(2010) 24, 1249–1257 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu REVIEW

The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis

P Burda1, P Laslo2 and T Stopka1,3

1Department of Pathophysiology and Center of Experimental Hematology, First Faculty of Medicine, Charles University, Prague, Czech Republic; 2Section of Experimental Haematology, Leeds Institute of Molecular Medicine, University of Leads, St James’s University Hospital, Leeds, UK and 31st Department of Medicine-Hematology, General University Hospital, Prague, Czech Republic

Hematopoiesis is coordinated by a complex regulatory network Additional domains include an N-terminal acidic domain and a of transcription factors and among them PU.1 (Spi1, Sfpi1) -rich domain, both involved in transcriptional activa- represents a key molecule. This review summarizes the tion, as well as a PEST domain involved in –protein indispensable requirement of PU.1 during hematopoietic cell fate decisions and how the function of PU.1 can be modulated interactions. PU.1 protein can be modified post-translationally by protein–protein interactions with additional factors. The by phosporylation at 41 (N-terminal acidic domain) and mutual negative regulation between PU.1 and GATA-1 is 142 and 148 (PEST domain), which results in augmented detailed within the context of normal and leukemogenic activity. hematopoiesis and the concept of ‘differentiation therapy’ to The PU.1 protein can physically interact with a variety of restore normal cellular differentiation of leukemic cells is regulatory factors including (i) general transcription factors discussed. Leukemia (2010) 24, 1249–1257; doi:10.1038/leu.2010.104; (TFIID, TBP), (ii) early hematopoietic transcription factors published online 3 June 2010 (GATA-2 and Runx-1), (iii) erythroid factor (GATA-1) and (iv) Keywords: PU.1; leukemia differentiation; GATA-1; chromatin; non-erythroid factors (C/EBPa, C/EBPb, IRF4/8 and c-Jun). differentiation therapy Interestingly, such protein–protein complexes can modify PU.1 overall transcriptional activity. As such, these interactions can be employed to modulate PU.1-dependent cell fate decisions and provides context-dependent decision to be dictated based on the specific nature of PU.1 interactions with other . PU.1 and its role in hematopoiesis PU.1 is key differentiation regulator that, in concert with its transcriptional partners, can modulate the expression of at least Hematopoiesis is a highly orchestrated multi-step process 3000 expressed in hematopoietic cells,2,6 including regulated by transcription factors that involves the proliferation, known cell-surface proteins (CD11b, CD16, CD18 and CD64), differentiation and maturation of a very small population of self- cytokines and their respective receptors (G-CSF, GM-CSF and renewing, pluripotent hematopoietic stem cells into various M-CSF) and many other targets expressed during myelo- specialized and distinct blood cell types. During this process, poiesis and hematopoietic stem cells (HSC) homing.7 Recent cellular specification is initiated by primary lineage determi- ChIP–Chip approaches using tiled promoter arrays have nants, such as the PU.1. At sub-threshold identified 41000 direct target genes of PU.1 within terminal levels, primary determinants can ‘transcriptionally prime’ multi- macrophages (murine RAW264 cell line) locating the majority potential progenitors to establish a low-level expression of a 1–3 of PU.1 occupied regions within the proximal core promoter mixed lineage pattern of . Upon reaching a region.8 Several examples document that some PU.1 binding critical threshold of activity, the primary determinants will sites are located close to the transcriptional start of TATA-less differentiate the multi-potential progenitors along a particular genes (for example, M-CSFR and FcgR1b) and have an important cell-fate and this commitment is regulated by secondary role in gene activation by recruiting the transcriptional regulators (transcription factors that cooperate or antagonize machinery.4,9 These data are supported by experimental with primary determinants) which function to ‘lock in’ the exchange of the PU.1 cis-binding site to that of a TATA box decision by repressing the alternate lineage and aide in 2,4 leading to activation of myeloid promoters by mechanism promoting the uni-lineage choice. involving direct interaction with TBP and with a set of TATA- PU.1 is encoded by the Sfpi1 (Spi-1) gene and produces a associated factors.10 Interestingly, PU.1 binding sites are often protein consisting of 272 amino acids (predicted MW of accompanied by other DNA cis-elements of known transcrip- 31 kDa), and is related to the Spi-B and Spi-C Ets-family factors tional hematopoietic co-regulators (for example, Sp1, IRF, (both sharing approximately 40% homology). PU.1 RUNX-1 and C/EBP) indicating gene regulation by a combina- contains various distinct functional domains namely an Ets torial manner consisting of various transcription factors.9,11–15 domain that shows a winged helix-turn-helix domain and 5 Examples of such combinatorial regulation include PU.1 in recognizes DNA sequence harboring the core GGAA motif. conjunction with Sp1 to regulate the c-fes gene16 or with IRF-4 during direct activation of immunoglobin light and heavy chain Correspondence: Dr T Stopka, Pathological Physiology and Center of genes.17,18 The nature of the PU.1 protein complexes formed at Experimental Hematology, First Faculty of Medicine, Charles the regulatory sites of target genes dictates the extent to which University in Prague, U Nemocnice 5, Prague 2, 12853, Czech Republic. E-mail: [email protected] genes are transcribed and thus altering the composition of the Received 30 November 2009; revised 18 March 2010; accepted 16 PU.1 protein complex can be used to regulate lineage-specific April 2010; published online 3 June 2010 gene expression of PU.1 target genes. PU.1 and GATA-1 in leukemia differentiation P Burda et al 1250 PU.1 mRNA and protein expression begins within HSC PU.1 and GATA-1 interact during early hematopoietic cell and is attenuated in common lymphoid and myeloid pro- decisions genitors and also within subsequent daughter cells. PU.1 is expressed in mature monocytes, , B and NK cells Work from laboratories of A. Skoultchi, D. Tenen and T. Graf yet absent in T cells, reticulocytes and .19,20 The showed that PU.1 and GATA-1 proteins physically interact with PU.1 gene itself is transcriptionally regulated by several each other within hematopoietic cells. It was in subsequent studies mechanisms. The proximal promoter of Spi1 contains binding that elucidated how this process is accomplished and, more sites for PU.1 indicating a self autonomous regulatory mechan- importantly, the molecular consequences of this protein–protein ism of gene expression21 as well as binding sites for Oct-1, Sp1, interaction during normal and pathological hematopoiesis.47–49 GATA-1 and Spi-B, which likely mediate initial regulation The initial findings that GATA-1 and PU.1 proteins are (activation or