Am J Res 2012;2(1):57-65 www.AJBlood.us /ISSN: 2160-1992/AJBR1110006

Review Article The role of transcription factors in the guidance of granulopoiesis

Katja Fiedler, Cornelia Brunner

Institute of Physiological Chemistry, University Ulm, Germany

Received October 31, 2011; accepted November 17, 2011; Epub January 1, 2012; Published January 15, 2012

Abstract: In recent years, the prospective isolation of hematopoietic stem and progenitor cells has identified the hier- archical structure of hematopoietic development and lineage-commitment. Moreover, these isolated cell populations allowed the elucitation of the molecular mechansims associated with lineage choice and revealed the indispensable functions of transcription factors as lineage determinants. This review summarizes current concepts regarding adult murine granulopoiesis and illustrates the importance of the transcription factors C/EBPα, PU.1 and GATA-2 for the development of , and .

Keywords: Granulopoiesis, transcription factors, C/EBPα

Introduction and activation of additional , macro- phages and T cells. In contrast, are Granulocytes are the most abundant type of resident in various organs such as the gastroin- myeloid cells in the blood stream and are char- testinal tract, mammary glands as well as bone acterized by two morphological features: the marrow and may contribute to tissue and im- multilobulated shape of the nucleus and the mune homeostasis. Only a minor part of the eponymous, excessive enrichment of storage eosinophils circulates with the blood stream vesicles, named granules, in the cytoplasm. and is recruited mainly upon T-helper 2-type Based on the staining properties of these gran- responses (TH2) into sites of , ules the granulocytes can be further subdivided where they produce several and lipid into neutrophils, eosinophils and . The mediators and release toxic proteins. most frequent granulocytes are neutrophils that Additionally, eosinophils are able to initiate and are constantly generated in a high number in amplify antigen-specific immune responses via the and circulate with the blood antigen presentation to naïve and memory T stream until activation in response to infections. cells. The least common granulocytes in the After the conquest of the physical barriers, pro- circulation are basophils. These cells are not vided by skin and mucus membranes and infil- only one of the main sources of histamine in tration of tissue by pathogens, neutrophils be- allergic reactions, but also associated with the come activated by signals that are provoked by differentiation of naïve T cells towards TH2 cells resident at the site of infection. during allergic and anti-parasitic immune re- The activated macrophages release cytokines sponses via antigen presentation and IL-4 pro- that in turn activate locally endothelial cells and duction. enable the capture of bypassing neutrophils to guide them into the infected tissue. Following Development of committed progenitors the arrival at the site of infection, neutrophils combat microorganism via , the Although the different types of granulocytes release of microbicidal proteins via degranula- have quite diverse immune functions, the differ- tion as well as by neutrophil extracellular trap entiation pathways of neutrophils, eosinophils formation. Furthermore, neutrophils are able to as well as basophils show huge similarities. Like regulate the immune response via recruitment all other blood leukocytes, granulocytes derive

Transcription factors in granulopoiesis

from hematopoietic stem cells (HSC) in the bone marrow. These HSC possess multi- potentiality as well as the ability for self-renewal and develop into multipotent progenitors (MPP), which are characterized by a more limited prolif- erative potential, but retained ability to differen- tiate into various hematopoietic lineages.

