Runx1

Runx1, previously known as AML-1, belongs to a family of highly conserved transcription factors that harbor a 128-amino-acid motif referred to as the Runt domain. The Runt domain functions in DNA binding, protein-protein interaction, adenosine triphosphate (ATP) binding, and nuclear localization. This family of transcription factors, known as the core binding factor (CBF) family, has been implicated in specification of cell fate and has roles in myeloid differentiation and lineage-specific granulocytic function.

Runx1 is the DNA-binding α-subunit of the CBF complex. Together with CBFβ, a widely expressed protein that enhances the DNA-binding affinity of the α-subunit, Runx1 binds the consensus DNA motif 5′ Pu ACCPuCA 3′ as a dimer. Disruption of the Runx1 gene in mice results in embryonic lethality due to failure of definitive hematopoiesis in the fetal liver. Although high levels of Runx1 expression have been reported in the early stages of myeloid differentiation, its expression decreases beyond the promyelocytic stage. Runx1 has been implicated in regulating a number of genes expressed early in the myeloid development pathway, including GM-CSF, macrophage colony-stimulating factor (M-CSF) receptor, MPO, NE, and IL-3. In addition to activating lineage-specific myeloid markers, Runx1 has been shown to stimulate the G1 to S transition in myeloid and lymphoid cell lines.

A significant percentage (10% to 20%) of human leukemia has been found to be associated with mutations in the RUNX1 gene (see Chapters 59 and 60). The most common is the t(8;21) translocation, which generates the fusion protein RUNX1/RUNX1T1 (pre viously known as AML1-ETO), where the Runt domain of Runx1 is fused in frame with the RUNX1T1 (ETO) transcriptional core pressor. This fusion protein is thought to function predominantly as a repressor that inhibits expression of genes normally activated by Runx1. For example, the tumor suppressor p14ARF, a critical Runx1 target encoded from the CDKN2A gene and which is necessary for p53 activation, is normally activated by Runx1 but is repressed by RUNX1-RUNX1T1. The mechanisms underlying Runx1 function through its target genes are not yet fully understood. However, studies in sea urchins have suggested that Runx1 regulates genes that con tribute to chromatin architecture during cell proliferation. Runx1 functions within a narrow window during development by assisting in the opening of chromatin associated with genes that are vital to hematopoietic development, and for the formation of transcription factor complexes on these genes.

Studies involving mouse knockin models of Runx1-RUNX1T1 expression have demonstrated that the fusion protein alone is not sufficient to cause leukemia. However, these animals are more susceptible to mutagen-induced AML, suggesting that Runx1-RUNX1T1 is part of a multistep process that contributes to leukemogenesis.

CCAAT Enhancer-Binding Protein Family of Transcription Factors.

 C/EBPs are a family of basic region-leucine zipper (b-ZIP) transcription factors that recognize the consensus DNA-binding sequence 5′TKN NGYAAK3′ (Y = C or T; K = T or G) within the regulatory regions of target genes. C/EBP family proteins bind DNA as either homodimers or heterodimers. This family of transcription factors, which plays a crucial role in hematopoiesis, includes C/EBPα, -β, -γ, -δ, -ε, and -ζ (CHOP-GADD 153). These proteins share highly homologous C-terminal leucine zipper dimerization domains and positively charged basic region DNA-binding motifs but differ in their N-terminal transactivation domains. CHOP-GADD 153 lacks this domain altogether and instead dimerizes with and inhibits trans activation by C/EBPα, -β, and -ε.

With the exception of C/EBPε, which is expressed exclusively in the late stages of granulopoiesis and in T lymphocytes, the C/EBP family members are expressed in a wide variety of tissues, including liver, adipose tissue, lung, intestine, adrenal gland, peripheral blood mononuclear cells, and placenta. Both C/EBPβ and C/EBPδ are expressed at high levels in late-stage granulocytes. The C/EBP family members are known to exert pleiotropic effects in the tissues in which they are expressed. This may be because of their tissue- and stage-specific expression, their ability to dimerize with members of their own family and of the Fos/Jun and ATF/CREB families of transcription factors, and their ability to interact with other transcription factors such as nuclear factor-κB (NF-κB) and specificity protein-1 (Sp-1).

The C/EBP factors have been implicated in regulating differentiation in a variety of tissues. C/EBPα plays a role in adipocyte differentiation: inhibition of C/EBPα blocks adipocyte differentiation, and overexpression of C/EBPα induces adipocyte differentiation. Regulation of constitutive hepatic genes as well as acute phase response genes in the liver involves several C/EBP family members, in particular C/EBPα. Modulation of myelomonocytic differentiation is also attributed to the activity of C/EBP family members. The importance of this family of transcription factors in myeloid differentiation has been further demonstrated by the study of hematopoietic abnormalities observed in mice with targeted disruption of C/EBPα, -β, and -ε.

