Myeloid-specific Gene Expression Research Articles

Natural Products with Antitumor Potential Targeting the MYB-C/EBPβ-p300 Transcription Module.

The transcription factor MYB is expressed predominantly in hematopoietic progenitor cells, where it plays an essential role in the development of most lineages of the hematopoietic system. In the myeloid lineage, MYB is known to cooperate with members of the CCAAT box/enhancer binding protein (C/EBP) family of transcription factors. MYB and C/EBPs interact with the co-activator p300 or its paralog CREB-binding protein (CBP), to form a transcriptional module involved in myeloid-specific gene expression. Recent work has demonstrated that MYB is involved in the development of human leukemia, especially in acute T-cell leukemia (T-ALL) and acute myeloid leukemia (AML). Chemical entities that inhibit the transcriptional activity of the MYB-C/EBPβ-p300 transcription module may therefore be of use as potential anti-tumour drugs. In searching for small molecule inhibitors, studies from our group over the last 10 years have identified natural products belonging to different structural classes, including various sesquiterpene lactones, a steroid lactone, quinone methide triterpenes and naphthoquinones that interfere with the activity of this transcriptional module in different ways. This review gives a comprehensive overview on the various classes of inhibitors and the inhibitory mechanisms by which they affect the MYB-C/EBPβ-p300 transcriptional module as a potential anti-tumor target. We also focus on the current knowledge on structure-activity relationships underlying these biological effects and on the potential of these compounds for further development.

MTG16 Variants Provide Context to Notch Signaling to Direct Leukemic Phenotype

Notch signaling guides hematopoietic stem and progenitor cells (HS/PCs) toward distinct cell fates and when dysfunctional, contributes to development of hematopoietic malignancies. Normally, Notch signaling favors T-cell fate and causes attenuated expression of myeloid-specific genes. In contrast, Notch pathway inactivation causes HS/PCs to diverge disproportionately toward a myeloid fate and is accompanied by T-lymphopenia. Mutations that constitutively activate Notch signaling trigger T-cell leukemia, while Notch inactivation is associated with myeloid leukemias. This suggests that Notch-regulated cell fate decisions are context dependent and that determinants downstream of Notch can influence Notch-dependent phenotypes in development and disease.Myeloid Translocation Gene (MTG) 16 binds the intracellular domain of Notch (NICD) and this interaction is necessary to establish CD4+/CD8+ (double positive-DP) T-cell fate. Mtg16 -/- progenitors give rise to GR1+/Mac1+ progeny in response to Notch activation. In ex vivo assays, normal DP T-cell development can be restored to Mtg16 -/- progenitors by enforced expression of MTG16, but not an MTG16 derivative which lacks NICD binding and mimics a naturally occurring Mtg16 splice variant (MTG16c). These findings suggest the status of expressed Mtg16 isoforms can determine lineage allocation in response to Notch signaling and intimates that Notch signaling could trigger alternate phenotypes depending upon the context in which it is activated.To assess impact of the NICD-MTG16 interaction as a determinant of malignant phenotype in the context of constitutive Notch signaling, we created bicistronic retroviral constructs whose NICD and MTG16 expression cassettes were separated by the “self-cleaving” 2A peptide of Foot and Mouth disease virus (FMDV). In each transduced progenitor cell, this insures concurrent stoichiometric expression of NICD and the chosen MTG16 derivative (wild type or NICD binding-deficient). Transduced Mtg16 -/- HS/PCs were then transplanted into irradiated, syngeneic recipients and leukemic phenotype established by flow cytometry. Mice transplanted with Mtg16 -/- cells transduced with vector only fail to reconstitute hematopoiesis in the recipient bone marrow due to a previously described HS/PC defect. Instead, these mice succumb to marrow failure by day 18 post-transplant. However, enforced expression of NICD alone enables survival of Mtg16 -/- progenitors, reconstitution of hematopoiesis and development of T-cell leukemia from transplanted donor elements. This same phenotype was observed when NICD was co-expressed with MTG16, indicating MTG16 is required for normal T-lymphopoiesis but is dispensible for NICD-driven T-cell leukemia. Notably, when Mtg16 -/- progenitors are transduced with NICD and a NICD binding deficient derivative of MTG16, recipients develop T-myeloid bilineal leukemia. This finding suggests that integration of NICD into transcriptional regulatory complexes involving MTG16 can alter the features of leukemic blasts along a continuum from lymphoid to myeloid phenotypes. To gain additional insights, we characterized promoter occupancy and Notch transcription complex (NTC) composition upon expression of MTG16 and its NICD binding deficient derivative. We find both MTG16 forms can exist in the same transcriptional complexes via their NHR2 oligomerization domains and can co-occupy the Notch-responsive Hes1 promoter. Given that NICD-MTG16 binding disrupts interactions between MTG16, histone deacetylases (HDACs) and other co-repressors, these data suggest that leukemic phenotype arising from constitutive Notch signaling may be influenced by combinatorial diversity among MTG16 splice variants and differential integration of the NICD into the NTC. DisclosuresNo relevant conflicts of interest to declare.