At the moment a defined model for the hierar- chy of multipotent progenitors is not available, because several studies have demonstrated different types of multipotent progenitors with myelo-lymphoid or myelo-erythroid potential, such as the lymphoid-primed multipotent pro- genitor (LMPP) [1] or a putative - -lymphoid progenitor (GMLP) [2]. Downstream of the HSC and MPP populations starts the hematopoietic differentiation process in hematopoiesis leading to oligopotent and later on to lineage-committed progenitors with a diminished proliferation but increased differen- tiation. The contemporary model of hematopoi- esis assumes that the decision for differentia- tion into the lymphoid/myeloid or megakaryo- cyte/erythrocyte lineages probably occurs very early in hematopoiesis. Several studies have demonstrated that multipotent progenitors like the LMPP retain only minor / erythrocyte lineage potential, whereas the vast majority of progenitors appears to be committed to the granulocyte/monocyte as well as the lym- Figure 1. Hierarchy of hematopoietic progenitors. The phoid lineage [1, 3]. developmental course shown in the scheme is proposed using results generated by prospective isolation and char- acterization of different progenitors in mouse. HSC, hema- In the next step of ongoing differentiation oli- topoietic stem cell; MPP, multipotent progenitor; LMPP, gopotent progenitors with differentiation capac- lymphoid-primed multipotent progenitor; CLP, common ity for severala hematopoietic lineages develop lymphoid progenitor; CMP, common myeloid progenitor; from an ancestor, the common lymphoid pro- MEP, megakaryocyte-erythrocyte progenitor; GMP, granu- genitor (CLP) [4] and the common myeloid pro- locyte- progenitor; MDP, monocyte-dendritic genitor (CMP) [5]. The CLP carries differentia- cell progenitor; BMCP, basophil- progenitor; EP, tion potential for all types of lymphoid cells in- erythroid progenitor; MCP, mast cell progenitor; EoP, eosi- cluding B cells, T cells and NK cells and is the nophil progenitor; BaP, basophil progenitor. Figure adapted from [2, 53, 54]. earliest population of oligopotent progenitors that upregulates the receptor for interleukin 7 (IL-7), an essential for T and B cell de- velopment. The CMP give rise to all types of myeloid colonies in clonogenic assays, while the gard to neutrophils a committed progenitor is granulocyte-monocyte progenitor (GMP) is re- not yet described (Figure 1). stricted to granulocytes and macrophages and the megakaryocyte-erythrocyte progenitor (MEP) Transcriptional regulation of myeloid progenitor is delimitated to and erythro- commitment cytes [5]. From the GMP derives the eosinophil lineage-committed progenitor (EoP) [6] and the The differentiation process from HSC into line- basophil/mast cell progenitor (BMCP), which in age-committed hematopoietic cells involves the turn gives rise to the mast cell progenitor (MCP) selective activation of lineage-specific genes as and the basophil progenitor (BaP) [7]. With re- well as the silencing of lineage-foreign genes

