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Mol Cell Biol. 2013 April; 33(8): 1687–1695.
PMCID: PMC3624254

Disruption of the Hbs1l-Myb Locus Causes Hereditary Persistence of Fetal Hemoglobin in a Mouse Model


The human β-globin locus is comprised of embryonic, fetal, and adult globin genes, each of which is expressed at distinct stages of pre- and postnatal development. Functional defects in globin proteins or expression results in mild to severe anemia, such as in sickle-cell disease or β-thalassemia, but the clinical symptoms of both disorders are ameliorated by persistent expression of the fetal globin genes. Recent genome-wide association studies (GWAS) identified the intergenic region between the HBS1L and MYB loci as a candidate modifier of fetal hemoglobin expression in adults. However, it remains to be clarified whether the enhancer activity within the HBS1L-MYB regulatory domain contributes to the production of fetal hemoglobin in adults. Here we report a new mouse model of hereditary persistence of fetal hemoglobin (HPFH) in which a transgene was randomly inserted into the orthologous murine Hbs1l-Myb locus. This mutant mouse exhibited typically elevated expression of embryonic globins and hematopoietic parameters similar to those observed in human HPFH. These results support the contention that mutation of the HBS1L-MYB genomic domain is responsible for elevated expression of the fetal globin genes, and this model serves as an important means for the analysis of networks that regulate fetal globin gene expression.


The human β-type globin locus consists of embryonic, fetal, and adult globin genes. The embryonic and fetal globin genes are typically expressed in the yolk sac and fetal liver, respectively (1). In the adult spleen and bone marrow, the adult globin gene is activated, while the embryonic and fetal globin genes are silenced (1). Two mechanisms have been proposed to regulate silencing of the embryonic and fetal globin genes in the adult: autonomous active silencing of the embryonic and fetal globin genes by gene autonomous recruitment of repressors and passive silencing due to competitive sequestration of locus control region (LCR) enhancer activity by the adult β-globin gene (1). Reactivation of the embryonic and/or fetal globin genes could be a critically useful ploy for establishing therapies for hereditary anemias and the associated pathophysiology caused by adult globin gene mutations, such as sickle-cell disease and β-thalassemia (25).

A couple of genome-wide association study (GWAS) experiments independently identified two candidate regions that might influence the expression of fetal hemoglobin in adults (6, 7). One is the BCL11A locus. BCL11A encodes a well-characterized hematopoietic transcription factor (8), and persistent expression of γ-globin was observed in BCL11A-deficient human and murine erythroid cells, demonstrating a contribution of BCL11A to hereditary persistence of fetal hemoglobin (HPFH) (9, 10). The other is the intergenic region between the human HBS1L and MYB genes. A 24-kbp linkage disequilibrium (LD) block located 33 kbp 5′ to HBS1L and 65 kbp 5′ to MYB was found to exhibit strong association with elevated levels of fetal hemoglobin (7), and the LD block was designated the HBS1L-MYB intergenic polymorphism block 2 (HMIP2). HPFH individuals with HMIP2 show slight macrocytic anemia and thrombocytosis, indicating that HMIP2 is also associated with erythrocyte and platelet traits (11). Several recent GWAS experiments further indicate a similar association of the HBS1L-MYB intergenic region not only in the European population but also in the Japanese population, indicating a common HBS1L-MYB-type HPFH phenotype arising independently in different human populations (1214). Chromatin immunoprecipitation on a microarray (ChIP-chip) and ChIP-sequencing (ChIP-seq) analyses identified erythroid-specific histone acetylation sites, GATA1-binding sites, and RNA polymerase II-binding sites in HMIP2, suggesting that HMIP2 contains regulatory elements for the HBS1L or MYB gene in erythroid cells (15, 16). However, molecular mechanisms underlying these genetic events in the HBS1L-MYB intergenic region remain to be elucidated.

We previously isolated a transgenic (Tg) mouse line harboring a transgene encoding the mouse erythropoietin receptor (Epor) cDNA driven by the Gata1 hematopoietic regulatory domain (G1HRD) (17, 18). This transgene was inserted into the intergenic region between the Hbs1l and Myb genes, and homozygous Tg mice (Hbs1l-MybTg/Tg) displayed a marked reduction of Myb expression in the megakaryocytic and erythroid lineages, including in megakaryocyte/erythrocyte progenitors (MEPs). Consequently, megakaryocyte/erythrocyte bifurcation of MEPs was skewed toward the megakaryocytic lineage, and therefore the Tg mice exhibited anemia and thrombocytosis (17). Given the location of the transgene insertion and its association with dyserythropoiesis, we surmised that there exists functional overlap between the hematopoietic phenotypes of these Tg mice (17) and those arising from mutation of the Hbs1l-Myb locus (11).