Transcriptome landscape of myeloid cells in human skin reveals diversity, rare populations and putative DC progenitors

BackgroundThe heterogeneous functions of dermal myeloid cells in antigen presentation, and scavenging pathogens and cell debris places them centrally in cutaneous inflammation. Single cell transcriptomics can provide new understanding of the heterogeneity and function of yet incompletely understood human dermal myeloid cell subsets. ObjectiveInvestigate the transcriptome landscape of myeloid cells in healthy human skin. MethodsSingle cell RNA-sequencing was performed on skin biopsies from ten healthy donors and analyzed to identify myeloid cell populations. ResultsOne LIN− HLA-DR+ cluster with expression of myeloid-specific genes was identified as a cluster of myeloid cells. Upon reanalysis of this cluster, we identified three macrophage subsets, marked by high expression of CCR1, MARCO or TREM2; and six dendritic cell subsets, marked by high expression of CLEC9A, CXorf21, MCOLN2, LAMP3, KIAA0101 and Langerin, representing respectively cDC1, two subsets of cDC2, a novel DC type, a cluster of proliferating DC, and a Langerhans cell subset. GO term analysis indicated specialized functions for the discrete rare populations of myeloid cells: TREM2 Mφ in lipid metabolism and LAMP3 DC as a mature cDC. Proliferating DCs appeared to represent cDC2 progenitors. ConclusionThe transcriptional landscape of myeloid cell populations in human skin indicates several, novel populations with specialized functions, as well as a rare proliferating DC population that likely accounts for local regeneration or expansion of dermal DCs. We provide robust gene expression markers for each of these populations that should permit better understandings of their roles in various homeostatic and pathologic immune processes in the skin.

Splitomicin, a SIRT1 Inhibitor, Enhances Hematopoietic Differentiation of Mouse Embryonic Stem Cells.

Background and ObjectivesEmbryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells.Methods and ResultsTreatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells.ConclusionsApplication of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.

The natural anti-tumor compound Celastrol targets a Myb-C/EBPβ-p300 transcriptional module implicated in myeloid gene expression.

Myb is a key regulator of hematopoietic progenitor cell proliferation and differentiation and has emerged as a potential target for the treatment of acute leukemia. Using a myeloid cell line with a stably integrated Myb-inducible reporter gene as a screening tool we have previously identified Celastrol, a natural compound with anti-tumor activity, as a potent Myb inhibitor that disrupts the interaction of Myb with the co-activator p300. We showed that Celastrol inhibits the proliferation of acute myeloid leukemia (AML) cells and prolongs the survival of mice in an in vivo model of AML, demonstrating that targeting Myb with a small-molecule inhibitor is feasible and might have potential as a therapeutic approach against AML. Recently we became aware that the reporter system used for Myb inhibitor screening also responds to inhibition of C/EBPβ, a transcription factor known to cooperate with Myb in myeloid cells. By re-investigating the inhibitory potential of Celastrol we have found that Celastrol also strongly inhibits the activity of C/EBPβ by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBPβ by disrupting the interaction of both transcription factors with p300. Myb, C/EBPβ and p300 cooperate in myeloid-specific gene expression and, as shown recently, are associated with so-called super-enhancers in AML cells that have been implicated in the maintenance of the leukemia. We hypothesize that the ability of Celastrol to disrupt the activity of a transcriptional Myb-C/EBPβ-p300 module might explain its promising anti-leukemic activity.