repression) of PU.1 during hematopoietic devel- components of a minimal gene regulatory network regulating the opment. Despite the presence of numerous cis-regulatory erythroid cell fate choice came from their unnatural co-expression elements, the proximal Spi1 promoter alone is insufficient to in erythroleukemic cells (MEL cells) of mice that harbor the friend drive PU.1 expression in a correct tissue-specific manner unless virus integration within the URE of the Spi-1 gene. This viral the distal regulatory enhancer (URE, upstream regulatory integration caused the aberrant and constitutive expression of PU.1 element) is also present.22,23 The distal enhancer is located throughout the erythroid compartment resulting in the erythroleu- within a DNAse I hypersensitivity site located 14 and 17 kb kemic pathology.50 These findings were subsequently corroborated upstream of the PU.1 transcription start site in mouse and by experiments with PU.1-overexpressing transgenic mice that humans, respectively.23 Although the URE is required for develop erythroleukemias at a high rate.51 optimal PU.1 expression, its molecular regulation of transcrip- MEL cells can be cultured in vitro and by treatment with tion is complex. Deletion of URE in the mouse has a detrimental certain chemical inducers (DMSO, HMBA) can accomplish the effect on the PU.1 protein level reducing it to 20% of normal process of terminal erythroid differentiation followed by a levels.24,25 Various transcription factors can bind to the URE proliferation block.52,53 During this differentiation process, PU.1 including PU.1 itself, Elf1, FLI-1, Runx-1 and C/EBP.11,23,26,27 expression declines whereas GATA-1 remains expressed. The URE regulates transcription of both sense as well as Similar to the chemical inducers, expression of exogenous antisense non-coding transcripts and this provides an addi- GATA-1 can induce MEL differentiation along the erythroid tional layer of modifying PU.1 activity via the regulation of lineage.54 Conversely, the transgenic overexpression of PU.1 in protein .28 MEL cells can block the GATA-1-mediated differen- The expression of PU.1 is regulated post-transcriptionally as tiation of MEL cells and induces an oncogenic .55 the 30untranslated region of PU.1 mRNA can be inhibited by the Collectively, these observations show that the dysregulation of microRNA miR-155,29 which is generated from a precursor pri- PU.1 expression is responsible for the oncogenic properties of miR-155 encoded by the BIC gene.30 miR-155 can negatively MEL cells. Despite these findings, it is curious to note that PU.1 regulate an array of approximately 400 target mRNAs with is mainly pro-differentiation factor in monocytes, granulocytes diverse function in hematopoietic progenitors, B and T-cells and B-lymphocytes and thus to explain its possible pro- and also in precursors of macrophages and granulocytes.29,31 oncogenic function within erythroid progenitors required further Previous studies have shown that miR-155-deficient mice detailed structural analyses. display defects in both B and T cell-mediated immune The ability of PU.1 and GATA-1 proteins to physically interact response31 and reduced numbers of B-cell germinal centers.30 was first shown using in vitro GST-pull down assay and by co- The requirement of PU.1 during hematopoiesis has been immunoprecipitation assays using overexpressed proteins within addressed by various experimental approaches, however, the mammalian cell lines.47 Using various sets of protein-deletion most definitive are those involving highly specific mouse models mutants (employed in the abovementioned assays) it was shown produced by genetic manipulation at the level of embryonic that PU.1 directly interacts with the zinc-finger region of GATA- stem cells. Deletion of PU.1 in mice results in a relatively late 1 through its C-terminal DNA-binding domain.47–49 Homologs embryonic lethality (around E17.5).32 PU.1À/À mice have a of GATA-1 (GATA-2 and GATA-3) and PU.1 (SPI-B) can also reduced pool of HSC and progenitors33–35 leading to the loss of interact with each other, however, display significantly lower mature macrophages, , B-cells, T-cells and mast affinities.47 cells32,36,37 and to the mild defect of .33 Interest- In reporter assays using MEL, as well as non-hematopoietic ingly, PU.1 null hematopoietic progenitors express diminished cells, PU.1 efficiently blocks GATA-1 transactivation of plasmid levels of the myeloid growth-factor receptors (G, GM, M) yet constructs and this effect is dependent on the presence of both can be expanded in vitro upon addition of the IL-3 cytokine DNA-binding as well as the transactivation domain of PU.1.47 indicating that their growth survival can be regulated in a PU.1- As PU.1 can block erythroid MEL differentiation55 and GATA-1 independent fashion.36,38,39 Conditional deletion of the PU.1 stimulates erythroid differentiation54 it seems quite possible gene in adult progenitors showed its requirement that the stoichiometry between PU.1 and GATA-1 determines for both monocyte and lymphoid progeny whereas granulocytic- whether MEL cells remain arrested in an erythroleukemic state like cells were increased indicating that PU.1 may inhibit adult or are able to be withdrawn from this block and consequentially in vivo granulopoiesis at latter stages of development.34,40 induce the gene expression program associated with erythroid Similarly, PU.1 has only a minor function in advanced differentiation. The mechanism of such stoichiometry can differentiation states of B cells25,34,35,41–43 and is not required operate in the absence of DNA-binding sites and therefore the in mature T cells.44–46 transcription factors can either mutually block each other’s Collectively the above-mentioned facts reveal an important accession to DNA56 or can coexist on DNA and block each and crucial role of PU.1 as a primary transcriptional determinant other’s transactivation by protein–protein interactions within of hematopoietic cell fate with current evidence showing its chromatin.49,57 substantial role in the cell fate control of HSC and progenitor Initially it was thought that PU.1 prevents binding of GATA-1 populations rather than the maturation of terminally differen- to DNA as shown by electrophoretic mobility shift assays.56 tiated cells. However, by using DNase-I footprinting such inhibition was

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1251

Figure 1 (a) Mechanism of repression of GATA-1 target genes by PU.1: GATA-1 recruits under normal erythroid differentiation a histone acetyltransferase (CBP) to stimulate active chromatin structure. PU.1 by interacting with GATA-1 on DNA recruits pRB and Suv39h (a histone H3K9 methyltransferase) and creates a repressed chromatin structure that is further occupied by H3K9Me3-binding protein HP1a.(b) Mechanism of repression of PU.1 target genes by GATA-1: PU.1 during myelopoiesis stimulates target gene transcription together with its c-Jun. GATA-1 interacts with PU.1 and blocks transcription by inhibiting histone H3K9 acetylation.