Therefore, in order to elucidate the functional contribution of the Hbs1l-Myb intergenic region, in this study we have examined globin gene expression in these Tg mice and the Myb contribution to globin gene regulation. We found that the mouse bearing the Tg insertion in the Hbs1l-Myb intergenic region expressed approximately 4- and 20-fold-higher levels of mouse embryonic εy- and βh1-globins, respectively, than littermate control mice did. The Tg mice also showed macrocytic anemia and thrombocytosis. These results thus demonstrate that the Hbs1l-Myb intergenic region is actually responsible for this persistent expression of embryonic globins after birth. Of note, in the mutant mouse, the expression levels of both flanking genes of HMIP2, Hbs1l, and Myb were reduced, as observed in human HPFH individuals, and mice with compound heterozygosity for the Tg insertion and Myb knockout showed elevated expression of embryonic globin genes. These results argue that Myb contributes to the HPFH-like phenotypes observed in the mice harboring the impaired Hbs1l-Myb intergenic region. We also found that Myb activates the DRED (direct repeat erythroid definitive) complex, a known repressor of embryonic and fetal globin genes in the adult (19, 20), suggesting the involvement of the DRED complex in this regulatory pathway.



The Hbs1l-Myb insertion mutant mouse (17) and the Myb knockout mouse (21) were described previously. The former mice harbored a transgene encoding the mouse erythropoietin receptor (Epor) cDNA driven by G1HRD (18). All mice were handled according to the regulations of the Standards for Human Care and Use of Laboratory Animals of Tohoku University (23) and Guidelines for Proper Conduct of Animal Experiments of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (24).

Quantification of genome copy number.

Genomic DNA was prepared from the tails of non-Tg and heterozygous mutant mice. Quantitative PCR (qPCR) was performed with these DNA samples in triplicate using Sybr green. The primers are described in Table S1 in the supplemental material.

Hematological analysis.

Hematological indices were measured using an automatic blood cell analyzer (Nihon Koden).

Flow cytometry and cell sorting.

Sorting and analyses of spleen cells were performed using the fluorescence-activated cell sorter (FACS) Aria (BD Biosciences). Single-cell suspensions were prepared from the spleens of neonates. The cells were incubated in 2% fetal bovine serum–phosphate-buffered saline containing Fc block (CD16/CD32) (BD Pharmingen) for 15 min on ice and stained with CD71-fluorescein isothiocyanate (FITC) and Ter119-phycoerythrin (PE) antibodies (eBioscience) for 30 min on ice.

RNA extraction and RT-PCR.

Total RNA was extracted with RNAiso (TaKaRa) and reverse transcribed with SuperScript III (Invitrogen) according to the manufacturer's instruction. Quantitative reverse transcription-PCR (qRT-PCR) was run on ABI7300 (Applied Biosystems). Primers are described in Table S1 in the supplemental material.

Cell culture.

Primary human CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center. Culture and differentiation of primary human CD34+ cells were performed as previously described (9, 25).

shRNA knockdown.

Lentiviral constructs for expression of short hairpin RNA (shRNA) targeting human MYB in the pLKO vector (TRCN0000040058 and TRCN0000009853) were obtained from Sigma-Aldrich. The empty vector pLKO.1 was used to produce control lentiviruses. Lentiviruses were prepared and infection of cells was carried out as described previously (26).

Statistical analysis.

Data are presented as means ± standard deviations (SD). The Student t test (two tailed) was used to calculate the statistical significance (P) of mouse analysis. The paired t test (two tailed) was used to calculate the P values for human CD34+ cell analysis.


A transgene insertion site is located within orthologous region of the HPFH-associated LD block.

We previously isolated a mouse line harboring a transgene in the intergenic region between the Hbs1l and Myb genes, which corresponded to the LD block within the human HBS1L-MYB intergenic region or HMIP2 (7) (Fig. 1A). In this study, we first characterized the insertion position and copy number of the transgene precisely in this line of mice (17). The copy number of transgenes was determined by quantitative detection of G1HRD within the transgene by qPCR (Fig. 1B, white bar). Because the Gata1 gene is located on the X chromosome, male and female mice have either one or two copies, respectively. Male and female Tg heterozygous mice were determined to have 5 or 6 copies of the Gata1 promoter, respectively, indicating that the initial transgene integration event resulted in the acquisition of 4 Tg copies (Fig. 1C).