Mirn23a Inhibits B Lymphopoiesis through Antagonism of the B Cell Transcription Factor Network

Transcription factors (TFs) regulate genetic networks to direct the differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature blood lineages. Historically studies have focused on identifying genes activated by TFs that act as master hematopoietic regulators. Recently, it has become appreciated that these regulatory TFs direct one distinct differentiation program while simultaneously directing the repression of genes associated with alternative blood lineages. MicroRNAs (miRNAs) are short (~22nt) non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level and are hypothesized to act downstream of lineage specific TFs to contribute to the silencing of genes associated with alternative cell fate adoption. MiRNAs are observed to direct cell fate choice in assays utilizing overexpression and knockdown approaches; however, experiments from mouse gene targeting have yet to confirm these results. Specifically no miRNA has been shown to direct the differentiation of multipotent progenitors (MPPs) to granulocyte monocyte progenitors (GMPs) or common lymphoid progenitors (CLPs). We previously observed that overexpression of the mirn23a gene (Codes for three mature miRNAs: miR-23a, miR-24-2, and miR-27a) in hematopoietic progenitors increased myelopoiesis at the expense of B lymphopoiesis both in vitro and in vivo. To follow up this observation we generated a mirn23a germline knockout mouse. Flow cytometry characterization of hematopoiesis in mirn23a-/- mice reveals a significant increase in B220+ B-lymphocytes in both the bone marrow (BM) and the spleen. Furthermore, this increase in B-lymphocytes was shown to be at the expense of myeloid cells, as CD11b+ cells were significantly decreased in both the BM and spleen. Analysis of the BM progenitor populations revealed a significant increase in the CLP population, due to an increase in the B-cell specific lymphoid progenitor (BLP, CLP+ Ly6D+) CLP subset. Consistent with decreased mature myeloid cells, we also observed a decrease in BM GMPs. The phenotype appears to be cell intrinsic as ex vivo culture of mirn23a-/- progenitor cells on OP9 stroma results in a significant increase in B220+ B-cells, and a decrease in CD11b+ myeloid cells compared to wild type cultures. Consistent with mirn23a promoting myeloid development, we observe that expression of mirn23a in 70z/3 pre-B cells increases myeloid specific gene expression programs. The upregulation of the myeloid genes may be due to the insufficient repression by B cell transcription factors Ebf1 and Pax5, which are downregulated in mirn23a expressing cells. Interestingly Ebf1 associates with the mirn23a promoter as shown by chromatin immunoprecipitation (ChIP) assay, and Ebf1 represses mirn23a promoter activity in luciferase reporter assays. Consistent with a role in repressing mirn23a, Ebf1 knockdown in A20 B Lymphoma cells increases miR-23a expression while overexpression of Ebf1 in NIH/3t3 cells decreases miR-23a expression. Previously we observed that the myeloid transcription factor PU.1 binds to the mirn23a promoter to positively regulate transcription. Additionally we observe that myeloid transcription factor C/EBP alpha associates with the mirn23a locus in ChIP assays. Data from our knockout mouse supports a critical role for mirn23a in determining immune cells fates. Our current results support the conclusion that myeloid transcription factors PU.1 and C/EBP alpha activate mirn23a in order to antagonize B cell gene regulation and promote myeloid cell fate adoption, whereas the essential B cell transcription factor Ebf1 represses mirn23a expression to avoid this repression during B cell specification. DisclosuresNo relevant conflicts of interest to declare.

The zinc finger transcription factor ZXDC activates CCL2 gene expression by opposing BCL6-mediated repression

The zinc finger X-linked duplicated (ZXD) family of transcription factors has been implicated in regulating transcription of major histocompatibility complex class II genes in antigen presenting cells; roles beyond this function are not yet known. The expression of one gene in this family, ZXD family zinc finger C (ZXDC), is enriched in myeloid lineages and therefore we hypothesized that ZXDC may regulate myeloid-specific gene expression. Here we demonstrate that ZXDC regulates genes involved in myeloid cell differentiation and inflammation. Overexpression of the larger isoform of ZXDC, ZXDC1, activates expression of monocyte-specific markers of differentiation and synergizes with phorbol 12-myristate 13-acetate (which causes differentiation) in the human leukemic monoblast cell line U937. To identify additional gene targets of ZXDC1, we performed gene expression profiling which revealed multiple inflammatory gene clusters regulated by ZXDC1. Using a combination of approaches we show that ZXDC1 activates transcription of a gene within one of the regulated clusters, chemokine (C-C motif) ligand 2 (CCL2; monocyte chemoattractant protein 1; MCP1) via a previously defined distal regulatory element. Further, ZXDC1-dependent up-regulation of the gene involves eviction of the transcriptional repressor B-cell CLL/lymphoma 6 (BCL6), a factor known to be important in resolving inflammatory responses, from this region of the promoter. Collectively, our data show that ZXDC1 is a regulator in the process of myeloid function and that ZXDC1 is responsible for Ccl2 gene de-repression by BCL6.

