Konference: 2007 49th ASH Annual Meeting - účast ČR
Kategorie:
Maligní lymfomy a leukémie
Téma: Simultaneous Session: Myeloid Differentiation and Neutropenia
Číslo abstraktu: 661
Autoři: Mgr. Vít Pospíšil; Mgr. Juraj Kokavec; Mgr. Pavel Burda, Ph.D.; Mgr. Nikola Čuřík, Ph.D.; Arthur I. Skoultchi, Ph.D.; Jiri Zavadil, Ph.D.; Doc. MUDr. Tomáš Stopka, Ph.D.
PU.1 (Sfpi1) is an ets family transcription factor required for the
proper generation of both myeloid (macrophages and neutrophils) and
lymphoid lineages (B and T lymphocytes)(Scott 1994, McKercher
1996). Graded expression of exogenous PU.1 in murine PU.1-deficient
fetal liver hematopoietic progenitors demonstrated that increased
levels of PU.1 are required to initiate development of macrophages
(DeKoter, 2000). We have studied the effects of graded expression
of PU.1 on its occupancy in chromatin and on the development of
myeloid cells in vitro. We measured changes in gene expression,
PU.1 occupancy and histone modifications in PU.1-null hematopoietic
progenitor cells stably expressing PU.1 fused to the ligand-binding
domain of the estrogen receptor (PU.1-ER) (Walsh 2002). The level
of active PU.1-ER was regulated with graded levels of the ER
inducer tamoxifen. In vitro, intermediate levels of tamoxifen
produced cells with granulocyte characteristics in the suspension
cell fraction and macrophage-like characteristics in the attached
fraction, whereas high levels of PU.1 produced mostly attached
macrophage-like cells. Expression of granulocyte-specific PU.1
target mRNAs including gelatinase B (Mmp9) and myeloperoxidase
(Mpo) were observed to be expressed only with intermediate levels
of tamoxifen. In contrast, expression of macrophage PU.1 target
mRNAs including Cd14, F4/80 and Cd68 mRNAs were observed to be
gradually upregulated upon PU.1-ER activation, with the maximum
expression at the highest levels of tamoxifen. Thus, the expression
levels of PU.1 target genes and phenotypic characteristics of the
cells are dependent on PU.1 levels. Interestingly, macrophage-like
cells can be produced from granulocytic-like cells by changing
tamoxifen levels and vice versa. Chromatin immunoprecipitation
analysis revealed specific PU.1 occupancy within regulatory regions
of the genes predominantly expressed in macrophages including Cd14
and Cd11b after treatment with high levels of tamoxifen. Specific
PU.1 occupancy within regulatory regions of the granulocyte
specific genes including MMP9 was observed at intermediate levels
of tamoxifen. Suprisingly, chromatin immunoprecipitation analysis
revealed specific PU.1 occupancy within regulatory regions of the
lymphocytic PU.1 target genes including Interleukin-7 receptor
(Il-7r) and RAG1 at intermediate levels of tamoxifen even though
expression of these genes was not detected. Accumulation of
acetylated K9 and methylated K4 of histone H3 in gene loci of
macrophage and granulocytic markers such as Cd14, Cd11b, and Mmp9
correlated with their mRNA expression. However, lymphocyte-specific
regulatory regions including that of Il-7r gene were hypoacetylated
in H3K9 despite a marked PU.1 recruitment suggesting additional
factors may be required for PU.1 mediated transactivation. To
identify these molecules we have tested PU.1-dependent
transcription factors: Egr2, Nab2, Cebpa and Gfi-1 and found that
upon increasing PU.1 levels, expression of Egr2/Nab2 and
Gfi-1/Cebpa changed in a reciprocal manner and these changes
preceded expression of the lineage specific markers. We are
currently testing if PU.1 directly regulates expression of Egr2,
Nab2, Cebpa and Gfi-1 during granulocytic/macrophage
differentiation.
Abstract #661 appears in Blood, Volume 110, issue 11, November 16,
2007
Keywords: PU.1|Granulocyte|Macrophage
Disclosure: No relevant conflicts of interest to declare.
Monday, December 10, 2007 3:30 PM
Session Info: Simultaneous Session: Myeloid Differentiation and
Neutropenia (3:30 p.m.-5:00 p.m.)
Datum přednesení příspěvku: 10. 12. 2007