observed at a non-physiological molar ratio (20:1) whereas at a activation of endogenous GATA-1 or inhibition of PU.1 1:1 ratio PU.1 actually altered the GATA-1-generated footprint expression in MEL cells.58 On the basis of these observations near the GATA-1-binding site thereby arguing for the possibility (as depicted in Figure 1, left panel), it is very likely that PU.1 that both PU.1 and GATA-1 coexists on DNA.57 The GATA-1 uses similar mechanism (as identified in MEL cells) to repress reporter construct was transfected into U2OS cells, a human the erythroid transcriptional program during normal myeloid/ osteosarcoma cell line, and in this in vivo assay it clearly lymphoid differentiation. responded positively to GATA-1 and negatively following the The mechanism of mutual GATA-1 and PU.1 repression has addition of PU.1.57 Chromatin immunoprecipitation studies been examined and corroborated in several different cellular showed the co-occupancy of both PU.1 and GATA-1 proteins to systems. In chicken GATA-1 downregulates the DNA57 and that this co-occupancy was dependent on the myeloid marker MHCIIg without altering PU.1 levels.49 presence of both a GATA-1 cis-binding site and the Ets domain Validating the earlier findings (as described above), on the PU.1 protein. Interestingly, the N-terminal portion of of GATA-1 C-terminal sequence could inhibit GATA-1-medi- PU.1 does not affect the occupancy of PU.1 and GATA-1 on its ated repression of PU.1 in luciferase reporter assays and DNA-binding site57 yet is required to bind with the retinoblas- furthermore, this same region is sufficient for the activation of toma protein (pRb) for repression of GATA-1.57 Conversely, GATA-1-mediated transcription.49 PU.1 is able to repress GATA-1-mediated transcription even Although the cross-antagonistic model between PU.1 and when the C-terminal zinc-finger region of GATA-1 was replaced GATA-1 is simplistic in its nature, the presence of additional by GAL4 DNA-binding domain, which does not interact with binding partners for both proteins create a far more complex PU.1.57 Data of several laboratories collectively show that PU.1 regulatory network that now includes additional inputs. For inhibits GATA-1 on DNA,57,58 although effects of PU.1-binding example, c-Jun, a bZIP transcription factor, which is part of the GATA-1 outside DNA cannot be excluded. AP-1 transcription factor complex, functions as a critical co- Despite these biochemical studies, the question still remained of PU.1 transactivation of various myeloid promoters as to how exactly is the repressive activity of PU.1 being including the M-CSF .59 Repression of the M-CSF generated and executed. Above-mentioned experiments identi- receptor can be caused by GATA-1 whereby it displaces c-Jun fied that the acidic subdomain of PU.1 binds the C-pocket of from PU.1 leading to the reduction of PU.1 activity.48 This pRb and that this interaction is required for repression of GATA- mechanism is supported by abilities of GATA-1 and c-Jun to 1-mediated erythroid differentiation.57 The most obvious ques- specifically bind to the Ets domain of PU.1 and therefore they tion arose as to how and with whom pRb mediates the can both compete for their respective interaction with the DNA- repressive activities of PU.1? Detailed analyses of both endo- binding domain of PU.1.48 genous and transgenic integrated GATA-1 DNA-binding sites Collectively these, and the aforementioned studies, strongly revealed that PU.1 represses GATA-1 by recruiting pRb, Suv39h support the molecular model that mutually interacting PU.1 and and HP1a leading to local histone H3K9 methylation.58 This GATA-1 proteins represent an integral component in the multi-protein complex is detected within close proximity of specification of multi-potential hematopoietic cells along the GATA-1-binding sites of important erythroid genes (for example, myeloid and erythroid lineages respectively and, furthermore, Hbb-b1 and HS2, HS3 of control region, Hbb-a2, Alase, functions as a minimal cross-antagonistic gene regulatory Zfpm1, Nfe2 and Eklf) and its formation requires the presence of network that dictates the development of early multi-potential GATA-1 protein.58 Derepression of GATA-1 target genes can, in progenitors to activate its own lineage-specific gene expression turn, be efficiently achieved following the inversion of H3K9 programs and concomitantly repress alternate-lineage gene methylation near repressed genes to those of H3K9 acetylation, expression profiles. Recent genetic studies have validated the a mark of transcriptionally active chromatin. Such exchange in proposed regulatory model by using GATA-1 and PU.1 endo- the modification of the histone proteins can be induced by both genous reporters in mouse.60 GATA-1 and PU.1 expression was

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1252 detected within multi-potential progenitors with the GATA-1- inflammatory macrophages, whereas PU.1 generated myeloid positive population displaying a restricted myelo-erythroid dendritic-like cells.75 Interestingly, the mechanism of the T-cell potential lacking the potential to produce lymphocytes. Alter- reprogramming by PU.1 and C/EBP requires downregulation of nately, PU.1-positive multi-potential progenitors displayed the primary T-cell determinants Notch1 and GATA-3.75 Recent /monocyte/lymphoid-restricted progenitor activity studies have shown that even the simple ectopic expression of lacking the developmental potential for / both PU.1 and C/EBPa within mesenchymal -derived erythroid differentiation. fibroblasts is sufficient to activate the myeloid program resulting It is important to note that the combinatorial effect of PU.1 in an induced macrophage-like phenotype capable of phago- and GATA factors (not only GATA-1, but also GATA-2 and cytosis.76 Finally, enforced expression of GATA-1 leads to re- GATA-3) represent highly cell context-dependent mechanism of programming of myeloblasts and CD34 þ bone marrow cells into cell fate specification. For example, despite their inclination for ,77,78 whereas within mouse granulocyte–monocyte cross-antagonism, during and development progenitors ectopic GATA-1 can reprogram these cells to adopt the GATA and PU.1 factors actually synergize together to erythroid, eosinophilic or basophile-like properties.79 promote differentiation.36,61 Additional factors that interact with The above-described observations show that PU.1 and GATA-1 or PU.1 can also modulate the cell-dependent speci- GATA-1 act as ‘the motors for reprogramming’ and this ficity of their activities. The nuclear factor FOG-1 (ZFPM1) is a correlation could be very useful with regard to the application coactivator of GATA-162–64 and is coexpressed with GATA-1 of ‘differentiation therapy’ whereby re-establishing the correct during within erythroid and megakar- expression of PU.1 and or GATA-1 within immature leukemic yocytic cells.65 Surprisingly, in eosinophil and mast cells, FOG- cells, or those attempting to mature into an unwanted lineage 1 can antagonize GATA-1 activity thereby functioning as a should reprogram and differentiate these cells back into their corepressor within this cellular-dependent context.62,66 respective cellular lineage. The widely explored model of cell fate decisions during hematopoietic stem and progenitor development is based on the cross-antagonistic relationship between the PU.1 and GATA-1 GATA-1 and PU.1 levels are determinants of leukemogenesis transcription factors. However, once multi-potential progenitors differentiate into latter precursor stages such as monocyte/ Several key studies have shown that dysregulation of either granulocyte development, GATA-1 is no longer expressed and PU.1 or GATA-1 activity can contribute to leukemogen- the process of cell specification depends on PU.1 and another esis.24,25,80,81,82 Targeted disruption of the URE in mice primary lineage determinant, CEBPa.67 The cellular commit- significantly reduces PU.1 expression and results in the ment into the monocyte or granulocyte lineage relies on the development of with frequent cytoge- engagement of counter-antagonistic secondary lineage determi- netic aberrations.24 The URE structure is dynamically regulated nants, Gfi-1, Egr-1/2 and Nab-2, in the process of activation and by chromatin loops associated with the repression of macrophage and genes, respectively.2,4 regulator SATB1 bound directly to the URE. SATB1 binds to Striking similarities are observed in the regulatory circuit special AT-rich sequences and its expression appears to be a described for the development and subsequent lineage differ- positive regulator of PU.1 expression requiring an intact URE.83 entiation from