58 Am J Blood Res 2012;2(1):57-65 Transcription factors in granulopoiesis

and developmental regulators in a defined or- vation potential of the counterpart [15]. Based der. The orchestration of such complex lineage- on these findings, GATA-1 is prospected as the determining programs is dependent on several erythroid/megakaryocyte lineage determinant, factors, but extensive research has emphasized whereas PU.1 is regarded as the myeloid/ the essential role of gene regulatory networks in lymphoid lineage determinant. directing cell fate choice and lineage restriction. These gene regulatory networks are composed Downstream of LMPP or GMLP, lineage choice of several master transcription factors that join embraces myeloid, as well as B or T lymphoid special features, such as mutual regulation of lineage and mainly depends on the gene regula- transcriptional activity by antagonism as well as tory network of the transcription factors PU.1, lineage-determining functions via activation of early B cell factor (EBF) and Notch. For myeloid lineage-specific genes and repression of lineage lineage restriction, a high expression level of -foreign genes. One example for such gene regu- PU.1 is necessary, whereas low levels of PU.1 latory networks is the choice for erythroid ver- plus EBF expression establish the B lymphoid sus myeloid-lymphoid lineage restriction at the lineage restriction and Notch instructs the T transition from MPP to LMPP or MEP that is lymphoid lineage choice. The B cell fate deter- regulated by the E-twenty six (Ets) family tran- minant EBF has been demonstrated to initiate scription factor PU.1 and the transcription factor the early B cell lineage program of gene expres- GATA-binding protein 1 (GATA-1). GATA-1 is ex- sion (B29, VpreB and Pax5) and to antagonize pressed in erythroid, megakaryocyte, mast as expression of myeloid lineage genes (Cebpa, well as eosinophil lineages and contains zinc Sfpi1 and Id2) [16]. Additionally, the same regu- fingers, which mediate DNA binding to the WGA- latory properties have been shown for the T cell TAR DNA sequence as well as protein-protein fate determinant Notch that inhibits B cell de- interaction [8, 9]. In contrast to GATA-1, PU.1 is velopment via repression of EBF function as restricted to monocyte as well as B lymphoid well as Pax5 expression, a secondary B cell line- lineages and consists of a N-terminal transacti- age determinant [15]. vation domain, a PEST-domain, and the epony- mous Ets-domain at the C-terminus that medi- The granulocyte versus monocyte lineage ates DNA binding to an 11 bp sequence with a choice central GGAA motif [10, 11]. Additionally, both transcription factors are detectable in MPP and Regarding granulopoiesis the transcription fac- gene disruption studies have demonstrated the tor CCAAT-enhancer binding protein α (C/EBPα) indispensable functions of GATA-1 and PU.1 for has to be enumerated, since studies have dem- megakaryocyte/erythrocyte and myeloid/ onstrated that conditional deletion of C/EBPα in lymphoid development, respectively. Analyses of bone marrow cells of mice using the Mx1-Cre systemic PU.1-deficient mice revealed a com- system leads to a total lack of mature granulo- plete loss of CMP, GMP and CLP populations cytes and a partial lack of due to a but normal numbers of MEP causing impaired differentiation block at the transition from CMP lymphoid as well as myeloid cell development to GMP [17]. C/EBPα is the prototype of the C/ without influencing megakaryocyte/erythrocyte EBP family and displays all characteristic fea- development [12]. In contrast, GATA-1-deficient tures of the transcription factor family, such as mice die between embryonic day 10.5 and 11.5 the N-terminal transactivation domain as well as due to severe anemia resulting from a matura- the C-terminal DNA-binding domain consisting tion arrest of erythroid cells [13]. Further sup- of a highly conserved basic region and a leucine port for the lineage instructive role of GATA-1 zipper dimerization domain. Prerequisite for originated from forced expression of GATA-1 in binding of C/EBPα to the cognate DNA-site is lineage-committed progenitors like GMP and the homo- or heterodimerization with another CLP exclusively leading to megakaryocyte/ transcription factor via the leucine zipper do- erythrocyte development [14]. Several other main that in turn allows the basic region to bind studies dealing with certain aspects of the mo- to the CCAAT motif [18, 19]. The need for C/ lecular interaction of PU.1 and GATA-1 as well EBPα during the transition from CMP to GMP is as their gene regulatory capacity revealed the possibly due to the transcriptional upregulation cross-antagonism between these proteins in- of PU.1, since forced C/EBPα expression in he- volving direct physical interaction of both fac- matopoietic progenitors favors monopoiesis and tors that results in an inhibition of the transacti- not granulopoiesis, whereas exogenous C/EBPα

