Reversing the Hemoglobin Switch
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clinical implications of basic research The new england journal of medicine n engl j med 363;23 nejm.org december 2, 2010 2258 Reversing the Hemoglobin Switch Vijay G. Sankaran, M.D., Ph.D., and David G. Nathan, M.D. Natural observations can often provide important clues for how therapies can be developed to ame- liorate disease. Disorders of β-hemoglobin, includ- ing sickle cell disease and β-thalassemia, are good examples of this maxim, since a variety of clini- cal observations have shown that increased lev- els of fetal hemoglobin can ameliorate the sever- ity of these diseases. This observation has led to the decades-long hunt for strategies to increase the expression of fetal hemoglobin in a targeted manner. There has been some success in develop- ing drugs to achieve such increased expression of fetal hemoglobin for therapeutic benefit, as ex- emplified by the use of hydroxyurea. However, the molecular basis for the efficacy of these agents remains obscure, and such drugs have been shown to be only moderately effective in a subgroup of patients. 1 Over the course of human gestation, the pre- dominant hemoglobin expressed in the fetus is fetal hemoglobin, which is composed of two adult α-globin chains and two γ-globin chains. Around the time of birth, there is a switch from fetal he- moglobin to adult hemoglobin expression, which is mediated by a shift from γ-globin to adult β-globin production at the β-globin gene locus (Fig. 1 ). By mid-to-late infancy, the production of γ-globin constitutes a minor fraction of the pro- duction of adult β-globin. As a result, fetal hemo- globin accounts for less than 1% of the total hemoglobin after this point. Although the molec- ular mechanisms mediating this switch in human ontogeny have been widely investigated, until re- cently the molecular basis for this switch re- mained obscure. A recent study by Zhou and colleagues 2 pro- vides new insight into the molecular mechanisms that mediate this developmental switch by show- ing that the transcription factor KLF1 (Krüppel- like factor 1) is an important regulator of this process. Genomewide association studies have spawned further studies to identify the molecu- lar mediators of this process. One locus that has been implicated is within the gene encoding tran- scription factor BCL11A (B-cell lymphoma/leu- kemia 11A), which has led to work showing that BCL11A is an important developmental regulator of the fetal-to-adult hemoglobin switch in humans by serving as a repressor of the γ-globin gene. 3 BCL11A appears to act directly within the β-globin locus, along with a set of partner proteins. How- ever, the factors that allow for the developmental regulation of BCL11A expression and that may also affect other aspects of globin gene regula- tion have remained elusive. Zhou and colleagues began by developing a mouse model that had low levels of expression of KLF1, circumventing the early lethality seen in Figure 1 (facing page). Molecular Rheostats of the Globin Switch. The human β-globin gene locus, which is located on chromosome 11, has a series of five genes that are de- velopmentally expressed at distinct stages of human ontogeny (bottom of Panel A). The ε-globin gene is re- stricted to the transiently expressed embryonic primi- tive lineage of red cells produced in the first several weeks of gestation. The γ-globin genes encode the genes that uniquely form fetal hemoglobin, which is the pre- dominant hemoglobin during the course of gestation. There is a switch to expression of the adult β-globin gene around the time of birth (Panel B). B-cell lym- phoma/leukemia 11A (BCL11A) and its protein part- ners, including GATA binding protein 1 (GATA1), zinc finger protein multitype 1 (ZFPM1, or FOG1), and the nucleosome remodeling and deacetylase (NuRD) com- plex, bind to sequences within the globin locus (shown as tan ovals in Panel A) and repress the expression of the γ-globin genes. Krüppel-like factor 1 (KLF1) inter- acts with this process by positively regulating the ex- pression of BCL11A (green arrow in Panel A) and also by directly binding to and promoting transcription of the adult β-globin gene. It is unknown whether KLF1 affects fetal hemoglobin expression through more global effects on erythropoiesis (top of Panel A), which may also be targeted by other factors associated with fetal hemoglobin (e.g., MYB). HSs denotes DNase I hypersensitive sites, and LCR locus control region. The New England Journal of Medicine Downloaded from nejm.org at PENN STATE UNIVERSITY on March 8, 2013. For personal use only. No other uses without permission. Copyright © 2010 Massachusetts Medical Society. All rights reserved.