megakaryocyte/erythroid progenitors. The gen- Mice deleted of the Satb1 gene showed lower levels of PU.1 in eration of the bipotential megakaryocyte/erythroid progenitors progenitor cells but not in the terminally differentiated progeny progenitor arises from the loss of PU.1 with the ensuing process indicating that Satb1 may cooperate in early stages of myeloid of erythrocytic versus megakaryocytic commitment dependent development.83 Satb1À/À mice, unlike UREÀ/À mutants, do not upon the EKLF and Fli-1 transcription factors, respectively.68 develop myeloid leukemia. However, it should be noted that Although the precise mechanism of how EKLF and Fli-1 can Satb1À/À mice die prematurely (early postnatal) and the limited direct lineage commitment remains to be determined, recent life-span may not be sufficient for any myeloid to studies have shown the EKLF-mediated repression of megakar- develop. Interestingly, a single polymorphism within yopoiesis by the inhibition of Fli-1 recruitment to megakaryo- the URE has been identified, which significantly diminishes cytic-specific promoters as well as to its own promoter.69 Based binding of SATB1. The single nucleotide polymorphism is on these observations, mutual cross-antagonism between frequently associated with poor prognosis of human leukemias83 transcription factors is a common and re-occurring mechanism and strongly suggests that the altered URE enhancer activity is in regulating cell fate choices during hematopoiesis. coupled with reduced PU.1 levels within developing myeloid PU.1 can regulate cell fate choice not only by (i) antagonistic progenitors thereby contributing to the pathogenesis of human or synergistic interplay with other primary determinants (GATA- myeloid leukemias. 1, C/EBPa), (ii) interaction with secondary lineage determinants Regulation of PU.1 at the post-transcriptional level by miR- but also by (iii) concentrations of its own expression where 155 provides an additional example of how dysregulation of substantial evidence strongly suggests that higher levels of PU.1 PU.1 expression could contribute to human leukemo/lympho- preferentially promotes myeloid development whereas lower magenesis. Elevated levels of miR-155 have been found in levels is required for efficient B-cell development.70–73 More- myeloproliferative and lymphoproliferative diseases such as over, lowering levels of PU.1 within multi-potential progenitors acute myeloid leukemia, chronic lymphocytic leukemia, diffuse can enhance B lymphopoiesis74 establishing a role for PU.1 large B-cell lymphoma, primary mediastinal B-cell lymphoma concentrations in the regulation of myeloid versus B lymphocyte and Hodgkin’s lymphoma.30,84–86 As PU.1 mRNA is a direct cell fate choice. target of miRNA-155 it is likely that this dysregulation of PU.1 PU.1 and GATA-1 are often described as ‘master regulators’ expression, among other crucial gene targets, contributes to the largely based on their hierarchical position during hematopoi- development of certain hematological malignancies. esis as well as their ability to induce cellular trans-differentiation of the GATA-1 gene can generate a truncated when expressed transgenically. The effects of ectopic expression protein, which contributes to development of transient myelo- of PU.1 and C/EBP was studied in committed T-cells proliferative disorder and acute megakaryoblastic leukemia in and revealed that C/EBPa/b reprogrammed these cells into patients with Down’s syndrome.80,81 Similarly, the knockdown

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1253 of GATA-1 expression to approximately 5% of its wild-type level combined with stable levels of GATA-1 create an excess of causes high incidence of erythroleukemia in mice.87 available PU.1, which is not paired by available GATA-1, thus Therefore the manipulation of either PU.1 or GATA-1 levels altering the stoichemistry between these two proteins resulting produces cellular phenotypic features of blocked differentiation in PU.1-mediated gene activation associated with acquisition of and unlimited proliferation potential. Collectively, the mole- active chromatin mark Acetyl H3K9 near the PU.1-binding sites cular interaction between GATA-1 and PU.1 occurs during early (Figure 1, right panels). The reciprocal mechanism is shown for lineage commitment of multi-potential progenitors and enable erythroid-specific genes such as Nfe2. These findings collec- rapid cell fate choices between the development of leukocytes tively show that manipulating the relative expression of or red cells from a common progenitor. Once this decision is transcription factor levels (for example, inhibition of GATA-1 made within erythroid cells, the PU.1 gene becomes repressed or activation of PU.1 (or vice versa), in erythroleukemias may and conversely in developing leukocytes the production of represent an efficient tool for inducing, and rescuing, leukemic GATA-1 is inhibited. Perturbations of this process can result in a blasts cells into a differentiated cell state.6,91 leukemic blockade. In summary, regulation of both PU.1 and GATA-1 expression As mentioned earlier, both PU.1 and GATA-1 proteins are levels are crucial for the correct cell fate determination of multi- coexpressed in MEL cells where the individual levels of each potential progenitors and that inappropriate regulation of these transcription factor are sufficient to initiate specific changes of primary determinants can cause a block of cell differentiation chromatin structure within their respective target genes. As such, and expansion of a clonal population of leukemic cells. The the promiscuous activity of PU.1 blocks erythroid differentiation developmental block seems to occur at the differentiation stage at a stage (Figure 2). This developmental arrest is where primary determinants are beginning to specify a cell fate relieved by either PU.1 inhibition or increasing GATA-1 activity lineage by activating key target transcription factors. This is culminating in the restoration of normal erythroid differentia- clearly exemplified within MEL cells where PU.1 inhibits GATA- tion.54,58 Reciprocally, inhibition of GATA-1 or increased PU.1 1-mediated transactivation of its target genes such as Fog1 and activity results in the non-erythroid differentiation of the MEL Nfe2, and concomitantly GATA-1 inhibits, albeit incompletely, into arrested cells with myelo- PU.1-mediated transactivation of its targets being additional lymphoid characteristics6 (Figure 2). Using this cellular system, primary myeloid lineage determinants such as C/EBPa and recent studies have identified key target genes that are directly CBFB.6 It is very important to note that most of the PU.1 target regulated as a consequence of manipulating the stoichiometric genes are repressed in MEL cells and thus targeting of C/EBPa balance between PU.1 and GATA-1. Gene expression profiling and CBFB by PU.1 likely represents crucial PU.1-dependent combined with chromatin immunoprecipitation identified determinants and reflects an intermediate developmental two key hematopoietic transcription factors, C/EBPa and Cbfb, process of the myelo-lymphoid cell fate. Conversely, Nfe2 and that are direct targets for both PU.1-mediated activation Fog1 are two very important GATA-1 direct targets and and GATA-1-mediated repression.6 Interestingly, both C/EBPa represent early GATA-1-dependent determinants. Understand- and CBFB are required for normal myeloid development and ing such molecular mechanisms can become useful tools for mutations in either can give rise to leukemia.88–90 The dynamic understanding of both normal hematopoiesis as well as the relationship between PU.1 and GATA-1 during both the pathogenesis of human leukemias. leukemic state of MEL cells and also upon MEL differentiation can be described in the following putative steps: at myeloid genes such as C/EBPa, PU.1 binds directly to DNA but its The concept of differentiation therapy by modulating transactivation is antagonized by GATA-1 binding directly to chromatin structure with GATA-1 and PU.1 PU.1 molecules on DNA. Activation of exogenous PU.1 The generation and establishment of a leukemic blockade is associated with extensive gene-specific changes in chromatin structure that represents both a genetic marker of a dysregulated cell as well as a tool for manipulating the gene expression to rescue the expression of a gene program back into a normal state. Hematopoietic transcription factors can initiate chromatin changes near their binding sites by recruiting chromatin modification enzymes including histone acetyltransferases and histone deacetylases (HDACs). The chromatin modifiers are not specific to any one transcription factor as both