Fig 1
The Tg insertion site is located within the HPFH-associated LD block. (A) Overview of the human HBS1L-MYB locus. The genome position was derived from the NCBI build 36/hg18 reference genome sequence. Mammalian conservation data were acquired from the ...

We next assessed the gene configuration in close proximity to the Tg insertion site. As has been reported (17), the Tg is inserted between the Hbs1l and Myb genes in mouse chromosome 10 (Fig. 1D, top panel). We have assigned the Hbs1l side of the gene by sequencing (bottom panel, right end of the hatched bar). However, we were unable to determine the insertion site relative to Myb by the gene sequencing. Therefore, we decided to assess the copy number of various DNA segments flanking the disruption point. To this end, copy numbers of six segments in the locus were examined (bottom panel, white bars 1 to 6) by qPCR using genomic DNA recovered from either heterozygous mutant (Hbs1l-MybTg/+) or wild-type mice. While two genomic copies were detected for segments 4 through 6, only a single copy of segments 1 through 3 was detected in the genome of Hbs1l-MybTg/+ mice (Fig. 1E), indicating that segments 1 to 3 are deleted in the Tg allele (Fig. 1D, dotted red line). The deleted segment contains a highly conserved noncoding sequence (CNS) (Fig. 1D, red box), whose human counterpart is located within HMIP2 (Fig. 1A, red box). These results thus demonstrate that the Tg insertion and genomic deletion have occurred in the HMIP2 orthologous region of the Tg-inserted allele.

Tg insertion mutant mice display an HPFH phenotype.

Tg homozygous (Hbs1l-MybTg/Tg) mice were born in the expected Mendelian ratios (Fig. 2A) but were pale in comparison to wild-type and Hbs1l-MybTg/+ littermates (Fig. 2B), suggesting neonatal anemia in the Hbs1l-MybTg/Tg pups. The hematological parameters of Hbs1l-MybTg/Tg neonates exhibited typical patterns of macrocytic anemia and thrombocytosis (Fig. 2C), which are similar to those observed in human HPFH individuals with the HBS1L-MYB polymorphism (11). The mRNA levels of embryonic εy-globin (Hbb-y) and βh1-globin (Hbb-bh1) in spleens taken from neonatal Hbs1l-MybTg/Tg mice were approximately 4-fold and 20-fold greater than those from wild-type mice, respectively (Fig. 2D). The expression level of βh1-globin was also significantly elevated in Hbs1l-MybTg/+ mice compared to wild-type mice (Fig. 2D). In contrast, the level of adult β-major-globin (Hbb-b1) expression was reduced in Hbs1l-MybTg/Tg mice to less than 80% of that in wild-type mice (Fig. 2D). Thus, these results indicate that Hbs1l-MybTg/Tg homozygous mutant mice recapitulate the phenotypes of human HPFH, demonstrating that in mice the Hbs1l-Myb intergenic region negatively regulates the expression of embryonic globin genes.

Fig 2
The insertional Tg mutant mouse recapitulates an HPFH phenotype. (A) Number of neonates born from Hbs1l-MybTg/+ intercrossing pairs. Percentages of Hbs1l-Myb+/+, Hbs1l-MybTg/+, and Hbs1l-MybTg/Tg neonates in total pups are shown in parentheses. (B) Representative ...

Impairment of HMIP2 orthologous region affects expression of Hbs1l and Myb genes in erythroid progenitors.

Spleen erythroid cells of mice are usually divided into four stages (fractions I to IV) by flow cytometry; fraction I corresponds to the most immature stage or proerythroblasts, while fractions II, III, and IV correspond to more mature erythroblasts (27). In order to clarify roles of the Hbs1l-Myb intergenic region during erythroid differentiation, we first analyzed splenic erythroid cells by flow cytometry. The numbers of mature erythroblasts in fractions III and IV were reduced in the Hbs1l-MybTg/Tg neonatal mouse spleen, implying that there might be defects in erythroid differentiation, proliferation, and/or survival (Fig. 3A and andBB).