C/EBPα

C/EBPα is thought to be a master regulator of the granulopoietic developmental program. It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid-specific gene promoters regulating gene expression at many different stages of myeloid maturation. Although Cebpa−/− mice die perinatally because of defects in gluconeogenesis that result in fatal hypoglycemia, they also have a selective early block in the differentiation of granulocytes without affecting either monocyte/macrophage maturation or the differentiation of other hematopoietic lineages. Myeloid cells of Cebpa−/− mice lack the G-CSFR, and it is thought that lack of mature neutrophils in these mice occurs as a result. However, the myeloid defect in Cebpa−/− mice is more severe than that seen in G-CSFR knockout mice, suggesting that C/ EBPα has additional functions vital to granulocytic maturation.

CEBPA is a single exon gene, but it is expressed as two isoforms that arise from alternate translation start sites giving rise to a full-length C/EBPα p42 and a truncated dominant negative C/EBPα p30 isoform. Translational control of C/EBPα isoform expression is orchestrated by a conserved upstream open reading frame (uORF) in the 5′ untranslated region (UTR). This region is thought to be responsive to the activities of the translation initiation factors eIF4E and eIF2 (reviewed by Khanna-Gupta) such that an increase in the activity of eIF4E or eIF2 results in an increase in expression of the shorter p30 isoform (reviewed by Calkhoven et al.).

Several groups have reported loss of function mutations in the CEBPA gene in a subset of patients (~10%) with normal-karyotype AML (reviewed by Muller and Pabst).

These mutations can be broadly classified into two categories. The first includes in-frame mutations clustered in the highly conserved C-terminus of the C/EBPα protein. The second category involves frameshift mutations at the N-terminus of C/EBPα resulting in the premature termination of the full-length C/EBPα p42 isoform while keeping the truncated C/EBPα p30 protein intact. The remaining C/EBPα p42 is thought to be rendered inactive by the dominant negative activity of the p30 isoform, although the mechanism is unknown. Mice that express a vector inducing overexpression of C/ EBPα p30 from the Cebpa locus develop AML with complete penetrance. Thus changes in the expression ratio of the two C/EBPα isoforms play a role in cell fate and in leukemogenesis (reviewed by Muller and Pabst and Kirstetter et al.).

The expression of C/EBPα is associated with growth arrest and differentiation of granulocyte precursor cells. This block in proliferation is thought to occur via the interaction of C/EBPα with, and inhibition of, the cyclin-dependent protein kinases cdk2 and cdk4. In addition, C/EBPα inhibits E2F-dependent transcription, which in turn leads to inhibition of cell proliferation and induction of differentiation associated with C/EBPα-induced granulopoiesis.

C/EBPβ

Expression of C/EBPβ increases during myeloid maturation and is important for monocyte/macrophage gene expression and development. Mice lacking the Cebpb gene demonstrate reduced B-cell numbers and defects in macrophage activation and function and increased susceptibility to microbial infections. Knockout studies reveal that C/ EBPβ is not essential for myeloid development, although knockin of Cebpb into the Cebpa locus of Cebpa−/− mice rescues granulopoiesis. Several monocyte/macrophage-specific genes are activated by C/ EBPβ, including G-CSFR, lysozyme, CD11c, monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, and nitric oxide synthase. As with C/EBPα, multiple isoforms of C/EBPβ are generated from a single transcript through three alternate translation initiation sites and a leaky ribosome scanning mechanism. The shortest of these isoforms, initiated at the most distal start codon, results in the formation of liver enriched inhibitory protein (LIP), which lacks the N-terminal activation domain present in full-length C/EBPβ and has been implicated as a negative regulator of C/EBPβ function. It has been suggested that the ratio of C/EBPβ to LIP may affect cellular proliferation and differentiation. The activity of C/EBPβ is regulated through protein-protein interactions and posttranslational modifications. For example, in early myeloid progenitor cells, C/EBPβ is found in an unphosphorylated state in the cytoplasm, whereas on differentiation, C/EBPβ becomes phosphorylated and translocates to the nucleus.

