Alan D. Friedman

Transcriptional regulation of granulocyte and monocyte development.

Granulocytes and monocytes develop from a common myeloid progenitor. Early granulopoiesis requires the C/EBPα, PU.1, RAR, CBF, and c-Myb transcription factors, and terminal neutrophil differentiation is dependent upon C/EBPε, PU.1, Sp1, CDP, and HoxA10. Monopoiesis can be induced by Maf-B, c-Jun, or Egr-1 and is dependent upon PU.1, Sp1, and ICSBP. Signals eminating from cytokine receptors modulate factor activities but do not determine cell fates. Orchestration of the myeloid developmental program is achieved via cooperative gene regulation, via synergistic and inhibitory protein–protein interactions, via promoter auto-regulation and cross-regulation, via regulation of factor levels, and via induction of cell cycle arrest: For example, c-Myb and C/EBPα cooperate to activate the mim-1 and NE promoters, PU.1, C/EBPα, and CBF, regulate the NE, MPO, and M-CSF Receptor genes. PU.1:GATA-1 interaction and C/EBP suppression of FOG transcription inhibits erythroid and megakaryocyte gene expression. c-Jun:PU.1, ICSBP:PU.1, and perhaps Maf:Jun complexes induce monocytic genes. PU.1 and C/EBPα activate their own promoters, C/EBPα rapidly induces PU.1 and C/EBPε RNA expression, and RARα activates the C/EBPε promoter. Higher levels of PU.1 are required for monopoiesis than for B-lymphopoiesis, and higher C/EBP levels may favor granulopoiesis over monopoiesis. CBF and c-Myb stimulate proliferation whereas C/EBPα induces a G1/S arrest; cell cycle arrest is required for terminal myelopoiesis, perhaps due to expression of p53 or hypo-phosphorylated Rb.

Multiple Functional Domains of AML1: PU.1 and C/EBPα Synergize with Different Regions of AML1

ABSTRACT Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which we can study synergistic, tissue-specific activation for two reasons. First, myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors which are crucial for normal hematopoiesis: PU.1, AML1, and C/EBPα. Second, these proteins interact in such a way as to demonstrate at least two examples of synergistic activation. We have shown that AML1 and C/EBPα activate the M-CSF receptor promoter in a synergistic manner. As we report here, AML1 also synergizes, and interacts physically, with PU.1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPα has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPα but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPα. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPα, and PU.1.

The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

ABSTRACT Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor.

Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb.

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer. Mutation of a core binding factor (CBF)-binding site at -288 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte colony-stimulating factor (G-CSF). A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.

CBFβ-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells

CBFβ-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBFβ-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBFβ-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBFβ-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBFβ. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBFβ-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBFβ-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBFβ-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor α or β subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBFβ-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBFβ-SMMHC by allowing its increased expression.

Cell cycle and developmental control of hematopoiesis by Runx1

Runx1 binds DNA in cooperation with CBFβ to activate or repress transcription, dependent upon cellular context and interaction with a variety of co‐activators and co‐repressors. Runx1 is required for emergence of adult hematopoietic stem cells (HSC) during embryonic development and for lymphoid, myeloid, and megakaryocyte lineage maturation from HSC in adult marrow. Runx1 levels vary during the cell cycle, and Runx1 regulates G1 to S cell cycle progression. Both Cdk and ERK phosphorylate Runx1 to influence its interaction with co‐repressors, and the Wnt effector LEF‐1/TCF also modulates Runx1 activities. These links likely allow cytokines and signals from adjacent cells to influence HSC proliferation versus quiescence and the rate of progenitor expansion, in response to developmental or environmental demands. J. Cell. Physiol. 219: 520–524, 2009.

C/EBPα:AP-1 leucine zipper heterodimers bind novel DNA elements, activate the PU.1 promoter and direct monocyte lineage commitment more potently than C/EBPα homodimers or AP-1

The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA binding. Members of the C/EBP, AP-1 and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind-specific DNA motifs. Here we demonstrate that C/EBPα also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPα zippers with c-Jun, JunB or c-Fos, but not with c-Maf or MafB. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. C/EBPαLZE:C/EBPαLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site and C/EBPαLZE:c-JunLZK a hybrid element identified as TTGCGTCAT by oligonucleotide selection. In murine myeloid progenitors, C/EBPα:c-Jun or C/EBPα:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPα or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. C/EBPα:cJun binds and activates the endogenous PU.1 promoter, providing one mechanism for induction of monopoiesis by this complex.

Exogenous cdk4 overcomes reduced cdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF - a model for overcoming inhibition of proliferation by CBF oncoproteins.

Core Binding Factor (CBF) is required for the development of definitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFβ-SMMHC are commonly expressed in subsets of acute leukemia. CBFβ-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this effect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is sufficient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFβ-SMMHC-induced inhibition of proliferation. Representational difference analysis (RDA) identified cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFβ-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16INK4a, overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These findings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins.

Cross-talk between regulators of myeloid development: C/EBPα binds and activates the promoter of the PU.1 gene

CCAAT/enhancer‐binding protein (C/EBP)α and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP‐binding sites. Gel‐shift assay demonstrated that C/EBPα expressed in 293T cells bound the site centered at –68 most potently. C/EBPα from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPβ did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The –68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the –68 C/EBP‐binding site in a −85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBPα and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPα‐estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPα DNA‐binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPα and C/EBPα‐ER with the PU.1 promoter. Finally PU.1 RNA is reduced several‐fold in immortalized C/EBPα (−/−) compared with (+/−) cells. Together, these findings indicate that C/EBPα binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPα may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.

Regulation of granulocyte and monocyte differentiation by CCAAT/enhancer binding protein α

CCAAT/enhancer binding protein alpha (C/EBPalpha)-ER induces 32Dcl3 neutrophilic differentiation and inhibits 32DPKCdelta maturation to macrophages in response to phorbol ester. In 32Dcl3 cells, C/EBPalpha-ER rapidly induces the PU.1 and C/EBPalpha RNAs even in the presence of cycloheximide, suggesting that these are direct C/EBPalpha genetic targets. C/EBPalpha strongly binds and modestly activates the murine PU.1 promoter via an evolutionarily conserved binding site. C/EBPalpha-ER variants incapable of binding DNA still slow G1 progression but do not induce differentiation. N-terminally truncated C/EBPalpha variants, including the p30 isoform expressed in a subset of AMLs, also retain the ability to slow 32D cl3 proliferation, whereas the C/EBPalpha(BRM2)-ER variant does not slow G1 progression, has a reduced capacity to induce early granulocytic markers, and does not induce terminal maturation. In 32DPKCdelta cells, C/EBPalpha-ER strongly inhibits endogenous or exogenous JunB induction, dependent upon the outer surface of the C/EBPalpha basic region, but does not inhibit c-Jun, PU.1, or C/EBPbeta expression. Exogenous JunB restores AP-1 DNA binding but does not overcome inhibition of monopoiesis by C/EBPalpha-ER. In summary, we propose that while C/EBPalpha is required for development of immature granulocyte-monocyte progenitors, C/EBPalpha subsequently inhibits monopoiesis, via inhibition of JunB express and via additional activities, and induces granulopoiesis, via induction of PU.1, C/EBPepsilon, and cell cycle arrest.