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in myeloid cell lines directs granulopoiesis [20]. miR-223 are the cell cycle regulator E2F1 and Moreover, lineage choice between monocytes the monocyte-lineage promoting gene Mef2c and granulocytes depends on the expression leading to suppression of proliferation and in- level of PU.1 and C/EBPα, which has been duction of granulocyte differentiation [31]. A shown by studies using different mouse as well similar mechanism has been recently described as in vitro models for diminished PU.1 expres- for miR-34a that is also increased expressed sion in the hematopoietic system. In all experi- during granulopoiesis by C/EBPα-mediated tran- mental setups, reduced expression of PU.1 is scription and targets the cell cycle regulator followed by an augmented granulopoiesis to the E2F3 [32]. disadvantage of monocyte development. In line with these findings, we have demonstrated that Taken together, the plethora of studies impli- loss-of-function mutations of Btk in myeloid cates the following model for monocyte versus cells diminshed the C/EBPα as well as PU.1 granulocyte lineage choice: First, C/EBPα is expression resulting consequently also in an needed for the transition from CMP to GMP by increased granulopoiesis at the expanse of mo- induction of PU.1 expression (Figure 2). High nopoiesis [21]. Additionally, gene expression protein levels of PU.1 induce monopoiesis via analyses of PU.1-deficient progenitors revealed interaction with other transcription factors like a decreased or even absent expression of sev- interferon regulatory factor 8 (IRF8) or activating eral monocyte-specific genes, like the macro- protein-1 family transcription factors (AP-1/Jun phage scavenger receptor or the M-CSF recep- proteins) and the transcriptional activation of tor. monocyte-specific genes [20]. However, AP-1 family transcription factors are also able to het- The indispensable functions of C/EBPα for erodimerize with C/EBPα [33] implicating an granulocyte development are additionally inhibition mechanism of PU.1 for granulocyte pointed out by the transcriptional upregulation development by sequestering the binding part- of several granulocyte-specific factors. One of ners of C/EBPα. In contrast to the high protein these factors is the transcriptional repressor levels of PU.1 that favor monopoiesis, insuffi- growth factor independent 1 (Gfi1), which is cient activation of PU.1 transcription allows C/ necessary for the repression of proliferation and EBPα to induce the granulopoiesis program ac- of monocyte lineage-promoting factors such as companied by suppression of monopoiesis. M-CSF [22]. Another lineage-determining mechanism displays the repression of the mi- The eosinophil versus basophil lineage choice croRNAs miR-21 and miR-196b Gfi-1 during granulopoiesis, since ectopic expression of both The lineage commitment towards the eosinophil miRNAs in myeloid progenitors results in a com- or basophil lineage downstream of the GMP plete block of G-CSF induced granulopoiesis mainly depends on the expression of GATA tran- [23]. Besides the upregulation of other tran- scription factors. First hints for the importance scriptional regulators, C/EBPα forces granulo- of GATA transcription factors revealed from cyte development additionally by transactivation gene expression analyses of eosinophils and of various genes, such as genes coding for the basophils demonstrating that GATA-1 as well as G-CSF receptor [24, 25] or for myeloperoxidase GATA-2 are elusively expressed in EoP, BMCP (MPO) [26], and downregulation of proliferation and BaP, but not in GMP [34, 35]. In addition by direct interaction with the cell cycle regulator the targeted deletion of the high-affinity GATA- E2F [27, 28]. In line with these experimental binding site in the GATA-1 promoter resulted in results is the association of inactivating C/EBPα the selective loss of eosinophils [36], whereas mutations with hematopoietic malignancies like ectopic expression of GATA-2 in GMP introduced acute myeloid leukemia and high-risk myelodys- exclusively the development of eosinophils [35]. plastic syndrome proposing that C/EBPα pos- Nevertheless, the GATA transcription factors are sesses the ability to arrest cell proliferation and not the solely transcription factors necessary for to drive terminal differentiation [29]. Moreover, the development of eosinophils and basophils, the transcription of miR-223 is activated by C/ since C/EBPα and PU.1 are also involved in line- EBPα that replaces the transcriptional repressor age commitment. The importance of C/EBPα for NFI-A upon activation of granulocytic differentia- eosinophil development has been shown by the tion [30] and results in the upregulation of miR- loss of the eosinophil lineage after the deletion 223 during granulopoiesis [30, 31]. Targets of of C/EBPα in mice [37] and the disruption of C/

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MBP (major basic protein) by GATA-1 and PU-1. In summary, the gene regulatory network causing eosinophil versus basophil line- age choice is supposed to be dependent on the level of expression as well as on the order of expression of the transcrip- tion factor GATA-1 or GATA-2, C/EBPα and PU.1. For example, the upregulation of GATA-1 or GATA-2 along with a sus- tained C/EBPα expression at the GMP stage results in the development of eosi- nophils progenitors. However, for lineage commitment of basophils and mast cells the upregulation of GATA-2 expression accompanied by the downregulation of C/EBPα expression is needed. Down- stream of the BMCP basophils as well as mast cells can develop depending on the expression level of C/EBPα: reintroduc- tion of C/EBPα expression leads to the development of basophils, whereas fur- ther downregulation of C/EBPα expres- sion results in mast cell development (Figure 2).