NUP98-HOXA9 Reprograms Embryonic Hematopoiesis, Suppresses Cellular Apoptosis, and Causes Malignant Tissue Infiltrates in Transgenic Zebrafish.

Abstract 3961Poster Board III-897The NUP98-HOXA9 fusion oncogene results from a t(7;11)(p15;p15) chromosomal translocation and is associated with inferior prognosis in de novo and treatment-related acute myeloid leukemia (AML), as well as blast crisis in chronic myeloid leukemia (CML). HOXA9 belongs to the highly-conserved HOX gene family of developmental transcription factors and is critical for vertebrate hematopoiesis. Upregulation of HOXA9 is a frequent phenomenon in human AML, implicating a central role in myeloid disease. Thus, elucidating the activity of oncogenic NUP98-HOXA9 may reveal universal mechanisms of leukemogenesis and lead to design of targeted therapies. The zebrafish is a robust model for studying vertebrate hematopoiesis and leukemogenesis, by virtue of its ex utero development, and conserved genetics and cell biology. We have engineered a Cre-lox inducible transgenic zebrafish harboring the human NUP98-HOXA9 fusion oncogene downstream of the zebrafish pu.1 promoter. NUP98-HOXA9 perturbed zebrafish embryonic hematopoiesis, leading to upregulated expression of myeloid-specific genes, pu.1, lysC, and l-plastin, and downregulation of the erythroid-specific gene, gata1. These changes in gene transcription affect both ‘primitive' and ‘definitive' waves of zebrafish hematopoiesis (including the recently-described erythro-myeloid progenitors [EMPs]), suggesting that NUP98-HOXA9 reprograms blood cell precursors to a predominantly myeloid fate. Preferential upregulation of pu.1 during ‘primitive' hematopoiesis further suggests an impaired potential for terminal myeloid differentiation. These effects appear developmentally mediated, as cell proliferation was not increased as measured by phosphorylated histone-H3 (pH3) staining. However, following ionizing irradiation (IR), NUP98-HOXA9 expression preserved cell proliferation and suppressed apoptosis in the presence of DNA damage. Irradiated transgenic embryos displayed wild-type levels of pH3, suggesting inhibition of cell cycle checkpoints. Irradiated embryos also displayed reduced levels of the conserved apoptotic marker, activated caspase-3, as well as downregulation of the puma pro-apoptotic gene, and upregulation of bcl2 and bcl-xL anti-apoptotic genes. These data postulate suppression of caspase-3-dependent apoptosis as a mechanism for NUP98-HOXA9 -mediated oncogenesis in vivo. Incomplete phenotypic ‘rescue' with bcl-xL morpholino suggests insufficiency of single-target therapy, with a need for multi-target interruptions to restore radiosensitivity. Between 20-24 months post fertilization, a proportion of these NUP98-HOXA9 transgenic zebrafish (n=9/19; 47%) presented with gross abdominal masses and/or abnormal swimming behavior. Thus far, histological sectioning has demonstrated enlarged kidneys and livers, with evidence of infiltrative, pleiomorphic, mitotically-active cells with myeloid morphology. This pathology is reminiscent of the myeloproliferative disease (MPD) observed in NUP98-HOXA9 transgenic mice. Taken together, these data provide insights into the mechanisms underlying NUP98-HOXA9-mediated leukemogenesis and provide an unprecedented opportunity for in vivo chemical modifier screens to identify promising therapeutic agents that restore a normal phenotype. Disclosures:No relevant conflicts of interest to declare.