GATA-1 and PU.1 can directly interact with either the histone acetyltransfer- ase CBP (CREB-binding protein) resulting in enhanced gene activation92,93 or with HDACs, which enables efficient gene repression and is often accompanied by histone H3K9 methyla- tion.94 Surprisingly, inhibition of the H3K9-methylating enzymes Suv39h or H3K9-binding protein HP1a within MEL cells does not lead to derepression of GATA-1 target genes unless PU.1 itself is present.58 However, changing the stoichio- metry between PU.1 and GATA-1 during induced erythroid Figure 2 Scheme of erythroleukemia and its differentiation. Upon differentiation of MEL cells have been shown to facilitate differentiation from multi-potential progenitor (MPP) the erythroid an exchange between modified (H3K9 methylated) H3 development may be blocked at the level of proerythroblasts as with unmodified H3.3 near derepressed GATA-1 DNA target exemplified by MEL cell model. Manipulation of either PU.1 or GATA- 58 1 levels (indicated by arrows) can relieve this block and restore genes. This shows the complexity in attempting to alter differentiation potential toward erythroid (basophilic erythroblast) or the expression of chromatin modifiers to ‘reset or reprogram’ mixed population of myelo-lymphoid cells with nuclear abnormalities. any gene regulatory network with current research intensely

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1254 focused on how specific histone marks can be efficiently receptor dimer complexes induces a conformational change that and rapidly reversed. Such mechanisms involves enzymatic displaces the existing corepressor proteins and recruits coacti- cleavage of modified histone residue, example includes HDACs vators, thus facilitating initiation of transcription.107 Conditional and histone demethylases.95 Demethylase domain proteins expression of PML–RARa (protein encoded by the t(15;17) are able to target active mark H3K4Me3 (often associated translocation) in HT93 acute promyelocytic cells suppressed with recruitment of RNA polymerase II) to repress transcription95 PU.1 expression, whereas treatment of APL cell lines and or to demethylate inactive mark H3K9 to activate primary APL leukocytes with ATRA was capable of restoring transcription.96 PU.1 expression and induced neutrophil differentiation.108 There are many ongoing clinical trials that use global- These observations strongly indicate that ATRA treatment was operating inhibitors of histone and DNA modifications to capable of inducing differentiation of the developmentally manipulate leukemic gene expression profiles. Histone deace- arrested leukemic cells through re-establishing the underlying tylase inhibitors (iHDAC) are widely studied anticancer agents, gene regulatory network. which not only inhibit tumor growth and survival but reinitiate The literature discussed in this review strongly supports the differentiation and or induce apoptosis.97 The molecular concept that dysregulation of transcription factors is a key mechanism of why tumor cells are more sensitive and suscep- contributing factor in the pathogenesis of myeloid leukemias. tible to iHDAC is not fully understood yet likely includes A greater understanding of the gene regulatory network that differences in transcriptional regulation of genes involved in cell orchestrates normal, as well as leukemogenic differentiation will cycle, apoptosis or survival. Notably, in the human breast be of fundamental importance in achieving critical goals for the carcinoma cell line MA-11 some iHDAC can cause cell cycle clinical application of these regulatory factors in myelodysplasia arrest in G1 phase mediated through the induction of cyclin- and myeloid leukemias. After all, it is not impossible to imagine dependent kinase p21,98 whereas other inhibitors (trichostatin) that under certain conditions, targeting transcription factors with induce a delay at the G(2)/M transition, mis- the aim of restoring their normal functional activity would result segregation and multi-nucleation and thereby induce cell death in the production of mature cells that would restore their normal by promoting exit from aberrant mitosis without spindle function within leukocytes and or red blood cells. checkpoint. Azacitidine is a member of drugs known as DNA- demethylating agents and its anticancer effects are believed to be twofold by its ability to demethylate DNA as well as prevent Acknowledgements the methylation of DNA. By demethylation mechanism, trans- cription of tumor suppressor genes can be restored, resulting in We apologize to the many authors whose work we could not cite recovery of the cell cycle control. Valproic acid also displays owing to space constraints. This work was supported by the grant iHDAC properties that can induce the in vitro differentiation of from Internal Grant Agency of Ministry of Health of the Czech transformed hematopoietic progenitors and leukemic blasts in Republic (NR9021-4/2006). Work in P Laslo’s laboratory is funded acute myeloid leukemia patients.99 by the Yorkshire Cancer Research and the Wellcome Trust. Despite the current drug regimes, the effects of HDAC inhibitors and DNA methyltransferase inhibitors in leukemic patients are not cell specific, do not lead to significant References improvement in overall survival and therefore still remain as an experimental approach. In this regard it seems that an 1 Hu M, Krause D, Greaves M, Sharkis S, Dexter M, Heyworth C alternate, and more effective approach for the clinical applica- et al. Multilineage gene expression precedes commitment in the tions of ‘differentiation therapy’, would be to specifically hemopoietic system. Genes Dev 1997; 11: 774–785. manipulate the expression and-or activities of targeted tran- 2 Laslo P, Spooner CJ, Warmflash A, Lancki DW, Lee HJ, scription factor(s) rather than the use of global inhibitors of Sciammas R et al. Multilineage transcriptional priming and chromatin modification enzymes, which have low gene determination of alternate hematopoietic cell fates. Cell 2006; specificity. 126: 755–766. 3 Cross MA, Enver T. The lineage commitment of haemopoietic Targeting transcription factor proteins and miRNA molecules progenitor cells. Curr Opin Genet Dev 1997; 7: 609–613. with the aim of restoring their normal functional activity has 4 Krysinska H, Hoogenkamp M, Ingram R, Wilson N, Tagoh H, significant potential in the ‘differentiation therapy’ of leukemias. Laslo P et al. A two-step, PU.1-dependent mechanism for Modification of gene-specific transcriptional activities within developmentally regulated chromatin remodeling and transcrip- tumor cells was first used using all-trans-retinoic acid (ATRA), tion of the c-fms gene. Mol Cell Biol 2007; 27: 878–887. the acid form of vitamin A, by Zhen Yi Wang of Shanghai 5 Kodandapani R, Pio F, Ni CZ, Piccialli G, Klemsz M, McKercher S et al. A new pattern for helix-turn-helix recognition revealed Medical School in the late 1980s in the treatment of acute by the PU.1 ETS-domain-DNA complex. Nature 1996; 380: 100 promyelocytic leukemia (APL). APL represent an acute 456–460. myeloid leukemia that is characterized by chromosomal trans- 6 Burda P, Curik N, Kokavec J, Basova P, Mikulenkova D, locations involving the transfer of sequence-encoding retinoic Skoultchi AI et al. PU.1 activation relieves GATA-1-mediated acid receptor a (RARa) and is unique from other acute myeloid repression of Cebpa and Cbfb during leukemia differentiation. leukemia subtypes in its specific responsiveness to ATRA Mol Cancer Res 2009; 7: 1693–1703. 7 Gangenahalli GU, Gupta P, Saluja D, Verma YK, Kishore V, therapy. By inhibiting HDACs and DNA methyltransferase Chandra R et al. Stem cell fate specification: role of master activities, pharmacological doses of ATRA can modify the regulatory switch transcription factor PU.1 in differential chromatin structure as well as the DNA methylation pattern at hematopoiesis. Stem Cells Dev 2005; 14: 140–152. specific DNA-binding sites on target genes for retinoic acid 8 Weigelt K, Lichtinger M, Rehli M, Langmann T. Transcriptomic resulting in terminal granulocytic differentiation of APL.101–106 profiling identifies a PU.1 regulatory network in macrophages. ATRA exert its effects by modulation of gene expression by two Biochem Biophys Res Commun 2009; 380: 308–312. 9 Ross IL, Yue X, Ostrowski MC, Hume DA. Interaction between distinct classes of nuclear receptors: retinoic acid receptors PU.1 and another Ets family transcription factor promotes (RAR) and retinoid X receptors. ATRA binds with high affinity to macrophage-specific Basal transcription initiation. J Biol Chem RARs, but not to retinoid X receptors. Binding of retinoids to the 1998; 273: 6662–6669.