C/EBPβ in Emergency Granulopoiesis

As short-lived cells, neutrophils must be continuously produced in the BM under normal steady-state conditions. The large storage pool of neutrophils is sufficient to provide an immediate increase in circulating neutrophils in response to acute infection. However, if there is a severe or persistent demand for neutrophils due to a prolonged or particularly severe infection, a switch from steady-state to emergency granulopoiesis occurs to meet this increased demand. This marked de novo increase in neutrophil production is defined as “emergency granulopoiesis” and is critical for survival of the host. As a rule, the emergency granulopoiesis pathway follows three distinct steps: pathogen sensing and alerting the innate immune system of infection, triggering of the molecular events that lead to increased neutrophil production in the BM, and finally, restoration of steady-state conditions following clearance of the pathogen from the system (reviewed by Dao et al.).

Transcriptional Networks in Emergency Granulopoiesis

The role of transcription factors in emergency granulopoiesis has only recently begun to be elucidated. In an elegant study, expression levels of C/EBP family members were measured in early granulocytic precursor cells following induction of emergency granulopoiesis with cytokines or Candida albicans infection. Although expression of C/ EBPα, C/EBPδ, and C/EBPε was downregulated in the granulocytic precursor cells, C/EBPβ levels remained elevated, suggesting that C/ EBPβ may play an important role in emergency granulopoiesis. C/ EBPα-null hematopoietic precursor cells are capable of generating neutrophils in substantial numbers upon cytokine stimulation or upon infection with C. albicans, but C/EBPβ-null mice were unable to support emergency granulopoiesis in response to cytokine stimulation, even though steady-state granulopoiesis remained unaffected. Based on these observations, C/EBPα and C/EBPβ appear to function specifically and antagonistically toward one another during steady state and emergency granulopoiesis. C/EBPα functions as a master regulator of steady-state granulopoiesis by limiting proliferation via inhibition of expression of the cyclin-dependent kinases, cdk2 and cdk4, and of c-Myc, and by promoting granulocytic differentiation. C/EBPβ may promote emergency granulopoiesis largely because it does not block the expression of cdk2, cdk4, or c-Myc and thereby allows for proliferation of granulocytic progenitors and increasing neutrophil numbers during emergency granulopoiesis.

According to current models, the switch from steady-state to emergency granulopoiesis is thought to be regulated by increased levels of cytokines such as G-CSF and, to less extent, GM-CSF and IL-6. These cytokines are known to be upregulated in response to pathogen invasion. G-CSF signaling activates the JAK/STAT pathway, in particular, STAT3, a known regulator of granulopoiesis that together with C/EBPβ binds to the MYC promoter and activates gene expression. This also prevents binding of C/EBPα to the MYC promoter, resulting in C/EBPβ-mediated gene expression prevailing over that of C/EBPα during emergency granulopoiesis.

C/EBPγ

C/EBPγ is a ubiquitously expressed C/EBP family member that was first identified by its affinity for cis-regulatory sites in the Ig heavy chain promoter and enhancer. C/EBPγ contains a C/EBP-like b-Zip domain but lacks an N-terminal transactivation domain and can inhibit transcriptional activation of other C/EBP members in some cell types. Impairment of natural killer (NK) cytotoxic activity and of interferon-γ production has been reported in Cebpg−/− mice.

C/EBPδ

C/EBPδ is expressed at low or undetectable levels in several tissues of adult mice and humans. Expression has been shown to dramatically increase upon induction with bacterial lipopolysaccharide (LPS) and inflammatory cytokines, suggesting a role for C/EBPδ in the acute phase and inflammatory response. Experiments with double knockout of C/EBPβ and C/EBPδ suggest a synergistic role for these two C/EBP family members in controlling terminal adipocyte differentiation. Both C/EBPβ and C/EBPδ are also expressed during late neutrophil development, suggesting a parallel role in late neutrophil gene expression.

C/EBPε

The human CEBPE gene resides on chromosome 14 and is transcribed under the control of two alternative promoters, Pα (thought to func tion in mature neutrophils) and Pβ (thought to function in BM). A combination of differential splicing and alternate promoter use results in four messenger RNA (mRNA) isoforms 2.6 kb and 1.3 to 1.5 kb in size, from which three proteins of size 32.2, 27.8, and 14.3 kDa have been identified. Cebpe−/− mice produce hyposegmented granulocytes that are functionally defective, and these mice develop myelodysplasia later in life. Absence of C/EBPε is thought to block the later steps in terminal differentiation of mature segmented granulocytes. Mutant mice usually survive 2 to 5 months and eventually succumb to low pathogenicity bacterial infections. C/EBPε thus plays a crucial role in terminal granulocytic differentiation.

