INTRODUCTION

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The development of the hematopoietic system is regulated by a series of lineage-restricted transcription factors that control critical cell fate decisions, including the formation of primitive and definitive hematopoietic stem cells from embryonic mesoderm and the survival, expansion, lineage commitment, and differentiation of more committed progenitors. Our laboratory (29) as well as those of others (42, 43) has demonstrated that the AML1/core binding factor  (CBF) transcription factor complex, the most common target of chromosomal translocations in human leukemia (reviewed in reference 23), is essential for the formation of the definitive hematopoietic system. Null mutations in either AML1 or CBF, or expression of the dominant inhibitory t(8;21)-encoded leukemia protein, AML1-ETO (27, 47), result in an embryonic lethal phenotype, with embryos dying during the midpoint of development from a complete absence of fetal liver-derived hematopoiesis and lethal central nervous system hemorrhages. Although primitive yolk sac-derived erythropoiesis appears normal in these mutants, no definitive hematopoietic progenitors of any lineage are present. Thus, AML1/CBF appears to function as a critical element that controls the development of hematopoietic cells of the definitive lineages. This function is achieved by binding of AML1/CBF to the core enhancer DNA sequence and regulating the transcription of essential target genes (reviewed in reference 39).

A large number of transcriptional targets of AML1/CBF have been identified, including granulocyte-macrophage colony-stimulating factor (GM-CSF), the receptor for CSF-1, myeloperoxidase, neutrophil elastase, interleukin 3, and the , , , and  subunits of the T-cell antigen receptor (reviewed in reference 39). Although each of these gene targets provide critical functions in hematopoietic cells, experimental data suggest that none are essential for the establishment of definitive hematopoiesis (20-22, 35). Therefore, it is clear that additional AML1-regulated target genes that play critical roles in the signaling pathways required for the establishment of definitive hematopoiesis must exist. In addition, the downstream mechanistic pathways through which the AML1/CBF-mediated transcription cascade functions remain incompletely defined.

To identify components of the downstream pathways initiated by AML1/CBF, we attempted to clone genes whose expression both occurs during the initial development of definitive hematopoietic progenitors and is dependent on the presence of a functional AML1/CBF transcription factor complex. To accomplish this, we performed representational difference analysis (RDA) using mRNA isolated from wild-type and AML1-deficient embryonic stem (ES) cells differentiated in vitro to a point in development at which the earliest commitment to definitive hematopoietic progenitors occurs. We and others have previously demonstrated that this in vitro ES cell differentiation assay accurately replicates the in vivo phenotype that results from the loss of AML1, that is, a complete lack of definitive hematopoiesis (29, 42). Using this approach, we cloned a novel erythroid cell-specific gene designated HERF1 (for hematopoietic RING finger 1), whose expression depends on the presence of functional AML1/CBF. Functional analysis indicates that HERF1 plays an important role in the maturation of definitive erythroid cells.

	MATERIALS AND METHODS

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EB cultures. The procedure for in vitro differentiation of embryoid bodies (EBs) was as previously described (29). In brief, 3 × 10² wild-type or AML1-deficient ES cells were plated in 1 ml of Iscove modified Eagle medium containing 1.2% methylcellulose, 15% fetal calf serum, 2 mM glutamine, 450 µM monothioglycerol, and a mixture of hematopoietic growth factors including human erythropoietin (2 U/ml; Amgen), murine stem cell factor (10 ng/ml; Genzyme), murine interleukin 3 (1.8 ng/ml; R&D Systems), and murine GM-CSF (10 ng/ml; Genzyme). Cultures were grown for 7 days at 37°C under humidified conditions with 5% CO₂. EBs from 30 individual cultures were then pooled, washed in phosphate-buffered saline (PBS), and used for RNA isolation. The morphology of the developing EBs was monitored by microscopic examination of cytocentrifuge preparations of single dispersed EBs.

RDA. Total RNA was extracted from pooled EBs by using a modified acid-guanidinium-thiocyanate-phenol-chloroform extraction method (5), and poly(A)⁺ RNA was isolated from the extracted nucleic acid by using a FastTrack 2.0 kit (Invitrogen) according to the manufacturer's directions. Oligo(dT)-primed double-stranded cDNA was synthesized from 5 µg of poly(A)⁺ RNA by using a cDNA synthesis system (GIBCO BRL) according to the manufacturer's instructions.

Library screening, cloning, Northern blot analysis, and in situ hybridization. A lambda ZAP II mouse spleen cDNA library (Stratagene) was screened with the [-³²P]CTP-labeled 0.49-kb HERF1 fragment isolated by RDA. Positive phage were isolated by plaque purification, and HERF1-containing pBluescript phagemid were purified from positive phage by excision in vivo from the lambda ZAP II vector, using ExAssist helper phage (Stratagene). The DNA sequence of a full-length HERF1 cDNA was obtained by the dideoxy-chain termination method. Throughout the complete coding region, at least two independent clones of the same region were sequenced on both strands. Northern blot analysis and in situ hybridization were performed essentially as described elsewhere (28, 36).