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1255 10 Eichbaum QG, Iyer R, Raveh DP, Mathieu C, Ezekowitz RA. 29 Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, Restriction of interferon gamma responsiveness and basal Kohlhaas S et al. microRNA-155 regulates the generation of expression of the myeloid human Fc gamma R1b gene is immunoglobulin class-switched plasma cells. Immunity 2007; mediated by a functional PU.1 site and a transcription initiator 27: 847–859. consensus. J Exp Med 1994; 179: 1985–1996. 30 Eis PS, Tam W, Sun L, Chadburn A, Li Z, Gomez MF et al. 11 Hoogenkamp M, Krysinska H, Ingram R, Huang G, Barlow R, Accumulation of miR-155 and BIC RNA in human B cell Clarke D et al. The Pu.1 locus is differentially regulated at the lymphomas. Proc Natl Acad Sci USA 2005; 102: 3627–3632. level of chromatin structure and noncoding transcription by 31 Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, alternate mechanisms at distinct developmental stages of Soond DR et al. Requirement of bic/microRNA-155 for normal hematopoiesis. Mol Cell Biol 2007; 27: 7425–7438. immune function. Science 2007; 316: 608–611. 12 Petrovick MS, Hiebert SW, Friedman AD, Hetherington CJ, 32 Scott EW, Simon MC, Anastasi J, Singh H. Requirement of Tenen DG, Zhang DE. Multiple functional domains of AML1: transcription factor PU.1 in the development of multiple PU.1 and C/EBPalpha synergize with different regions of AML1. hematopoietic lineages. Science 1994; 265: 1573–1577. Mol Cell Biol 1998; 18: 3915–3925. 33 Kim HG, de Guzman CG, Swindle CS, Cotta CV, Gartland L, 13 Hagemeier C, Bannister AJ, Cook A, Kouzarides T. The Scott EW et al. The ETS family transcription factor PU.1 is activation domain of transcription factor PU.1 binds the necessary for the maintenance of fetal liver hematopoietic stem retinoblastoma (RB) protein and the transcription factor TFIID cells. Blood 2004; 104: 3894–3900. in vitro: RB shows sequence similarity to TFIID and TFIIB. 34 Iwasaki H, Somoza C, Shigematsu H, Duprez EA, Iwasaki-Arai J, Proc Natl Acad Sci USA 1993; 90: 1580–1584. Mizuno S et al. Distinctive and indispensable roles of PU.1 in 14 Rehli M, Niller HH, Ammon C, Langmann S, Schwarzfischer L, maintenance of hematopoietic stem cells and their differentia- Andreesen R et al. Transcriptional regulation of CHI3L1, a tion. Blood 2005; 106: 1590–1600. marker gene for late stages of macrophage differentiation. J Biol 35 Dakic A, Metcalf D, Di Rago L, Mifsud S, Wu L, Nutt SL. PU.1 Chem 2003; 278: 44058–44067. regulates the commitment of adult hematopoietic progenitors 15 Liu J, Ma X. Interferon regulatory factor 8 regulates RANTES and restricts granulopoiesis. J Exp Med 2005; 201: 1487–1502. gene transcription in cooperation with interferon regu- 36 Walsh JC, DeKoter RP, Lee HJ, Smith ED, Lancki DW, latory factor-1, NF-kappaB, and PU.1. J Biol Chem 2006; 281: Gurish MF et al. Cooperative and antagonistic interplay between 19188–19195. PU.1 and GATA-2 in the specification of myeloid cell fates. 16 Heydemann A, Juang G, Hennessy K, Parmacek MS, Simon MC. Immunity 2002; 17: 665–676. The myeloid-cell-specific c-fes promoter is regulated by Sp1, 37 McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, PU.1, and a novel transcription factor. Mol Cell Biol 1996; 16: Baribault H et al. Targeted disruption of the PU.1 gene results 1676–1686. in multiple hematopoietic abnormalities. EMBO J 1996; 15: 17 Eisenbeis CF, Singh H, Storb U. PU.1 is a component of a 5647–5658. multiprotein complex which binds an essential site in the murine 38 DeKoter RP, Walsh JC, Singh H. PU.1 regulates both cytokine- immunoglobulin lambda 2-4 enhancer. Mol Cell Biol 1993; 13: dependent proliferation and differentiation of granulocyte/ 6452–6461. macrophage progenitors. EMBO J 1998; 17: 4456–4468. 18 Pongubala JM, Nagulapalli S, Klemsz MJ, McKercher SR, 39 Olson MC, Scott EW, Hack AA, Su GH, Tenen DG, Singh H Maki RA, Atchison ML. PU.1 recruits a second nuclear factor et al. PU.1 is not essential for early myeloid gene expression but to a site important for immunoglobulin kappa 30 enhancer is required for terminal myeloid differentiation. Immunity 1995; activity. Mol Cell Biol 1992; 12: 368–378. 3: 703–714. 19 Back J, Allman D, Chan S, Kastner P. Visualizing PU.1 activity 40 Dakic A, Wu L, Nutt SL. Is PU.1 a dosage-sensitive regulator of during hematopoiesis. Exp Hematol 2005; 33: 395–402. haemopoietic lineage commitment and leukaemogenesis? 20 Nutt SL, Metcalf D, D’Amico A, Polli M, Wu L. Dynamic Trends Immunol 2007; 28: 108–114. regulation of PU.1 expression in multipotent hematopoietic 41 Medina KL, Pongubala JM, Reddy KL, Lancki DW, Dekoter R, progenitors. J Exp Med 2005; 201: 221–231. Kieslinger M et al. Assembling a gene regulatory network for 21 Chen H, Ray-Gallet D, Zhang P, Hetherington CJ, Gonzalez DA, specification of the B cell fate. Dev Cell 2004; 7: 607–617. Zhang DE et al. PU.1 (Spi-1) autoregulates its expression in 42 Ye M, Ermakova O, Graf T. PU.1 is not strictly required for B cell myeloid cells. Oncogene 1995; 11: 1549–1560. development and its absence induces a B-2 to B-1 cell switch. 22 Li Y, Okuno Y, Zhang P, Radomska HS, Chen H, Iwasaki H et al. J Exp Med 2005; 202: 1411–1422. Regulation of the PU.1 gene by distal elements. Blood 2001; 98: 43 Nutt SL, Eberhard D, Horcher M, Rolink AG, Busslinger M. Pax5 2958–2965. determines the identity of B cells from the beginning to the end 23 Okuno Y, Huang G, Rosenbauer F, Evans EK, Radomska HS, of B-lymphopoiesis. Int Rev Immunol 2001; 20: 65–82. Iwasaki H et al. Potential autoregulation of transcription factor 44 Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. PU.1 by an upstream regulatory element. Mol Cell Biol 2005; The macrophage and B cell-specific transcription factor PU.1 is 25: 2832–2845. related to the ets oncogene. Cell 1990; 61: 113–124. 24 Rosenbauer F, Wagner K, Kutok JL, Iwasaki H, Le Beau MM, 45 Hromas R, Orazi A, Neiman RS, Maki R, Van Beveran C, Moore J Okuno Y et al. Acute myeloid leukemia induced by graded et al. Hematopoietic lineage- and stage-restricted expression of the reduction of a lineage-specific transcription factor, PU.1. ETS oncogene family member PU.1. Blood 1993; 82: 2998–3004. Nat Genet 2004; 36: 624–630. 46 Nelsen B, Tian G, Erman B, Gregoire J, Maki R, Graves B et al. 25 Rosenbauer F, Owens BM, Yu L, Tumang JR, Steidl U, Kutok JL Regulation of lymphoid-specific immunoglobulin mu heavy et al. Lymphoid cell growth and transformation are suppressed chain gene enhancer by ETS-domain proteins. Science 1993; by a key regulatory element of the gene encoding PU.1. 261: 82–86. Nat Genet 2006; 38: 27–37. 47 Rekhtman N, Radparvar F, Evans T, Skoultchi AI. Direct 26 Hoogenkamp M, Lichtinger M, Krysinska H, Lancrin C, interaction of hematopoietic transcription factors PU.1 and Clarke D, Williamson A et al. Early chromatin unfolding by GATA- 1: functional antagonism in erythroid cells. Genes Dev RUNX1: a molecular explanation for differential requirements 1999; 13: 1398–1411. during specification versus maintenance of the hematopoietic 48 Zhang P, Behre G, Pan J, Iwama A, Wara-Aswapati N, gene expression program. Blood 2009; 114: 299–309. Radomska HS et al. Negative cross-talk between hematopoietic 27 Yeamans C, Wang D, Paz-Priel I, Torbett BE, Tenen DG, regulators: GATA proteins repress PU.1. Proc Natl Acad Sci USA Friedman AD. C/EBPalpha binds and activates the PU.1 distal 1999; 96: 8705–8710. enhancer to induce monocyte lineage commitment. Blood 49 Nerlov C, Querfurth E, Kulessa H, Graf T. GATA-1 interacts with 2007; 110: 3136–3142. the myeloid PU.1 transcription factor and represses PU.1- 28 Ebralidze AK, Guibal FC, Steidl U, Zhang P, Lee S, Bartholdy B dependent transcription. Blood 2000; 95: 2543–2551. et al. PU.1 expression is modulated by the balance of functional 50 Moreau-Gachelin F, Tavitian A, Tambourin P. Spi-1 is a putative sense and antisense RNAs regulated by a shared cis-regulatory oncogene in virally induced murine erythroleukaemias. Nature element. Genes Dev 2008; 22: 2085–2092. 1988; 331: 277–280.

