism, IVS-I-108 (T−−>C), and a new beta-thalassemia mutation, -27 (A−−>T), discovered in the course of a prenatal diagnosis
.
Hemoglobin.
1999
;
23
(
4
):
339
-
344
.
62.
Moassas
F
,
Alabloog
A
,
Murad
H
.
Description of a rare β-globin gene mutation: -86 (C>G) (HBB: c.-136C>G) observed in a Syrian family
.
Hemoglobin.
2018
;
42
(
3
):
203
-
205
.
63.
Gilman
JG
,
Manca
L
,
Frogheri
L
, et al
.
Mild beta+(-87)-thalassemia CACCC box mutation is associated with elevated fetal hemoglobin expression in cis
.
Am J Hematol.
1994
;
45
(
3
):
265
-
267
.
64.
Carter
D
,
Chakalova
L
,
Osborne
CS
,
Dai
YF
,
Fraser
P
.
Long-range chromatin regulatory interactions in vivo
.
Nat Genet.
2002
;
32
(
4
):
623
-
626
.
65.
Okamura
E
,
Matsuzaki
H
,
Campbell
AD
,
Engel
JD
,
Fukamizu
A
,
Tanimoto
K
.
All of the human beta-type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice
.
FASEB J.
2009
;
23
(
12
):
4335
-
4343
.
66.
Cho
SW
,
Xu
J
,
Sun
R
, et al
.
Promoter of lncRNA gene PVT1 is a tumor-suppressor DNA boundary element
.
Cell.
2018
;
173
(
6
):
1398
-
1412.e22
.
67.
Chen
H
,
Levo
M
,
Barinov
L
,
Fujioka
M
,
Jaynes
JB
,
Gregor
T
.
Dynamic interplay between enhancer-promoter topology and gene activity
.
Nat Genet.
2018
;
50
(
9
):
1296
-
1303
.
68.
Fukaya
T
,
Lim
B
,
Levine
M
.
Enhancer control of transcriptional bursting
.
Cell.
2016
;
166
(
2
):
358
-
368
.
69.
Lower
KM
,
Hughes
JR
,
De Gobbi
M
, et al
.
Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition
.
Proc Natl Acad Sci USA.
2009
;
106
(
51
):
21771
-
21776
.
70.
Ivaldi
MS
,
Diaz
LF
,
Chakalova
L
,
Lee
J
,
Krivega
I
,
Dean
A
.
Fetal γ-globin genes are regulated by the BGLT3 long noncoding RNA locus
.
Blood.
2018
;
132
(
18
):
1963
-
1973
.
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