1: J Cell Physiol. 1999 Sep;180(3):390-401. 

Increased expression of the distal, but not of the proximal, Gata1 transcripts
during differentiation of primary erythroid cells.

Vannucchi AM, Linari S, Lin CS, Koury MJ, Bondurant MC, Migliaccio AR.

Division of Hematology, University of Florence and Azienda Ospedale Careggi,
Italy.

Gata1 is expressed from either one of two alternative promoters, the erythroid
(proximal to the AUG) and the testis (distal to the AUG) promoter, both used by
hemopoietic cells. To clarify the role of the distal and proximal Gata1
transcripts in erythroid differentiation, we determined by specific reverse
transcriptase-polymerase chain reactions their relative levels of expression
during the differentiation of erythroid precursors purified from the spleen of
mice treated with phenylhydrazine (PHZ) or infected with the anemia-inducing
strain of the Friend virus (FVA cells). PHZ cells are erythroid precursors that
progress in vivo to erythroblasts in 3 days. Both PHZ and FVA cells
synchronously proliferate and differentiate in vitro in the presence of
erythropoietin (EPO). The levels of total and of distal, but not of proximal,
Gata1 transcripts increased by five- to eightfold during in vivo and in vitro
differentiation of FVA and PHZ cells. The increase in expression was temporally
associated with an increase in the expression of Eklf, Scl, and Nfe2, three
genes required for erythroid differentiation, and preceded by 24 h the
repression of Gata2 and Myb expression. The day 1 PHZ cells that survived 18 h
in the absence of EPO do not express globin genes and express detectable levels
of distal but not of proximal Gata1 transcripts. These cells activate the
expression of the globin genes within 2 h when exposed to EPO. Therefore, during
erythroid differentiation of primary cells, increased expression of distal Gata1
transcripts underlies the increase in the expression of total Gata1 associated
with the establishment of the erythroid differentiation program.

PMID: 10430179 [PubMed - indexed for MEDLINE]


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2: Blood. 1993 Sep 15;82(6):1900-6. 

Erratum in:
    Blood 1995 Feb 1;85(3):862.

Single-copy transduction and expression of human gamma-globin in K562
erythroleukemia cells using recombinant adeno-associated virus vectors: the
effect of mutations in NF-E2 and GATA-1 binding motifs within the
hypersensitivity site 2 enhancer.

Miller JL, Walsh CE, Ney PA, Samulski RJ, Nienhuis AW.

Clinical Hematology Branch, National Heart, Lung and Blood Institute, National
Institutes of Health, Bethesda, MD 20892.

The use of recombinant adeno-associated virus (rAAV) vectors provides a new
strategy to investigate the role of specific regulatory elements and
trans-acting factors in globin gene expression. We linked hypersensitivity site
2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*)
mutationally marked to allow its transcript to be distinguished from endogenous
gamma-globin mRNA. The vector also contains the phosphotransferase gene that
confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2
motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the
GATA-1 motif prevented binding of the factor. Several NeoR K562 clones
containing a single unrearranged RAAV genome with the A gamma* gene linked to
the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1
HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells,
mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones,
compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin
induction of mut-NFE2 clones did not result in an increase in the A gamma*
signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A
gamma* mRNA at the same level as WT clones in both uninduced and induced cells.
Thus, GATA-1 binding to this site does not appear to be required for the
enhancing function of HS2 in this context. This single-copy rAAV transduction
model is useful for evaluating the effects of specific mutations in regulatory
elements on the transcription of linked genes.

PMID: 8400240 [PubMed - indexed for MEDLINE]


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