Transcriptional regulation of terminal neutrophil differentiation

Terminal neutrophil granulopoiesis starts with the and Figure 2. Transcriptional regulation of granulocyte lineage state, where the switch from proliferation commitment. The scheme displays a simplified overview of to differentiation takes place, displayed gene regulatory networks, which have a major influence on by the loss of ability for cell division after hematopoietic lineage choice during hematopoiesis. Supposed the promyelocyte state. Moreover, the (dashed lines) and proved (continuous lines) cross- formation of the first granules starts, antagonisms between key transcription factors which function which are named primary or azurophilic to regulate binary cell fate choices are noted in the scheme. granules. Primary granules are defined by Additionally, transcription factors that are important for the a high content of myeloperoxidase (MPO), generation of particular intermediates are noted in white. HSC, ; MPP, multipotent progenitor; LMPP, bactericidal permeability-increasing pro- lymphoid-primed multipotent progenitor; CLP, common lym- tein, defensins, and a family of structur- phoid progenitor; CMP, common myeloid progenitor; MEP, ally related serine proteases, namely megakaryocyte-erythrocyte progenitor; GMP, granulocyte- cathepsin G, neutrophil elastase (ELANE) macrophage progenitor; EoP, eosinophil progenitor; MDP, and proteinase 3 [38]. The most impor- monocyte- progenitor; BMCP, basophil-mast cell tant transcription factors at myeloblast/ progenitor; MCP, mast cell progenitor; BaP, basophil progeni- promyelocyte stage are C/EBPα and Gfi1, tor. Figure adapted from [15]. which are necessary for the suppression of monocyte development and prolifera- tion as well as for the transcriptional acti- vation of granulocyte-specific genes like EBPα expression in BMCP leading exclusively to MPO, ELANE or CEBPE [22, 28, 29]. The impor- mast cell development at the expanse of baso- tance of Gfi1 and ELANE has been demon- phils [7]. Furthermore, Du and colleagues have strated by studies analyzing the genetic back- demonstrated the synergistic transactivation of ground of severe congenital (SCN) the promoter of the eosinophil-specific gene and other forms of neutropenia. These studies

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Figure 3. Transcription factors involved in terminal granulopoiesis. The terminal granulopoiesis that is characterized by sequential formation of different granule types and segmentation of the nucleus starts at the myeloblast/ promyelocyte stage and ends with mature neutrophils. Granule types not only differ in the time point at which they are formed, but also in their specific content, which is described at the bottom of the figure. Above the line in the boxes matrix content is depicted and beneath the proteins that are located to the vesicle membrane. At different stages of terminal granulopoiesis several transcription factors, which are indicated on top of the figure, are important for the regulation of maturation and timed expression of granule proteins. Figure adapted from [22, 38, 44].

revealed that one of the major causes for loss lopoiesis and resulted in mature neutrophils of neutrophil differentiation beyond promyelo- due to direct regulation of C/EBPα expression cyte state are mutations in the ELANE gene [39, [43]. 40], but in rare cases of SCN also mutations of the GFI1 gene have been described [41]. De- Ongoing differentiation beyond tailed analyses of Gfi1 in mice further supported leads to the development of and the function of Gfi1 as molecular switch towards , which are defined by the be- granulocyte development by suppression of ginning of nuclear segmentation and the ap- monocyte-specific genes, like Csf1 (M-CSF) and pearance of secondary (also called specific) Csf1r (M-CSFR) [42]. Recently, Skokowa and granules as well as the exit from cell cycle. Sec- colleagues described another transcription fac- ondary granules are characterized by high tor important for terminal neutrophil differentia- amounts of lactoferrin, cathelicidin, collagenase tion - the lympohid enhancer-binding factor 1 as well as leukolysin and lysozyme in the matrix (LEF-1) - that is highly expressed in promyelo- [38, 44]. The regulation of secondary granule cytes and associated with severe congenital protein expression and the exit from cell cycle neutropenia (CN). Analyses of bone marrow mainly depends on the transcription factor C/ samples of CN patients revealed a differentia- EBPε, whose expression peaks in myelocytes tion block at the promyelocytic stage and a and metamyelocytes [28, 45] (Figure 3). Based greatly reduced or even absent expression of on studies using C/EBPε-deficient mice, which LEF-1 in comparison to bone marrow samples of displayed neutrophil-specific defects including healthy individuals. Moreover, reconstitution of bilobed nuclei, abnormal respiratory burst, com- LEF-1 in early hematopoietic progenitors of pa- promised bactericidal activity as well as im- tients with CN corrected the defective granu- paired chemotaxis [46, 47], the genetic cause