Stem cells: the challenge and opportunities

Recent studies have clearly identified that hematopoietic stem cells (HSC) are part of a hierarchical organization defined by cells able to initiate long-term repopulation (LT-HSC) of injured BM followed by populations with transient repopulation ability. As HSCs are able to supply blood cells for the lifespan of the individual, LT-HSCs undergo an aging process. The consequences of aging are an increase in the stem cell pool size, increased self-renewal and production of cells with expression of myeloid-specific genes and genes commonly expressed in myeloid leukemia. The age-related increased incidence of myeloproliferative disorders is therefore not surprising. Marrow cells circulate and migrate to a number of different organs. Previously assumed tissue and organ specificities appear to be less stringent and primitive cells residing in one organ may contribute to the structural and functional regeneration of other organs. It is intriguing to speculate that cells and cytokines used in appropriate concentrations may be able to repair damaged organs by using the defective organ as a scaffold, thus reducing the need for the transplantation of solid organs.

The Fes tyrosine kinase: a signal transducer that regulates myeloid-specific gene expression through transcriptional activation

The c- fps/ fes protooncogene encodes a 92-kDa protein tyrosine kinase that is involved in myeloid cell development and immune responses of granulocytes and macrophages. To help define its biological role and mechanism of action, we have developed a gain of function allele of Fes that has potent biological activity in myeloid cells. Introduction of constitutively active Fes into myeloid progenitors induced the appearance of fully differentiated macrophages or granulocytes depending on the lineage commitment of the transduced cells. We found that Fes-induced macrophage differentiation correlated with activation of the ets family transcription factor PU.1, which is essential for macrophage development. On the other hand, granulocyte differentiation by Fes was mediated through activation of CCAAT/enhancer-binding protein alpha (C/EBP-α) and STAT3, two transcription factors that are critical for granulocytic differentiation. We postulate that Fes transduces inductive signals for terminal macrophage and granulocyte differentiation, and that this biological activity is mediated through the activation of lineage-specific transcription factors.

Chromatin immunoprecipitation (ChIP) studies indicate a role for CCAAT enhancer binding proteins alpha and epsilon (C/EBPα and C/EBPε) and CDP/cut in myeloid maturation-induced lactoferrin gene expression

In vitro models of granulopoiesis involving the inducible expression of either CCAAT enhancer binding protein alpha (C/EBP alpha) or C/EBP epsilon in myeloid cells have been shown to lead to the induction of a granulocytic maturation program accompanied by the expression of myeloid-specific genes. Since members of the C/EBP family of transcription factors recognize and bind to similar DNA-binding motifs, it has been difficult to elucidate the specific role of each of the C/EBP family members in eliciting myeloid gene expression. In order to address this issue, we focused on the expression of the lactoferrin (LF) gene. LF expression is transcriptionally regulated in a C/EBP-dependent manner in myeloid cells. Using chromatin immunoprecipitation (ChIP) analysis we demonstrate that C/EBP alpha binds to the LF promoter in nonexpressing cells. Upon induction of maturation, C/EBP epsilon binds to the LF promoter, which correlates with LF expression. Lack of LF expression in the acute promyelocytic leukemia cell line NB4, which harbors the t(15;17) translocation, cannot be correlated with aberrant binding at the C/EBP site in the LF promoter. It is, however, associated with the persistent binding of the silencer CCAAT displacement protein (CDP/cut) to the LF promoter in these cells. We conclude that C/EBP alpha, C/EBP epsilon, and CDP/cut all play definitive roles in regulating late gene expression during normal myeloid development.

Expression of myeloid-specific genes in childhood acute lymphoblastic leukemia - a cDNA array study.

Several specific cytogenetic changes are known to be associated with childhood acute lymphoblastic leukemia (ALL), and many of them are important prognostic factors for the disease. Little is known, however, about the changes in gene expression in ALL. Recently, the development of cDNA array technology has enabled the study of expression of hundreds to thousands of genes in a single experiment. We used the cDNA array method to study the gene expression profiles of 17 children with precursor-B ALL. Normal B cells from adenoids were used as reference material. We discuss the 25 genes that were most over-expressed compared to the reference. These included four genes that are normally expressed only in the myeloid lineages of the hematopoietic cells: RNASE2, GCSFR, PRTN3 and CLC. We also detected over-expression of S100A12, expressed in nerve cells but also in myeloid cells. In addition to the myeloid-specific genes, other over-expressed genes included AML1, LCP2 and FGF6. In conclusion, our study revealed novel information about gene expression in childhood ALL. The data obtained may contribute to further studies of the pathogenesis and prognosis of childhood ALL.