Fig 3
Insertion mutagenesis in the Hbs1l-Myb intergenic region affects expression of embryonic globin genes as well as the two flanking genes, Hbs1l and Myb. (A) The CD71 and Ter119 expression patterns in the spleens of neonates were examined by flow cytometry. ...

We also examined expression profiles of globin genes, along with Myb and Hbs1l genes, at each stage. For this purpose, cells were sorted into fractions I to IV, and the mRNA level of the individual globin gene was examined by qRT-PCR. We observed no significant difference in the levels of α- and β-major-globin mRNAs between wild-type and Hbs1l-MybTg/Tg mice in more mature fractions II to IV, while the levels were increased in fraction I of Hbs1l-MybTg/Tg mice (Fig. 3C and andD).D). In contrast, the levels of εy- and βh1-globin mRNAs were higher in fraction II-to-IV erythroblasts of Hbs1l-MybTg/Tg mice (Fig. 3E and andF).F). Especially, the βh1-globin mRNA level was markedly elevated in Tg homozygous mutant mice.

We next investigated the expression of both flanking genes of the murine HMIP2 orthologous region, Hbs1l and Myb. Hbs1l encodes a GTP-binding elongation factor-like protein of currently unknown function (28), and Myb encodes a transcription factor that is essential for the maintenance of hematopoietic stem cells and differentiation of erythroid cells, megakaryocytes, and lymphocytes (21, 2931). The levels of expression of the Hbs1l and Myb genes were both lower in flow-sorted fraction I of Hbs1l-MybTg/Tg mice (Fig. 3G and andH),H), and this observation shows very good agreement with a report analyzing human HPFH (32). Of note is the fact that Myb expression in fractions I and II of Hbs1l-MybTg/Tg mice was significantly reduced, by 74.1% and 85.4%, of its level in wild-type mice, respectively (Fig. 3H), while the reduction in expression of the Hbs1l gene was moderate (Fig. 3G). These results thus indicate that the HMIP2 orthologous region in mouse harbors a positive regulatory element that affects the expression of Myb and Hbs1l, and Myb gene expression is more severely affected by the impairment of the orthologous region than Hbs1l gene expression is.

Myb contributes to embryonic globin gene silencing.

We surmised that the reduced expression of Myb, rather than that of Hbs1l, was responsible for the elevated embryonic globin gene expression observed in the mutant mice, as the contribution of MYB to human embryonic and fetal globin induction has been noticed (25, 32). To test this hypothesis, we selectively deleted the Myb gene in the Tg heterozygous background by generating the compound heterozygotes for the Tg insertion allele and the Myb knockout allele (21) (Hbs1l-MybTg/ mice) and investigated the expression levels of β-type globin genes. We expected that this experiment would allow us to differentiate the contribution of Myb from that of Hbs1l.

We crossed Hbs1l-MybTg/+ mice with Myb knockout heterozygous mice and obtained mice harboring both the Hbs1l-MybTg allele and the Myb heterozygous knockout allele, along with Hbs1l-Myb+/+, Hbs1l-MybTg/+, and Myb+/− mice (Fig. 4A). We referred to the former mice with both the Tg allele and knockout allele as Hbs1l-MybTg/ mice in this study (Fig. 4A). The number of Hbs1l-MybTg/ pups was smaller than that of Mendelian expected number or those of pups of the other genotypes (Fig. 4B). Hematopoietic parameters of the Hbs1l-MybTg/ mice exhibited macrocytic anemia and thrombocytosis, as observed in the Hbs1l-MybTg/Tg mice (Fig. 4C). The Myb+/− mice also showed slightly higher MCH (Fig. 4C). Of note is the fact that the expression levels of εy- and βh1-globin genes were significantly higher in the Hbs1l-MybTg/ mice than those in the rest of the mice, whereas β-major globin expression was lower in the Hbs1l-MybTg/ mice (Fig. 4D). Since the Myb gene status of the allele that does not harbor transgene insertion is the primary difference between the Hbs1l-MybTg/+ and Hbs1l-MybTg/ mice, the results that the latter display more abundant expression of εy- and βh1-globin genes than the former give rise to the compelling genetic evidence that Myb contributes to the silencing of εy- and βh1-globin genes. On the other hand, the expression level of the βh1-globin gene in the Hbs1l-MybTg/+ mice is moderately but significantly higher than that in the Myb+/− mice, suggesting that Hbs1l might contribute to the embryonic βh1-globin gene expression (Fig. 4D).

