1: Blood. 2002 Nov 15;100(10):3828-31. Epub 2002 Jul 5. 

Immortalization of yolk sac-derived precursor cells.

Yu WM, Hawley TS, Hawley RG, Qu CK.

Department of Hematopoiesis and the Flow Cytometry Facility, Jerome H. Holland
Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD 20855,
USA.

Hematopoiesis initiates in the extraembryonic yolk sac. To isolate various types
of precursor cells from this blood cell-forming tissue, yolk sac cells were
immortalized by retroviral-mediated expression of the HOX11 homeobox-containing
gene. Among the cell lines derived, some were able to spontaneously generate
adherent stromal-like cells capable of taking up acetylated low-density
lipoprotein, and they could be induced to form tubelike structures when cultured
on Matrigel. Although these cell lines were negative for hematopoietic cell
surface markers, they gave rise to hematopoietic colonies--containing cells
belonging to the monocytic, megakaryocytic, and definitive erythroid
lineages--when plated in methylcellulose medium supplemented with hematopoietic
growth factors. Low amounts of Flk-1 mRNA could be detected in these cells, and
they showed significant responsiveness to vascular endothelial growth factor,
stem cell factor, basic fibroblast growth factor, and interleukin 6. They also
expressed the transcription factors SCL, GATA2, GATA1, PU.1, and c-myb. These
yolk sac-derived cell lines may represent a transitional stage of early
hematopoietic development.

PMID: 12393673 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


2: J Biol Chem. 2001 Feb 2;276(5):3674-82. Epub 2000 Nov 9. 

Increased affinity of c-Myb for CREB-binding protein (CBP) after CBP-induced
acetylation.

Sano Y, Ishii S.

Laborartory of Molecular Genetics, RIKEN Tsukuba Institute and the CREST (Core
Research for Evolutional Science and Technology) Research Project of JST (Japan
Science and Technology Corporation), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074,
Japan.

The c-myb proto-oncogene product (c-Myb) is a sequence-specific DNA-binding
protein that functions as a transcriptional activator. The transcriptional
coactivator CREB-binding protein (CBP) binds via its KIX domain to the
activation domain of c-Myb and mediates c-Myb-dependent transcriptional
activation. CBP possesses intrinsic histone acetyltransferase activity, and can
acetylate not only histones but also certain transcriptional factors such as
GATA1 and p53. Here we demonstrate that the C/H2 domain of CBP, which is
critical for the acetyltransferase activity, also directly interacts with the
negative regulatory domain (NRD) of c-Myb. Consistent with this observation, CBP
acetylated c-Myb in vitro at Lys(438) and Lys(441) within the NRD. In addition,
CBP acetylated c-Myb in vivo not only at the sites found in this study but also
at the p300-induced acetylation sites reported recently. Replacement of lysine
by arginine at all of these sites dramatically decreased the trans-activating
capacity of c-Myb. The results of transcriptional activation assays with c-Myb
acetylation site mutants suggested that acetylation of c-Myb at each of these
five sites synergistically enhances c-Myb activity. Mutations of these
acetylation sites reduced the strength of the interaction between c-Myb and CBP.
Thus, acetylation of c-Myb by CBP increases the trans-activating capacity of
c-Myb by enhancing its association with CBP. These results demonstrate a novel
molecular mechanism of regulation of c-Myb activity.

PMID: 11073948 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


3: 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]


---------------------------------------------------------------


4: Blood. 1996 Mar 15;87(6):2221-34. 

Induction of hematopoietic commitment and erythromyeloid differentiation in
embryonal stem cells constitutively expressing c-myb.

Melotti P, Calabretta B.

Departments of Microbiology and Immunology, Jefferson Cancer Institute, Thomas
Jefferson University, Philadelphia, PA USA.

To provide insight into the mechanisms by which c-myb regulates hematopoiesis,
we analyzed the expression of markers for multiple hematopoietic lineages in
differentiating parental embryonic stem (ES) cells and in ES cells transfected
with c-myb or with a mutant c-myb deficient in DNA binding and assessed the
ability of these cells to undergo hematopoietic commitment and colony formation.
Undifferentiated ES cells transfected with intact c-myb, but not cells
transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as
well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of
GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells.
Transient expression assays suggested transactivation of gene expression
dependent on interaction with Myb binding sites in the CD34 and GATA1 5'
flanking regions. Undifferentiated parental and c-myb mutant-transfected ES
cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid
colonies in methylcellulose cultures in the absence of added hematopoietic
growth factors and, at higher frequency, in the presence of kit and flt-3
ligands. Colony formation was suppressed by treatment with antisense
oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression.
These findings indicate that c-myb regulates hematopoietic commitment and
progenitor cell proliferation and differentiation through the activation of
certain genes that define the stem/progenitor cell compartment.

PMID: 8630382 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------