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1256 51 Moreau-Gachelin F, Wendling F, Molina T, Denis N, Titeux M, 71 Nerlov C, Graf T. PU.1 induces myeloid lineage commitment in Grimber G et al. Spi-1/PU.1 transgenic mice develop multistep multipotent hematopoietic progenitors. Genes Dev 1998; 12: erythroleukemias. Mol Cell Biol 1996; 16: 2453–2463. 2403–2412. 52 Marks PA, Sheffery M, Rifkind RA. Induction of transformed cells 72 DeKoter RP, Singh H. Regulation of B lymphocyte and to terminal differentiation and the modulation of gene expres- macrophage development by graded expression of PU.1. sion. Cancer Res 1987; 47: 659–666. Science 2000; 288: 1439–1441. 53 Marks PA, Rifkind RA. Erythroleukemic differentiation. Annu 73 Zou GM, Chen JJ, Yoder MC, Wu W, Rowley JD. Knockdown of Rev Biochem 1978; 47: 419–448. Pu.1 by small interfering RNA in CD34+ embryoid body cells 54 Choe KS, Radparvar F, Matushansky I, Rekhtman N, Han X, derived from mouse ES cells turns cell fate determination to Skoultchi AI. Reversal of tumorigenicity and the block to pro-B cells. Proc Natl Acad Sci USA 2005; 102: 13236–13241. differentiation in erythroleukemia cells by GATA-1. Cancer 74 Spooner CJ, Cheng JX, Pujadas E, Laslo P, Singh H. A recurrent Res 2003; 63: 6363–6369. network involving the transcription factors PU.1 and Gfi1 55 Rao G, Rekhtman N, Cheng G, Krasikov T, Skoultchi AI. orchestrates innate and adaptive immune cell fates. Immunity Deregulated expression of the PU.1 transcription factor blocks 2009; 31: 576–586. murine erythroleukemia cell terminal differentiation. Oncogene 75 Laiosa CV, Stadtfeld M, Xie H, de Andres-Aguayo L, Graf T. 1997; 14: 123–131. Reprogramming of committed T cell progenitors to macrophages 56 Zhang P, Zhang X, Iwama A, Yu C, Smith KA, Mueller BU et al. and dendritic cells by C/EBP alpha and PU.1 transcription PU.1 inhibits GATA-1 function and erythroid differentiation by factors. Immunity 2006; 25: 731–744. blocking GATA-1 DNA binding. Blood 2000; 96: 2641–2648. 76 Feng R, Desbordes SC, Xie H, Tillo ES, Pixley F, Stanley ER et al. 57 Rekhtman N, Choe KS, Matushansky I, Murray S, Stopka T, PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage- Skoultchi AI. PU.1 and pRB interact and cooperate to repress like cells. Proc Natl Acad Sci USA 2008; 105: 6057–6062. GATA-1 and block erythroid differentiation. Mol Cell Biol 2003; 77 Hirasawa R, Shimizu R, Takahashi S, Osawa M, Takayanagi S, 23: 7460–7474. Kato Y et al. Essential and instructive roles of GATA factors in 58 Stopka T, Amanatullah DF, Papetti M, Skoultchi AI. PU.1 eosinophil development. J Exp Med 2002; 195: 1379–1386. inhibits the erythroid program by binding to GATA-1 on DNA 78 Kulessa H, Frampton J, Graf T. GATA-1 reprograms avian and creating a repressive chromatin structure. EMBO J 2005; 24: myelomonocytic cell lines into eosinophils, thromboblasts, and 3712–3723. erythroblasts. Genes Dev 1995; 9: 1250–1262. 59 Behre G, Whitmarsh AJ, Coghlan MP, Hoang T, Carpenter CL, 79 Heyworth C, Pearson S, May G, Enver T. Transcription factor- Zhang DE et al. c-Jun is a JNK-independent coactivator of the mediated lineage switching reveals plasticity in primary PU.1 transcription factor. J Biol Chem 1999; 274: 4939–4946. committed progenitor cells. EMBO J 2002; 21: 3770–3781. 60 Arinobu Y, Mizuno S, Chong Y, Shigematsu H, Iino T, Iwasaki H 80 Wechsler J, Greene M, McDevitt MA, Anastasi J, Karp JE, Le et al. Reciprocal activation of GATA-1 and PU.1 marks initial Beau MM et al. Acquired mutations in GATA1 in the specification of hematopoietic stem cells into myeloerythroid megakaryoblastic leukemia of . Nat Genet and myelolymphoid lineages. Cell Stem Cell 2007; 1: 416–427. 2002; 32: 148–152. 61 Du J, Stankiewicz MJ, Liu Y, Xi Q, Schmitz JE, Lekstrom-Himes 81 Li Z, Godinho FJ, Klusmann JH, Garriga-Canut M, Yu C, Orkin JA et al. Novel combinatorial interactions of GATA-1, PU.1, and SH. Developmental stage-selective effect of somatically mutated C/EBPepsilon isoforms regulate transcription of the gene leukemogenic transcription factor GATA1. Nat Genet 2005; 37: encoding eosinophil granule major basic protein. J Biol Chem 613–619. 2002; 277: 43481–43494. 82 Shimizu R, Kobayashi E, Engel JD, Yamamoto M. Induction of 62 Cantor AB, Iwasaki H, Arinobu Y, Moran TB, Shigematsu H, hyperproliferative fetal megakaryopoiesis by an N-terminally Sullivan MR et al. Antagonism of FOG-1 and GATA factors truncated GATA1 mutant. Genes Cells 2009; 14: 1119–1131. in fate choice for the mast cell lineage. J Exp Med 2008; 205: 83 Steidl U, Steidl C, Ebralidze A, Chapuy B, Han HJ, Will B et al. 611–624. A distal single nucleotide polymorphism alters long-range 63 Sugiyama D, Tanaka M, Kitajima K, Zheng J, Yen H, Murotani T regulation of the PU.1 gene in acute myeloid leukemia. J Clin et al. Differential context-dependent effects of friend of GATA-1 Invest 2007; 117: 2611–2620. (FOG-1) on mast-cell development and differentiation. Blood 84 Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A. High 2008; 111: 1924–1932. expression of precursor microRNA-155/BIC RNA in children 64 Tsang AP, Fujiwara Y, Hom DB, Orkin SH. Failure of with Burkitt lymphoma. Genes Cancer 2004; 39: megakaryopoiesis and arrested erythropoiesis in mice lacking 167–169. the GATA-1 transcriptional cofactor FOG. Genes Dev 1998; 12: 85 van den Berg A, Kroesen BJ, Kooistra K, de Jong D, Briggs J, 1176–1188. Blokzijl T et al. High expression of B-cell receptor inducible 65 Cantor AB, Orkin SH. Coregulation of GATA factors by the gene BIC in all subtypes of Hodgkin lymphoma. Genes Friend of GATA (FOG) family of multitype zinc finger proteins. Chromosomes Cancer 2003; 37: 20–28. Semin Cell Dev Biol 2005; 16: 117–128. 