Fig 4
The Myb gene contributes to embryonic globin silencing in the insertional mutant Tg mouse. (A) Mating strategy of compound heterozygotes between the Tg insertion mutant mice and the Myb knockout mice. +/+, Hbs1l-Myb+/+; Tg/+, Hbs1l-MybTg/+; +/−, ...

DRED lies downstream in the pathway of Hbs1l-Myb-type HPFH.

Our analyses have revealed that both Hbs1l and Myb genes are abundantly expressed in early erythroid cell stages (flow-sorted fraction I), while expression of the β-type globin genes abruptly increases in fraction II and remains at high levels in subsequent stages. This chronological discrepancy implied that elevated expression of the embryonic and fetal globin genes might be an indirect effect of reduced expression of Hbs1l and Myb. Therefore, we examined whether DRED and Bcl11a, two repressors of embryonic and fetal globin genes in the adult (9, 19, 20), are involved in the dysregulated fetal globin gene expression in neonatal Hbs1l-MybTg/Tg mice. TR2 and TR4 (generated from the Nr2c1 and Nr2c2 loci) are the DNA binding subunits of DRED.

We found that TR2, TR4, and BCL11A were abundantly expressed at earlier differentiation stages corresponding to fractions I and II, and their expression levels diminished during maturation into peripheral red blood cells (Fig. 5A to toC)C) (data not shown). The abundance of TR2 and TR4 mRNAs was lower in Hbs1l-MybTg/Tg erythroid progenitors than those of wild-type mice (Fig. 5A and andB),B), while there was no significant difference in levels of Bcl11a expression in wild-type and homozygous mutant erythroid progenitors (Fig. 5C). We also examined the expression level of Klf1 (also known as Eklf), an activator of the Bcl11a gene (33, 34), in neonatal Hbs1l-MybTg/Tg mice, since the KLF1 gene was reported to be upregulated by MYB (35). There was no significant difference in Klf1 gene expression between wild-type and Hbs1l-MybTg/Tg mice (Fig. 5D). Thus, HPFH in Hbs1l-MybTg/Tg appears to lie in the DRED repression pathway, but does not appear to intersect the KLF1-BCL11A pathway, in its contribution to mouse embryonic globin gene regulation.

Fig 5
DRED lies downstream in the pathway of Hbs1l-Myb type HPFH. (A to D) The abundance of Tr2 (A), Tr4 (B), Bcl11a (C), and Klf1 (D) mRNAs in fractions I and II (from Fig. 3A) from Hbs1l-Myb+/+ (n = 6), Hbs1l-MybTg/+ (n = 5), and Hbs1l-MybTg/Tg (n = 6) mice ...

MYB represses fetal globin gene by upregulating both DRED and KLF1/BCL11A pathways in human erythroid cells.

Finally, to test whether reduction of TR2 and TR4 accompanies fetal globin induction by MYB deficiency in human erythroid progenitors, we examined the expression levels of TR2 and TR4 in MYB knockdown primary human erythroid progenitors. To this end, we introduced lentiviral vectors expressing two types of shRNA (shRNAs 1 and 2) for MYB into CD34+ cells derived from human adult bone marrow and induced ex vivo erythroid differentiation as described previously (9, 25). In this culture method, the CD34+ cells differentiate into proerythroblasts on day 5 and basophilic erythroblasts on day 7 (9). Expression levels of the MYB gene in primary human CD34+-derived cells transduced with pLKO.1 carrying shRNAs 1 and 2 were reduced to 46.7% and 29.7% of the control level on day 5, respectively (Fig. 6A). The expression levels of embryonic (ε) and fetal (γ) globin genes were significantly increased in MYB knockdown cells (Fig. 6B). Expectedly, the expression level of TR2 was significantly decreased on days 5 and 7 (Fig. 6C). Reduction of TR4 expression level was observed on day 7 (Fig. 6D). These results were in a good agreement with the mouse results of this study. We further examined BCL11A and KLF1 expression levels in human MYB knockdown cells (Fig. 6E and andF).F). Unexpectedly, the expression levels of BCL11A and KLF1 were significantly reduced on days 5 and 7. These results thus suggest that under normal differentiation conditions, MYB represses fetal globin gene expression by upregulating both the TR2/TR4 and KLF1/BCL11A pathways in human erythroid cells, but upon the downregulation of MYB, both the TR2/TR4 and KLF1/BCL11A pathways are suppressed, and this activates the fetal globin gene expression (Fig. 6G).