Cebpe−/− mice have wild-type levels of the G-CSFR, and the defects manifested in these mice are confined to late-stage gene expression associated with the function of the mature neutrophil. It has been shown that the ability of G-CSF to regulate myeloid differentiation is dependent on the induction of C/EBPε. Verbeek and colleagues demonstrated that the mRNAs of several genes, including p47 phox (a component of the neutrophil–NADPH oxidase complex), as well as the secondary granule protein genes, are either absent or abnormal in the BM of Cebpe−/− mice. These observations suggest that C/EBPε also plays a critical role in the regulation of host antimicrobial defense.

Neutrophils from Cebpe−/− mice have morphologic and biochemical features similar to those observed in patients with neutrophil SGD. SGD is an extremely rare congenital disorder that is characterized by frequent and severe bacterial infections. Patients with SGD have defects in neutrophil function, including atypical nuclear morphology, impaired bactericidal activity, and abnormalities in neutrophil migration; they also lack both neutrophil and eosinophil secondary granule proteins. Sequence analysis of genomic DNA from two patients with SGD revealed mutations within the CEBPE gene, resulting in the expression of a mutant protein lacking the dimerization and DNA-binding domains and with impaired transcriptional activity. Loss of C/EBPε activity is thought to underlie the observed pathology in these patients.

C/EBPζ

C/EBPζ-C/EBP homologous protein (CHOP) is a C/EBP family member that was originally identified as a gene product induced in response to DNA-damaging agents. It has subsequently been shown to also be induced by various extracellular or intracellular (endoplasmic reticulum [ER]) stresses. The basic region of CHOP is less well conserved than that of the other C/EBP family members, and CHOP does not seem to bind to canonical C/EBP cis elements. CHOP has been shown to interfere with the transcriptional activity of C/EBPβ in a manner dependent on its leucine zipper.

PU.1

 PU.1 (encoded on the SPI1 gene) is a member of the Ets family of transcription factors and is expressed abundantly in B cells and macrophages. Expression of PU.1 has also been reported in granulocytes and eosinophils as well as in CD34+ hematopoietic progenitor cells. High levels of PU.1 expression in fetal mouse liver preferentially direct macrophage development, whereas low levels of PU.1 result in B-cell development. c-Jun, another member of the b-Zip family of transcription factors, serves as a coactivator of PU.1, and overexpression of c-Jun in myeloid progenitor cells results in macrophage development. Downregulation of c-Jun by C/EBPα is necessary for granulocytic maturation and appears to be a primary mechanism through which C/EBPα blocks macro phage development. C/EBPα not only binds to the promoter of the JUN gene and decreases its expression but also binds PU.1, thereby inhibiting its activity.

PU.1-binding sites have been reported in almost all myeloid specific promoters reported to date, including those for M-CSF, GM-CSF, and G-CSF receptors, all of which play critical roles in myeloid cell development. PU.1 activity is modulated both by protein-protein interactions and posttranslational modifications; phosphorylation of PU.1 by casein kinase II or by JNK kinase leads to increased transcriptional activity.

Abrogation of PU.1 expression in PU.1-null mice results in peri natal lethality accompanied by the absence of mature monocytes/ macrophages and B cells and delayed and reduced granulopoiesis. Following in vitro differentiation, embryonic stem (ES) cells derived from PU.1-null blastocysts fail to express mature myeloid cell markers, suggesting that PU.1 is not essential for the initial events associated with myeloid lineage commitment but is necessary for the later stages of development.

Growth Factor Independence-1

The GFI1 gene was first identified as a target of proviral insertion following infection with Moloney murine leukemia virus (MoMuLV) resulting in IL-2 factor independence in a rat lymphoma cell line (reviewed by van der Meer et al.). GFI1 is a highly conserved gene that encodes a 55-kDa nuclear proto-oncogene that harbors six C2 H2-type zinc finger domains at the C-terminus and a 20-amino acid stretch at the N-terminus known as the SNAG domain. The SNAG domain appears to be conserved in the Snail/Slug family of proteins and confers transcriptional repressor activity on Gfi-1. The human GFI1 gene is located on chromosome 1p22, and its closely related paralog GFI1B maps to chromosome 9q34. Gfi-1 is expressed at high levels in the thymus and BM, whereas Gfi-1b expression is confined to the BM and spleen. Homozygous knockout of Gfi-1b results in embryonic lethality at day E15, despite the fact that normal myelopoiesis occurs. Death in these mice has been attributed instead to a failure of erythropoiesis and megakaryopoiesis.