Crystals of ternary protein-DNA complexes composed of DNA-binding domains of c-Myb or v-Myb, C/EBPalpha or C/EBPbeta and tom-1A promoter fragment.

c-Myb and the C/EBP family are transcriptional regulatory factors that act in concert to regulate the expression of myeloid-specific genes. v-Myb encoded by avian myeloblastosis virus (AMV) is a mutated form of c-Myb that contains point mutations which disrupt the cooperation with C/EBPs. To understand the mechanism of the transcriptional synergy between c-Myb and C/EBPs and the effect of the v-Myb mutations on that synergy, knowledge based on their three-dimensional structures is essential. Crystals of ternary complexes, in which various combinations of the DNA-binding domains of c-Myb or v-Myb and C/EBPalpha or C/EBPbeta are bound to a DNA fragment from tom-1A promoter, were obtained by the vapour-diffusion method. Complete diffraction data sets were obtained from each native crystal and two types of iodine-derivative crystals. A three-wavelength MAD data set was also obtained from a bromine-derivative crystal.

Identification and characterization of the promoter for the gene encoding the human myeloid IgA Fc receptor (FcalphaR, CD89).

The Fc receptor for IgA (FcalphaR, CD89), a transmembrane glycoprotein, is expressed exclusively on human phagocytic cells including monocytes, macrophages, eosinophils, and neutrophils, and is capable of triggering various effector functions. In the present study, we identified and characterized, for the first time, the FcalphaR promoter. A 929-bp fragment of FcalphaR 5'-flanking sequence directed expression of a reporter gene specifically in monocytic cell line U937. Deletion analyses localized element(s) directing tissue-specific reporter gene expression to the 259 bp proximal to the translation initiation site. Within the region, the sequence between 59 and 197 bp downstream of the major transcription start site was shown to be essential for promoter activity. This sequence contains multiple potential binding sites for transcription factors which have been reported to function in myeloid-specific gene expression, including three CCAAT enhancer-binding protein (C/EBP)-binding sites, an NF-kappaB-binding site, an Spl site, an Ets family protein consensus-binding site, and a Myb-binding site. In addition, we identified two polymorphisms (C-->T transition) at the positions 114 bp upstream and 56 bp downstream of the major transcription start site, and demonstrated that the FcalphaR promoter region carrying both the -114T and +56T alleles had significantly lower promoter activity than that harboring the C alleles at both sites. Characterization of this promoter will facilitate further analyses of activation stimuli and transcription factors involved in FcalphaR-mediated immune system, and provide new insights into the mechanism(s) underlying altered FcalphaR expression associated with diseases such as allergic diseaes and IgA nephropathy.

Current issues in the management of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is caused by one of four genetic lesions that disrupt the alpha receptor for retinoic acid, RAR alpha. The fusion protein responsible for greater than 99% of APL cases, PML-RAR alpha, inhibits PML-dependent apoptotic pathways in a dominant negative fashion and blocks myeloid differentiation by direct transcriptional inhibition of retinoic acid target genes. This transcriptional inhibition is mediated by recruitment of co-repressor proteins and resultant deacetylation of histones in the promoter regions of genes (yet to be identified) that control promyelocyte development. In the presence of high levels of all-trans retinoic acid (ATRA), both PML-dependent apoptotic mechanisms and myeloid-specific gene expression programs are reactivated. In the clinic, the combination of anthracycline-based chemotherapy plus ATRA cures approximately 80% of APL patients, and a high percentage of relapsed patients can achieve second remissions with arsenic trioxide. With the publication of results from the European APL 93 trial, the 'standard-of-care' for induction treatment of APL now includes ATRA plus concurrent anthracycline-based chemotherapy. The amount and type of consolidation therapy necessary for an individual APL patient remains somewhat of an open question, but at present should include at least two cycles of chemotherapy. Based on recent trials that demonstrate a benefit of maintenance ATRA (+/- low-dose chemotherapy), all APL patients should probably receive some type of maintenance therapy. While the above approach currently cures the majority of APL patients, future improvements in the treatment of this disease will require risk-adapted protocols that incorporate real-time molecular monitoring and appropriate introduction of novel therapeutic agents.