Fig 6
MYB represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34+-derived cells transduced with pLKO.1 control or pLKO.1 with shRNAs targeting ...


Induction of fetal hemoglobin in adults is one of the key issues in developing therapies for sickle-cell disease and β-thalassemia, and elucidation of the molecular mechanisms detailing how fetal hemoglobin expression is regulated should provide important insights into strategies by which this end is achieved. In this study, we demonstrated that the intergenic region between Hbs1l and Myb is responsible for the persistence of murine embryonic globin gene expression after birth by utilizing an HPFH mouse model, which has been established through a serendipitous Tg insertion into the orthologous region of human HMIP2 LD block. We found that levels of expression of the genes flanking the LD block, Hbs1l and Myb, are reduced in the mutant erythroid progenitors. Analyses of the Tg insertion and Myb knockout compound heterozygous mice revealed that Myb contributes substantially to the embryonic globin gene silencing. Furthermore, expression of the DNA binding subunits of the DRED complex was diminished in erythroid progenitors of this Tg mutant mouse. These results thus support our contention that the disruption of the Hbs1l-Myb intergenic region by the Tg insertion suppresses the Myb level and leads to the persistence of embryonic globin gene expression, and the DRED complex appears to be involved in this regulation.

We found that a small segment (approximately 800 bp) has been deleted from the Tg allele, and the deleted segment contains a conserved noncoding sequence. Therefore, there is a possibility that this deleted region may contain an enhancer activity for the Myb gene. However, we think that this possibility is unlikely based on the following observations. First, the human orthologous region of the deleted region does not display DNase I hypersensitivity in human erythroid K562 cell lines (DNase I hypersensitivity by digital DNase I; ENCODE Project, University of Washington, Seattle; [data not shown]). Second, in the Hbs1l-Myb intergenic region, several enhancer domains for the Myb gene have been identified, and they are located at 36, 61, 68, 81, and 109 kb upstream of the Myb gene transcription start site (15). However, the deleted region in the Tg allele does not coincide with any of these known enhancers. Therefore, these observations imply that the small deleted region is unlikely to contain erythroid-specific regulatory activity.

The Tg insertion site (red box in Fig. 1D) is located approximately 3 kb upstream (the Hbs1l side) of the 81-kb site (blue box in Fig. 1D) (15). In mouse erythroleukemia (MEL) cells and primary erythroid cells, the 81-kb site was occupied by the GATA1/LDB1/TAL1/ETO2 complex and KLF1 (15). On the other hand, the inserted Tg consists of an Epor cDNA linked to a genomic promoter/enhancer fragment of the Gata1 gene (G1HRD). A single copy of G1HRD contains at least three GATA-binding enhancers: the Gata1 gene hematopoietic enhancer, a promoter double-GATA site, and the GATA-GACT repeat, which are cooperatively responsible for the high-level Gata1 gene expression in erythroid cells (18, 22, 3639). Therefore, we surmise that in erythroid cells the function of GATA-binding erythroid enhancers within the Hbs1l-Myb locus, especially around the 81-kb site, could be compromised because of preferential sequestration of GATA factors by the inserted Tg. Indeed, a 3-bp deletion between a GATA-binding sequence and E-box, which are predicted to disrupt GATA1-TAL1 complex formation, in the human orthologous region of the 81-kb site is identified as a polymorphism associated with a high level of fetal hemoglobin (40).

Alternatively, since four copies of the Tg insertion integrate more than 40 kb DNA into the locus, the Tg insertion separates the 109-kb element away from the Myb promoter. The 109-kb element, as well as other enhancer elements, has been shown to interact with the Myb promoter by forming a chromatin loop (15). Therefore, the Tg insertion may interfere with the interaction between the 109-kb element and Myb promoter and may give rise to the decrease of Myb gene expression. Similarly, there exists the Hbs1l gene on the opposite side of the Myb gene, and we found that the Hbs1l gene expression correlates well with the Myb gene expression in our Tg/Tg mouse analysis. This shows very good agreement with the analysis of human erythroid cells (32). Therefore, we surmise that the Tg insertion may also affect the Hbs1l gene expression. In fact, the βh1-globin expression in our Hbs1l-MybTg/+ mice is higher than that in the Myb+/− mice, suggesting that Hbs1l also contributes to the repression of fetal globin in adult mouse erythroid progenitors. These two possibilities remain to be elucidated.

