title: Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal β-like globin genes.
abstract: The clinical symptoms of hemoglobin disorders such as β-thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ-globin after birth. This knowledge has driven the discovery of important regulators that silence γ-globin postnatally. Improved understanding of the γ- to β-globin switching mechanism holds the key to devising targeted therapies for β-hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183-kb human β-globin locus, in which the (G)γ- and β-globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)-mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ-globin, measured by DsRed fluorescence and qRT-PCR (P<0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε-globin, (up to 20% of total β-like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ-globin expression (up to 90% of total β-like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human β-like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal β-like globin genes in the adult erythroid environment.
authors.affiliation: 1Cell and Gene Therapy Group, Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Rd., Parkville, VIC 3052, Australia. jim.vadolas@mcri.edu.au.
authors.forename: Mark
authors.initials: M
authors.lastname: Roosjen
authors.forename: Bradley
authors.initials: B
authors.lastname: McColl
authors.forename: Betty
authors.initials: B
authors.lastname: Kao
authors.forename: Linden J
authors.initials: LJ
authors.lastname: Gearing
authors.forename: Marnie E
authors.initials: ME
authors.lastname: Blewitt
authors.forename: Jim
authors.initials: J
authors.lastname: Vadolas
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D004920:Erythropoiesis / Q000235:genetics; D005319:Fetal Hemoglobin / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000235:genetics; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism; D055547:epsilon-Globins / Q000235:genetics / Q000378:metabolism; D055545:gamma-Globins / Q000235:genetics / Q000378:metabolism
chemical_list: C407733:Bcl11a protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D020598:Proto-Oncogene Proteins c-myb; D012097:Repressor Proteins; D055544:beta-Globins; C485184:enhanced green fluorescent protein; D055547:epsilon-Globins; C413663:fluorescent protein 583; D055545:gamma-Globins; D049452:Green Fluorescent Proteins; D005319:Fetal Hemoglobin; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse
keywords: RNA interference; epigenetic repressor; molecular mechanism; β-hemoglobinopathies