Transcript of Reversing the Hemoglobin Switch
clinical implications of basic research
T h e n e w e ngl a nd j o u r na l o f m e dic i n e
n engl j med 363;23 nejm.org december 2, 20102258
Reversing the Hemoglobin SwitchVijay G. Sankaran, M.D., Ph.D., and David G. Nathan, M.D.
Natural observations can often provide important clues for how therapies can be developed to ame-liorate disease. Disorders of β-hemoglobin, includ-ing sickle cell disease and β-thalassemia, are good examples of this maxim, since a variety of clini-cal observations have shown that increased lev-els of fetal hemoglobin can ameliorate the sever-ity of these diseases. This observation has led to the decades-long hunt for strategies to increase the expression of fetal hemoglobin in a targeted manner. There has been some success in develop-ing drugs to achieve such increased expression of fetal hemoglobin for therapeutic benefit, as ex-emplified by the use of hydroxyurea. However, the molecular basis for the efficacy of these agents remains obscure, and such drugs have been shown to be only moderately effective in a subgroup of patients.1
Over the course of human gestation, the pre-dominant hemoglobin expressed in the fetus is fetal hemoglobin, which is composed of two adult α-globin chains and two γ-globin chains. Around the time of birth, there is a switch from fetal he-moglobin to adult hemoglobin expression, which is mediated by a shift from γ-globin to adult β-globin production at the β-globin gene locus (Fig. 1). By mid-to-late infancy, the production of γ-globin constitutes a minor fraction of the pro-duction of adult β-globin. As a result, fetal hemo-globin accounts for less than 1% of the total hemoglobin after this point. Although the molec-ular mechanisms mediating this switch in human ontogeny have been widely investigated, until re-cently the molecular basis for this switch re-mained obscure.
A recent study by Zhou and colleagues2 pro-vides new insight into the molecular mechanisms that mediate this developmental switch by show-ing that the transcription factor KLF1 (Krüppel-like factor 1) is an important regulator of this process. Genomewide association studies have spawned further studies to identify the molecu-
lar mediators of this process. One locus that has been implicated is within the gene encoding tran-scription factor BCL11A (B-cell lymphoma/leu-kemia 11A), which has led to work showing that BCL11A is an important developmental regulator of the fetal-to-adult hemoglobin switch in humans by serving as a repressor of the γ-globin gene.3 BCL11A appears to act directly within the β-globin locus, along with a set of partner proteins. How-ever, the factors that allow for the developmental regulation of BCL11A expression and that may also affect other aspects of globin gene regula-tion have remained elusive.
Zhou and colleagues began by developing a mouse model that had low levels of expression of KLF1, circumventing the early lethality seen in
Figure 1 (facing page). Molecular Rheostats of the Globin Switch.
The human β-globin gene locus, which is located on chromosome 11, has a series of five genes that are de-velopmentally expressed at distinct stages of human ontogeny (bottom of Panel A). The ε-globin gene is re-stricted to the transiently expressed embryonic primi-tive lineage of red cells produced in the first several weeks of gestation. The γ-globin genes encode the genes that uniquely form fetal hemoglobin, which is the pre-dominant hemoglobin during the course of gestation. There is a switch to expression of the adult β-globin gene around the time of birth (Panel B). B-cell lym-phoma/leukemia 11A (BCL11A) and its protein part-ners, including GATA binding protein 1 (GATA1), zinc finger protein multitype 1 (ZFPM1, or FOG1), and the nucleosome remodeling and deacetylase (NuRD) com-plex, bind to sequences within the globin locus (shown as tan ovals in Panel A) and repress the expression of the γ-globin genes. Krüppel-like factor 1 (KLF1) inter-acts with this process by positively regulating the ex-pression of BCL11A (green arrow in Panel A) and also by directly binding to and promoting transcription of the adult β-globin gene. It is unknown whether KLF1 affects fetal hemoglobin expression through more global effects on erythropoiesis (top of Panel A), which may also be targeted by other factors associated with fetal hemoglobin (e.g., MYB). HSs denotes DNase I hypersensitive sites, and LCR locus control region.