86 O’Connell RM, Rao DS, Chaudhuri AA, Boldin MP, Taganov 66 Querfurth E, Schuster M, Kulessa H, Crispino JD, Doderlein G, KD, Nicoll J et al. Sustained expression of microRNA-155 in Orkin SH et al. Antagonism between C/EBPbeta and FOG in hematopoietic stem cells causes a myeloproliferative disorder. eosinophil lineage commitment of multipotent hematopoietic J Exp Med 2008; 205: 585–594. progenitors. Genes Dev 2000; 14: 2515–2525. 87 Shimizu R, Kuroha T, Ohneda O, Pan X, Ohneda K, Takahashi S 67 Dahl R, Walsh JC, Lancki D, Laslo P, Iyer SR, Singh H et al. et al. Leukemogenesis caused by incapacitated GATA-1 func- Regulation of macrophage and neutrophil cell fates by the tion. Mol Cell Biol 2004; 24: 10814–10825. PU.1:C/EBPalpha ratio and granulocyte colony-stimulating 88 Mueller BU, Pabst T. C/EBPalpha and the pathophysiology of factor. Nat Immunol 2003; 4: 1029–1036. acute myeloid leukemia. Curr Opin Hematol 2006; 13: 7–14. 68 Starck J, Cohet N, Gonnet C, Sarrazin S, Doubeikovskaia Z, 89 Kirstetter P, Schuster MB, Bereshchenko O, Moore S, Dvinge H, Doubeikovski A et al. Functional cross-antagonism between Kurz E et al. Modeling of C/EBPalpha mutant acute myeloid transcription factors FLI-1 and EKLF. Mol Cell Biol 2003; 23: leukemia reveals a common expression signature of committed 1390–1402. myeloid leukemia-initiating cells.[see comment]. Cancer Cell 69 Bouilloux F, Juban G, Cohet N, Buet D, Guyot B, 2008; 13: 299–310. Vainchenker W et al. EKLF restricts megakaryocytic differentia- 90 Miller J, Horner A, Stacy T, Lowrey C, Lian JB, Stein G et al. The tion at the benefit of erythrocytic differentiation. Blood 2008; core-binding factor beta subunit is required for bone formation 112: 576–584. and hematopoietic maturation. Nat Genet 2002; 32: 645–649. 70 Cross MA, Heyworth CM, Murrell AM, Bockamp EO, Dexter 91 Papetti M, Skoultchi AI. Reprogramming leukemia cells to TM, Green AR. Expression of lineage restricted transcription terminal differentiation and growth arrest by RNA interference of factors precedes lineage specific differentiation in a multi- PU.1. Mol Cancer Res 2007; 5: 1053–1062. potent haemopoietic progenitor cell line. Oncogene 1994; 9: 92 Blobel GA, Nakajima T, Eckner R, Montminy M, Orkin SH. 3013–3016. CREB-binding protein cooperates with transcription factor

Leukemia PU.1 and GATA-1 in leukemia differentiation P Burda et al 1257 GATA-1 and is required for erythroid differentiation. Proc Natl 101 Fazi F, Zardo G, Gelmetti V, Travaglini L, Ciolfi A, Di Croce L Acad Sci USA 1998; 95: 2061–2066. et al. Heterochromatic gene repression of the retinoic acid 93 Manabe N, Yamamoto H, Yamada T, Kihara-Negishi F, pathway in acute myeloid leukemia. Blood 2007; 109: Hashimoto Y, Mochizuki M et al. Prevention of PU 1-induced 4432–4440. growth inhibition and apoptosis but not differentiation block in 102 Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M murine erythroleukemia cells by overexpression of CBP. Int J et al. Methyltransferase recruitment and DNA hypermethylation Oncol 2003; 22: 1345–1350. of target promoters by an oncogenic transcription factor. Science 94 Kouzarides T. Chromatin modifications and their function. 2002; 295: 1079–1082. Cell 2007; 128: 693–705. 103 Grignani F, De Matteis S, Nervi C, Tomassoni L, Gelmetti V, 95 Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA et al. Cioce M et al. Fusion proteins of the -alpha Histone demethylation mediated by the nuclear amine oxidase recruit in promyelocytic leukaemia. Nature homolog LSD1. Cell 2004; 119: 941–953. 1998; 391: 815–818. 96 Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, 104 Lin RJ, Nagy L, Inoue S, Shao W, Miller Jr WH, Evans RM. Role of Peters AH et al. LSD1 demethylates repressive histone marks to the histone deacetylase complex in acute promyelocytic leukae- promote androgen-receptor-dependent transcription. Nature mia. Nature 1998; 391: 811–814. 2005; 437: 436–439. 105 He LZ, Guidez F, Tribioli C, Peruzzi D, Ruthardt M, Zelent A 97 Bhalla KN. Epigenetic and chromatin modifiers as targeted et al. Distinct interactions of PML-RARalpha and PLZF-RARalpha therapy of hematologic malignancies. J Clin Oncol 2005; 23: with co-repressors determine differential responses to RA in APL. 3971–3993. Nat Genet 1998; 18: 126–135. 98 Nome RV, Bratland A, Harman G, Fodstad O, Andersson Y, 106 He LZ, Tribioli C, Rivi R, Peruzzi D, Pelicci PG, Soares V et al. Ree AH. Cell cycle checkpoint signaling involved in histone Acute leukemia with promyelocytic features in PML/RARalpha deacetylase inhibition and radiation-induced cell death. transgenic mice. Proc Natl Acad Sci USA 1997; 94: 5302–5307. Mol Cancer Ther 2005; 4: 1231–1238. 107 Allenby G, Bocquel MT, Saunders M, Kazmer S, Speck J, 99 Gottlicher M, Minucci S, Zhu P, Kramer OH, Schimpf A, Rosenberger M et al. Retinoic acid receptors and retinoid X Giavara S et al. Valproic acid defines a novel class of HDAC receptors: interactions with endogenous retinoic acids. Proc Natl inhibitors inducing differentiation of transformed cells. EMBO J Acad Sci USA 1993; 90: 30–34. 2001; 20: 6969–6978. 108 Mueller BU, Pabst T, Fos J, Petkovic V, Fey MF, Asou N et al. 100 Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L et al. Use of ATRA resolves the differentiation block in t(15;17) acute all-trans retinoic acid in the treatment of acute promyelocytic myeloid leukemia by restoring PU.1 expression. Blood 2006; leukemia. Blood 1988; 72: 567–572. 107: 3330–3338.

Leukemia