C/EBPa is a key mediator of myeloid development, and mutation of C/EBPa is common on AML. Mice lacking C/EBPa are defective in the CMP to GMP transition, but the subsequent role of C/EBPa in monopoiesis versus granulopoiesis is not defined. We find that exogenous C/EBPa directs lineage-negative marrow progenitors along the monocytic pathway and that C/EBPa has the capacity to zipper and bind DNA as a heterodimer with c-Jun or c-Fos but not c-Maf or MafB. Endogenous C/EBPa co-ips with endogenous c-Jun or c-Fos. To study the role of specific heterodimers, we developed acid and basic leucine zippers (LZE, LZK) and find that C/EBPa:c-Jun or C/EBPa:c-Fos induce monopoiesis with far greater potency than does C/EBPa:C/EBPa homodimers or c-Jun:c-Fos. Oligonucleotide selection identified a consensus C/EBPa:AP-1 DNA element, and C/EBPa:c-Jun binds and activates the PU.1 promoter via a related site. Microarray studies using an inducible cell line, combined with ChIP data, suggest that the EGR2 promoter is an additional C/EBPa:AP-1 genetic target. Egr-2, like elevated PU.1, favors monopoiesis. Comparing MCSFR with GCSFR signals in lineage- negative murine marrow myeloid cells, we find that while G-CSF specifically activates STAT3, M-CSF preferentially activates ERK and thereby stabilizes c-Fos. ERK signaling is also known to induce the FOS and EGR1/2 genes and to favor myeloid over lymphoid development. We hypothesis that monocyte lineage commitment results from synergy between transcriptional induction of PU.1 and EGR1/2 by C/EBPa:c-Jun and C/EBPa:c-Fos combined with MCSFR signals that activate ERK to induce c-Fos and EGR1/2. To test this model, we propose:
AIM 1. To determine whether C/EBPa:c-Jun or C/EBPa:c-Fos heterodimers specifically induce monocytic commitment of myeloid progenitors in vivo, either by transplanting cells transduced with retroviral vectors or with tet-regulated lentiviral vectors followed by analysis 1-4 months later or be generation and breeding of MRP8 transgenic mice.
AIM 2 : To determine whether C/EBPa:AP-1 heterodimers directly regulate transcription of the EGR2 or related EGR1 genes and whether EGR1/2 or PU.1 induction is required for C/EBPa:AP-1 to direct monopoiesis. Direct interaction with and induction of the EGR1 or EGR2 promoters will be assessed and mapped, including studies with normal cells. The effect of dominant-inhibition of Egr-1 and Egr-2 or of diminished expression of PU.1 in mice lacking the PU.1 distal enhancer on the ability of C/EBPa:AP-1 complexes to induce monopoiesis will be assessed.
AIM 3. To determine whether MCSFR signals stabilize c-Fos and induce FOS and EGR1/2 transcription via ERK activation more effectively than GCSFR signals. Lineage-negative marrow cells will be used to compare M-CSF and G-CSF for ERK activation, c-Fos phosphorylation and protein stabilization, and FOS and EGR1/2 RNA expression at various time points and in response to ERK inhibition. The ability of ERK inhibition to alter myeloid lineage specification using FACS and RNA analysis of lineage markers will also be assessed.
Monocytes and granulocytes are different types of white blood cells that help fight infections and also can become transformed into acute leukemias. Proposed experiments using mouse bone marrow cells will determine how C/EBPa, c-Jun, c-Fos, Egr-2 and the M-CSF receptor direct development of monocytes instead of granulocytes from a common ancestor cell. These studies will guide future efforts to develop novel methods to provide white blood cells to patients with infections and to treat acute myeloid leukemias.
|Kim, H-G; LeGrand, J; Swindle, C S et al. (2017) The assembly competence domain is essential for inv(16)-associated acute myeloid leukemia. Leukemia 31:2267-2271|
|Guo, Hong; Ma, Ou; Friedman, Alan D (2014) The Cebpa +37-kb enhancer directs transgene expression to myeloid progenitors and to long-term hematopoietic stem cells. J Leukoc Biol 96:419-26|
|Ma, Ou; Hong, SunHwa; Guo, Hong et al. (2014) Granulopoiesis requires increased C/EBP? compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells. PLoS One 9:e95784|
|Guo, Hong; Ma, Ou; Speck, Nancy A et al. (2012) Runx1 deletion or dominant inhibition reduces Cebpa transcription via conserved promoter and distal enhancer sites to favor monopoiesis over granulopoiesis. Blood 119:4408-18|
|Hong, SunHwa; Skaist, Alyza M; Wheelan, Sarah J et al. (2011) AP-1 protein induction during monopoiesis favors C/EBP: AP-1 heterodimers over C/EBP homodimerization and stimulates FosB transcription. J Leukoc Biol 90:643-51|
|Paz-Priel, Ido; Friedman, Alan (2011) C/EBP? dysregulation in AML and ALL. Crit Rev Oncog 16:93-102|
|Dooher, Julia E; Paz-Priel, Ido; Houng, Simone et al. (2011) C/EBP?, C/EBP? oncoproteins, or C/EBP? preferentially bind NF-?B p50 compared with p65, focusing therapeutic targeting on the C/EBP:p50 interaction. Mol Cancer Res 9:1395-405|
|Paz-Priel, Ido; Houng, Simone; Dooher, Julia et al. (2011) C/EBP? and C/EBP? oncoproteins regulate nfkb1 and displace histone deacetylases from NF-?B p50 homodimers to induce NF-?B target genes. Blood 117:4085-94|
|Zhang, Li; Friedman, Alan D (2011) SHP2 tyrosine phosphatase stimulates CEBPA gene expression to mediate cytokine-dependent granulopoiesis. Blood 118:2266-74|
|Jack, Graham D; Zhang, Li; Friedman, Alan D (2009) M-CSF elevates c-Fos and phospho-C/EBPalpha(S21) via ERK whereas G-CSF stimulates SHP2 phosphorylation in marrow progenitors to contribute to myeloid lineage specification. Blood 114:2172-80|