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of a very rare congenital disorder named neutro- fates depending on the presence of other tran- phil specific granule deficiency (SGD) has been scription factors co-expressed in the cell or on delineated to the CEBPE locus [48]. Additional the level and order of protein expression. There- studies revealed essential functions of C/EBPε fore, the granulocyte differentiation process for the expression of secondary and tertiary clearly demonstrates the importance of regula- granule proteins [46, 49] and demonstrated the tory networks that integrate several intrinsic direct interaction of C/EBPε with E2F1 and Rb and extrinsic signals for the outcome of differ- protein, finally leading to cell cycle exit [50]. entiation and cell fate decisions.

The last step of terminal granulopoiesis, the Address Correspondence to: Dr. Cornelia Brunner, differentiation into band and segmented neutro- Institute of Physiological Chemistry, Ulm University, phils leads to mature neutrophils with finally Albert-Einstein-Allee 11, D-89081 Ulm, Germany Tel: segmented nuclei and tertiary as well as secre- 0049-731-500-33846; Fax: 0049-731-500-22892; E -mail: [email protected] tory granules. Tertiary granules are mainly de- fined by a high content of gelatinase as well as References leukolysin and lysozyme, whereas secretory granules only contain plasma proteins in their [1] Adolfsson J, Mansson R, Buza-Vidas N, matrix. The most important feature of a secre- Hultquist A, Liuba K, Jensen CT, Bryder D, tory vesicle is its membrane that is highly en- Yang L, Borge OJ, Thoren LA, Anderson K, Sit- riched for receptors such as Mac-1/CD11b, nicka E, Sasaki Y, Sigvardsson M and CD14, FcγRII (CD16) or formyl-peptide receptor Jacobsen SE. Identification of Flt3+ lympho- (fMLP-R) [44]. In the course of neutrophil termi- myeloid stem cells lacking erythro- nal differentiation, C/EBPα expression gradually megakaryocytic potential a revised road map diminishes during the myeloblast stage. C/EBPε for adult blood lineage commitment. Cell peaks at the / stage, 2005; 121: 295-306. [2] Iwasaki H, Akashi K. Myeloid lineage commit- whereas the expression level of the transcrip- ment from the hematopoietic stem cell. Immu- tion factors PU.1, C/EBPβ, C/EBPδ and C/EBPγ nity 2007; 26: 726-740. increases continously during development to [3] Iwasaki H, Akashi K. Hematopoietic develop- the metamyelocyte stage [45]. However, gene mental pathways: on cellular basis. Oncogene deletion studies using C/EBPβ- or C/EBPδ- 2007; 26: 6687-6696. deficient mice revealed no hematopoietic ab- [4] Kondo M, Weissman IL and Akashi K. Identifi- normalities with regard to terminal granulopoi- cation of clonogenic common lymphoid pro- esis. Still, Hirai and colleagues have demon- genitors in mouse bone marrow. Cell 1997; strated the indispensable role of C/EBPβ during 91: 661-672. [5] Akashi K, Traver D, Miyamoto T and Weissman emergency granulopoiesis in response to cyto- IL. A clonogenic common myeloid progenitor kine treatment or fungal infection. In contrast, that gives rise to all myeloid lineages. Nature C/EBPα and C/EBPε were not upregulated un- 2000; 404: 193-197. der these conditions [51]. In the case of the [6] Iwasaki H, Mizuno S, Mayfield R, Shigematsu transcription factor PU.1, a conditional gene H, Arinobu Y, Seed B, Gurish MF, Takatsu K deletion model revealed a PU.1-dependent and Akashi K. Identification of eosinophil line- transcriptional activation of gp91phox, a compo- age-committed progenitors in the murine bone marrow. J Exp Med 2005; 201: 1891-1897. nent of the cytochrome b558 of the NADPH oxi- dase, as well as of Mac-1/CD11b [52]. [7] Arinobu Y, Iwasaki H, Gurish MF, Mizuno S, Shigematsu H, Ozawa H, Tenen DG, Austen KF

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