Genomic Organization and Chromosomal Localization of the Human CD163 (M130) Gene: A Member of the Scavenger Receptor Cysteine-Rich Superfamily

The human protein CD163 (M130) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, which is exclusively expressed by monocytes and macrophages. Here, we investigated the genomic organization and the chromosomal localization of the human CD163 gene. The CD163 gene is composed of 17 exons and 16 introns and spans over 35 kb. Each of its nine SRCR domains is encoded by a separate exon, which is similar to other members of the group B SRCR subfamily. Two cytoplasmic variants of CD163 arise from alternative splicing of intron 15, while a truncated and an extracellular variant results from alternative splicing of intron 5 or intron 7, respectively. Using fluorescence in situ hybridization we mapped this gene to the human chromosome 12p13. The transcription initiation sites of the CD163 gene were determined and the 5′-flanking region was sequenced. The nucleotide analysis revealed several putative binding sites for transcription factors, which have been shown to play an important role in myeloid specific gene expression. In addition, we identified a L1 element located 1.4 kb upstream of the major transcription initiation site.

Transcription factors that regulate monocyte/macrophage differentiation.

Although all the cells in an organism contain the same genetic information, differences in the cell phenotype arise from the expression of lineage-specific genes. During myelopoiesis, external differentiating signals regulate the expression of a set of transcription factors. The combined action of these transcription factors subsequently determines the expression of myeloid-specific genes and the generation of monocytes and macrophages. In particular, the transcription factor PU.1 has a critical role in this process. We review the contribution of several transcription factors to the control of macrophage development.

Myeloid-specific gene expression.

The mononuclear phagocytes are recruited from bone marrow precursors to most tissues of the body, particularly during inflammation or immune stimulation. This combination of accessibility as stem cells and heterogenity of tissue locations makes the myeloid cell potentially important as a carrier of therapeutic agents. Understanding the regulation of transcription in myeloid cells is necessary for any future design of tissue-specific gene targeting vectors, particularly if there are inherent size limitations. Identified members of the C/EBP, Runt/PEBP2/CBF, and Ets families of transcription factors are critical for myeloid-specific gene expression and may have myeloid-restricted expression or myeloid-specific regulation in the hematopoietic system. AP-1, Sp1, and Myb appear to be important for myeloid-restricted expression in some cases. In addition, factors involved in the up-regulation of the level of gene expression when macrophages are activated by agents such as interferon-gamma and bacterial products have been identified. Some of the sequences to which these transcription factors bind in myeloid-restricted genes have been tested in cell lines and in transgenic mice and it is now possible to make an attempt to describe the characteristics of a myeloid-specific promoter.

Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression.

The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelomonocytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in bone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to -16) that did not include the cluster of c-Myb sites. A 4-bp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22) including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4-bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super-shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.

PU.1 (Spi-1) and C/EBPα Regulate the Granulocyte Colony-Stimulating Factor Receptor Promoter in Myeloid Cells

Cytokines, important for lineage commitment and differentiation during hematopoiesis, exert their influence by binding specific receptors. Receptor expression is tightly regulated and examining the factors that govern their expression will allow better understanding of the events that determine lineage commitment. The granulocyte colony-stimulating factor (G-CSF) receptor is expressed exclusively in myeloid cells and the placenta. We show here that the G-CSF receptor transcription start site is identical in each of these tissues. A 1,391-bp fragment of the G-CSF receptor promoter is both active in myeloid cell lines and tissue specific. We have also found two regions that are important for G-CSF receptor promoter activity. One region, located at bp -49, contains a GCAAT site that specifically binds the C/EBP alpha transcription factor in myeloid nuclear extracts. Mutation of this site prevents C/EBP alpha binding and reduces promoter activity by 60%. The other functionally important region of the G-CSF receptor promoter is in the 5' untranslated region, at bp +36 and +43, where there are two sites for the ets family member PU.1. Mutation of these sites prevents PU.1 binding and reduces promoter activity by 75%. These results reinforce the importance of both PU.1 and C/EBP alpha in the expression of myeloid-specific genes and neutrophil development.