The expression levels of Tr2 and Tr4 genes, encoding the DNA binding subunits of DRED, are significantly diminished in the mutant erythroid cells, suggesting that reduction of DRED may mediate HPFH caused by the mutations in the Hbs1l-Myb intergenic region. The DRED complex has been reported to repress the expression of embryonic and fetal globin genes by binding directly to direct repeat (DR) elements in εy- and βh1-globin gene promoters, the binding sites for numerous naturally occurring nondeletion HPFH mutants (19, 20). Embryonic and fetal globin gene promoters have two DR elements and one DR element, respectively, with different affinities for DRED (20). These different affinities for DRED may account for the phenotype of Hbs1l-MybTg/+ mice in which a significant increase of the expression level of βh1-globin, but not εy-globin, was observed.

In this study, we detected significant reductions of TR2 and TR4 in both erythroid progenitors of the Hbs1l-MybTg/Tg mice and human MYB knockdown cells. Meanwhile, reduction of KLF1 and BCL11A was observed in human MYB knockdown cells, but not in the Hbs1l-MybTg/Tg erythroid progenitors, despite the fact that the knockdown efficiencies of the Myb gene were similar in human and mouse cells. These results suggest that the MYB-KLF1-BCL11A pathway is efficient in human but not in mouse erythroid progenitors. A pathway where KLF1 activates BCL11A expression, resulting in fetal globin silencing, has been verified in both human and mouse cells (33, 34). In contrast, a pathway where MYB activates KLF1 expression has been proved only in human cells (35). Therefore, we surmise that the discrepancy between human and mouse cells in our current analysis indicates that the MYB-KLF1 pathway is not efficient in mouse cells.

This Hbs1l-Myb-type HPFH model mouse exhibited macrocytic anemia and thrombocytosis. Human HPFH individuals associated with the HBS1L-MYB locus consistently exhibit the same, albeit milder, hematopoietic deficiencies (11). These observations suggest that the HBS1L-MYB intergenic region is responsible for macrocytic anemia and thrombocytosis. We previously reported that megakaryocyte/erythrocyte bifurcation of MEPs was skewed to megakaryopoiesis in this Tg mutant mouse (17). Since abnormal biasing of MEPs could not fully account for macrocytic anemia, additional mechanisms must be in effect. In the present study, we observed that the number of mature erythroid progenitors, rather than immature erythroid progenitors, was reduced in Hbs1l-MybTg/Tg mouse spleen, suggesting defects in differentiation, proliferation, or survival in mature erythroid cells. MYB has been reported to activate genes involved in proliferation (e.g., MYC, CCNA1, CCNB1, and CCNE1) and survival (e.g., BCL2, HSPA5, and HSP70) (41). Thus, the proliferation and survival defects of erythroid progenitors may give rise to the kind of macrocytic anemia observed in these mutant mice. In considering the development of therapies for sickle-cell disease and β-thalassemia, induction of only fetal globin expression, without macrocytic anemia, would ideally be required. To this end, it may therefore be essential to clarify the molecular mechanism of not only fetal globin induction but also macrocytic anemia in HPFH associated with HBS1L-MYB.

In summary, this HPFH mouse model, created by insertional mutagenesis of a Gata1-directed transgene into the Hbs1l-Myb intergenic region, recapitulates persistence of embryonic globin expression and hematopoietic traits that are also observed in human HPFH individuals. We believe that detailed analysis of this mouse model would make a significant contribution to deepening our understanding of the genetic networks that regulate globin gene expression.

Supplementary Material

Supplemental material:


We thank Masanobu Morita and Ritsuko Shimizu for discussion, Hiromi Suda and Carmen Yu for technical assistance, and the Biomedical Research Core and Center for Laboratory Animal Research of Tohoku University Graduate School of Medicine for technical support.

This work was supported in part by Grants-in-Aid from the NIH (HL24415 to J.D.E.), JSPS (KAKENHI 19GS0312 to M.Y. and 22790269 to M.S.), the Tohoku University Global COE for Conquest of Signal Transduction Diseases with Network Medicine (to M.Y.), the NAITO Foundation (to M.Y.), and the Takeda Science Foundation (to M.Y.). H.Y. is a JSPS Research Fellow.


Published ahead of print 19 February 2013

Supplemental material for this article may be found at


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