Induction of globin synthesis in K562 cells is associated with differential expression of transcription factor genes.
Globin gene switching may be mediated by proteins expressed during different stages of development. Their identification may clarify the mechanisms of the conversion from fetal to adult globin production and lead to new approaches to reversing or retarding the gamma- to beta-globin gene switch. To explore this hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25, 2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and 72 h. Erythroid differentiation was verified by benzidine staining and by measuring the activity of a transduced A gamma-globin gene promoter linked to a luciferase reporter gene. Using differential display polymerase chain reaction (PCR), total mRNA extracted from induced cells at each time point of induction was reverse transcribed in the presence of A, G, and C anchored primers and 16 arbitrary primers, calculated to amplify approximately 50% of expressed genes. Amplified mRNAs from induced and uninduced cells were separated in polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to represent either up- or downregulated mRNA species in induced K562 cells were identified. Sixty-four of these fragments had more than 95% homology to known GenBank sequences. Seventeen fragments with characteristics of transcription factors were cloned. These include differentiation-related gene-1 (drg-1), PAX 3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), alpha-NAC transcriptional coactivator, LIM domain protein, and trophoblast hypoxia regulating factor. Differential expression of all 17 fragments over 72 h was confirmed by reverse Northern dot blot analysis, semiquantitative PCR using nested primers, and Northern analysis. Erythroid maturation in induced K562 cells is associated with differential expression of numerous genes. Some encode transcription factors that could effect the initiation of HbF synthesis. Almost half of the differentially expressed clones contained cDNAs of unidentified open reading frames and these are the object of continued study.
Plonczynski
M
M
G.V. (Sonny) Montgomery Department of Veterans Affairs Medical Center, Jackson, MS, USA.
Hardy
C L
CL
Safaya
S
S
Harrell
A
A
McCoy
L
L
Brinson
A
A
Agwarangbo
L
L
Steinberg
M H
MH
D015152:Blotting, Northern; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D005319:Fetal Hemoglobin / Q000235:genetics; D005786:Gene Expression Regulation; D005812:Genes, Switch; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA; D014157:Transcription Factors / Q000235:genetics*
D018076:DNA, Complementary; D014157:Transcription Factors; D005914:Globins; D005319:Fetal Hemoglobin