The New England Journal of Medicine Downloaded from nejm.org at PENN STATE UNIVERSITY on March 8, 2013. For personal use only. No other uses without permission.
Copyright © 2010 Massachusetts Medical Society. All rights reserved.
n engl j med 363;23 nejm.org december 2, 2010 2259
clinical implications of basic research
The New England Journal of Medicine Downloaded from nejm.org at PENN STATE UNIVERSITY on March 8, 2013. For personal use only. No other uses without permission.
Copyright © 2010 Massachusetts Medical Society. All rights reserved.
clinical implications of basic research
n engl j med 363;23 nejm.org december 2, 20102260
mouse models that have a complete knockout of this gene. On knocking down KLF1 expression in mice, the investigators observed a marked in-crease in the expression of the mouse embryonic globin genes (which are evolutionarily related to fetal hemoglobin genes4) and human γ-globin genes (from a human β-globin locus transgene).2 The expression of BCL11A in these mice was markedly repressed, as compared with expression of the protein in wild-type controls. This obser-vation, together with the finding that KLF1 binds directly to the BCL11A promoter, suggests that KLF1 positively regulates BCL11A expression. Moreover, KLF1 also binds directly within the β-globin locus, suggesting that it may directly reg-ulate globin gene expression. Zhou et al. also test-ed the effect of KLF1 knockdown in human ery-throid progenitor cells and observed a reduction in BCL11A expression and a concomitant increase in γ-globin expression — a reassuring finding, given that aspects of globin gene regulation are divergent between mice and humans.4
Also reassuring is an observation by Borg and colleagues5 that a nonsense mutation in KLF1 is associated with hereditary persistence of fetal he-moglobin in a large Maltese family, suggesting that haploinsufficiency of KLF1 increases fetal hemoglobin production in vivo without ostensi-bly disturbing erythropoiesis. However, carriers of this mutation had extremely variable levels of fetal hemoglobin, ranging from 3 to 20%, sug-gesting that the role of KLF1 in these changes may be complicated by the presence of other fac-tors that probably influence this phenotype.
Lowering the levels of KLF1 may be an impor-
tant way to increase fetal hemoglobin in patients. It is possible that small molecules or small inter-fering RNAs targeting KLF1 may be effective in inducing fetal hemoglobin production in humans. KLF1 is a particularly attractive candidate, given the exclusive expression in the erythroid lin-eage. However, caution is necessary. KLF1 has a broad role in erythropoiesis, and it is possible that disruption of its function may interfere with this process. With this caveat in mind, it is clear that further work is necessary to deter-mine the best means of disrupting the regulatory axis involving KLF1, BCL11A, and fetal hemoglo-bin. Further investigation of this pathway and other factors, such as the transcription factor MYB, that have been implicated in fetal hemo-globin silencing in genetic studies may fuel tar-geted strategies for fetal hemoglobin induction.
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
From Children’s Hospital Boston, Dana–Farber Cancer Insti-tute, and Harvard Medical School — all in Boston.
1. Platt OS. Hydroxyurea for the treatment of sickle cell anemia. N Engl J Med 2008;358:1362-9.2. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching. Nat Genet 2010;42:742-4.3. Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglo-bin expression is regulated by the developmental stage-specific repressor BCL11A. Science 2008;322:1839-42.4. Sankaran VG, Xu J, Ragoczy T, et al. Developmental and species-divergent globin switching are driven by BCL11A. Nature 2009;460:1093-7.5. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsuffi-ciency for the erythroid transcription factor KLF1 causes heredi-tary persistence of fetal hemoglobin. Nat Genet 2010;42:801-5.Copyright © 2010 Massachusetts Medical Society.
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