The essential role of Gfi-1 in neutrophil differentiation became apparent following two reports of gene disruption in mice. Gfi-1–null mice are severely neutropenic and eventually succumb to bacterial infections. In addition, these mice lack mature neutrophils and their granulocyte precursors are unable to differentiate into mature neutrophils upon induction with G-CSF. These cells also lack second ary granule protein expression, as is seen in Cebpe−/− granulocytes. Gfi1−/− BM contains an atypical Gr1+Mac1+ myeloid precursor cell that shares characteristics of both granulocyte and macrophage pre cursors. Ectopic expression of Gfi-1 in ex vivo–sorted Gfi1−/− progenitor cells restores G-CSF–mediated neutrophil maturation to these cells. These observations provide evidence for the critical role of Gfi-1 in the neutrophil maturation program. Other studies have further demonstrated that Gfi-1 synergizes with C/EBPε to transactivate the promoters of late myeloid genes. This synergy is lost in patients with SGD who have a heterozygous substitution mutation in the CEBPE gene and decreased levels of Gfi-1 in the BM.

Heterozygous dominant negative mutations in the GFI1 gene have been described in two patients with severe congenital neutropenia (SCN), underscoring the role of Gfi-1 in the neutrophil maturation pathway. It has been suggested that mutant Gfi-1 in these patients alters the expression of NE, mutations in which are commonly associated with SCN (see later). This observation confirms the vital role that Gfi-1 plays in human granulopoiesis.

CCAAT Displacement Protein

CCAAT displacement protein (CDP)/cut is a ubiquitously expressed, highly conserved, homeodomain (HD) protein with extensive homology to the Drosophila cut protein. CDP acts as a repressor of developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene (gp91 phox, also known as Nox2 or cytochrome b-245 heavy chain), which is expressed exclusively in differentiating granulocytes (reviewed by Nepveu22). Overexpression of CDP in 32Dcl3 myeloid cells blocks G-CSF–induced expression of SGP genes without blocking phenotypical maturation. CDP therefore acts as a negative regulator of stage-specific expression of both early and late neutrophil-specific genes.

The CDP homeobox protein contains three highly conserved DNA-binding repeats referred to as cut repeats (CR1, CR2, CR3) and an HD, each of which is capable of recognizing and binding specific DNA motifs in target genes. This may explain why the CDP molecule as a whole does not have a well-defined consensus DNA binding sequence; cut repeats cannot bind DNA as monomers but in combination exhibit high DNA-binding affinity. It has further been suggested that CDP-binding activity is restricted to proliferating cells, in which CDP target genes are repressed. These targets are upregulated as cells undergo cell cycle arrest and terminal differentiation, in association with a decrease in CDP binding. Target genes of CDP are numerous and include MYC, MOS, thymidine kinase (TK), cdk inhibitor p21 (WAF1/CIP1), cystic fibrosis transmembrane conductance regulator (CFTR), transforming growth factor-β (TGF-β) receptor 2 (TGFBR2), gp91 phox (NOX2/CYBB), major histocompatibility complex (MHC) class I locus, and neutrophil SGP genes.

During myeloid differentiation, CDP binding has been shown to regulate genes that are expressed at widely disparate stages of differentiation. For example, CDP represses the CYBB gene, which is expressed at a much earlier time in myelopoiesis than is the case for the LF gene. The mechanisms by which CDP mediates repression and modulates stage-specific gene expression at different stages of differentiation within a single lineage are not fully understood. CDP is reported to have repressive activity associated with its ability to be displaced by a positive trans-acting factor involving the CR1 and CR2 cut repeats. However, other modes of repressive activity involving the two active repression domains within the C-terminus of CDP have also been reported. CDP has been shown to function as a repressor of transcription via chromatin modification by recruitment of histone deacetylases (HDACs), consistent with the hypothesis that transcriptional silencing is associated with hypoacetylated histone markers. Both acetylation and phosphorylation of CDP are posttranscriptional modifications that have been postulated to regulate CDP function. Thus differential modification, by phosphorylation or acetylation, of CDP-DNA complexes bound to the promoters of target genes could result in the observed differential repression exerted by CDP during neutrophil development.

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المزيد

مواضيع ذات صلة

Cytokine Regulation of Myeloid Proliferation and Differentiation

Neutrophil Granules and Their Content Proteins

Stages of Neutrophil Differentiation

T Lymphocytes, Transfer of Safety Genes, and Strategies to Improve Tumor Cell Specificity

Optimizing T-Cell Trafficking and Overcoming Tumor Immune Evasion

Allogeneic Banked Cells

Clinical Results of Chimeric Antigen Receptor T Cells in Hematologic Malignancies

Types of Cellular Immunotherapy

Adoptive Immunotherapy of Cancer

Therapeutic Manipulation of T-Cell-Mediated Immunity

T-Cell Exhaustion: Impaired Response after Chronic Antigen Exposure

T-Cell Memory