Tetracycline-regulated expression of HERF1 and -sense HERF1. The mouse erythroleukemia cell line MEL (41) was maintained in complete medium (Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and 2 mM glutamine). MEL cells were stably transfected by electroporation with 5 µg of BstXI-linearized pTET.TAK.HYG vector (generous gift of Brian Van Ness, University of Minnesota, Minneapolis) containing a tetracycline-inducible fusion of the tet repressor DNA binding domain and the VP16 activation domain, (Tet-VP16 fusion protein) as well as a constitutively expressed hygromycin-selectable marker. Cells were electroporated at 975 µF and 276 V with a Gene Pulser (Bio-Rad, Richmond, Calif.) in a 0.4 cm cuvette at a density of 2 × 10⁷ cells/ml and a volume of 0.4 ml of medium. Cells were allowed to recover for 24 h before selection in hygromycin (1 mg/ml; Calbiochem) at a density of 5 × 10⁴ cells/ml in the presence of tetracycline (0.5 µg/ml; Sigma) in a 1 ml volume in 24-well plates. Inducible expression of the Tet-VP16 fusion protein was assayed by Western blot analysis of protein extracts from hygromycin-resistant clones cultured in the presence and absence of tetracycline. The polyclonal antibody used for Western blot analysis was raised against the yeast GAL4-VP16 activation domain (Upstate Biotechnology Inc.). Clones expressing low levels of uninduced protein and high levels of induced activity were identified, and one was selected for subsequent stable transfection with AhdI-linearized pTET.HERF1.NEO or pTET.HERF1.NEO, both of which were derived from the pTET.TAK.NEO vector. Conditions for selection of stable integration of these vectors were identical to those described above except that selection was carried out in the presence of G418 (0.8 mg/ml; Life Technologies), hygromycin (1 mg/ml), and tetracycline (0.5 µg/ml). Clones expressing inducible HERF1 and antisense (-sense) HERF1 were identified by Northern and Western blot analyses.

Preparation of HERF1 antisera and Western blot analysis. A unique SalI site was created immediately 5' to the ATG codon in the HERF1 cDNA by PCR, and a 698-bp SalI-BglII restriction fragment corresponding to the N-terminal 230 amino acids was cloned in frame into the pGEX-4T-2 vector (Gibco). The resultant plasmid encodes a glutathione S-transferase (GST)-HERF1 fusion protein that contains a novel alanine residue at the point of fusion. Escherichia coli DH5 was transformed with this plasmid, and the GST-HERF1 fusion protein was isolated by a modification of established methods (38). Briefly, a 2-liter culture of bacteria in log growth phase was induced with 0.1 mM IPTG (isopropyl--D-thiogalactopyranoside) and grown for an additional 20 h at 25°C. The bacterial cell pellets were then collected and resuspended in 80 ml of bacterial protein extraction reagent (Pierce) containing 1 mM phenylmethylsulfonyl fluoride, 2 µg of aprotinin per ml, 10 mM -glycerophosphate, 1 mM NaF, and 5 µg of leupeptin per ml. The bacterial suspension was sonicated and then added to 2 ml of a 50% slurry of glutathione-Sepharose 4B (Pharmacia Biotech), and the mixture was incubated at 4°C for 30 min with constant rocking. The Sepharose beads were then washed three times with bacterial protein extract reagent and three times with cold PBS and finally resuspended in a final volume of 2 ml of PBS. Cleavage of the HERF1 protein from GST was achieved by incubating the beads with 100 U of thrombin (Pharmacia Biotech) at 22°C for 16 h with constant rocking. The purified polypeptide was injected into New Zealand White rabbits to produce anti-HERF1 polyclonal antibodies (Rockland). Western blot analysis was performed as previously described (27).

Nucleotide sequence accession number. The HERF1 DNA sequence has been submitted to the GenBank database (accession no. BankH258518 AF134811).

	RESULTS

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Isolation of HERF1. To clone genes whose expression requires functional AML1/CBF, we performed RDA using an in vitro ES cell differentiation assay that accurately mimics the in vivo definitive hematopoietic defect resulting from the loss of AML1 (17, 29, 42). In this assay, differentiation of either wild-type or AML1-deficient ES cells as EBs for 6 days results in the development of primitive hematopoietic progenitors. By contrast, when EBs are grown for 10 days, wild-type but not AML1-deficient ES cells generate definitive hematopoietic cells. We therefore took advantage of this system to clone genes that were expressed in wild-type but not AML1-deficient cells. To accomplish this, RDA was performed on RNA isolated from EBs grown for 7 days in methylcellulose-containing cultures. At this point in development, primitive erythropoiesis is well established, whereas only the earliest commitment to definitive hematopoiesis has occurred (17).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Structural organization of selected members of the RING (R), B box (B), coiled-coil (C-C) family of proteins. RAR, retinoic acid receptor alpha.

Pattern of HERF1 gene expression. As an initial approach to define the pattern of HERF1 expression, we performed Northern blot analysis using RNA isolated from adult mouse tissues. As shown in Fig. 2A, HERF1 was expressed as a single 2.3-kb transcript in the spleen but was not detected in any of the nonhematopoietic tissues examined (heart, brain, lung, liver, skeletal muscle, and kidney). A closer examination of hematopoietic tissues revealed HERF1 expression in the bone marrow and spleen but not in hematopoietic organs that were composed primarily of lymphoid cells, including lymph node, thymus, and peripheral blood (Fig. 2A). Thus, these data suggest that HERF1 is expressed exclusively in hematopoietic tissues that contain developing myeloid, erythroid, and/or megakaryocytic progenitors.

View larger version (163K): [in this window] [in a new window]   FIG. 2.   Expression pattern of HERF1. Northern blots of adult tissues (A), murine embryos (B), or MEL cells following treatment with the differentiation-inducing agent DMSO (D) were hybridized with a full-length HERF1 cDNA. Filters were stripped and rehybridized with a probe for glycerol-3-phosphate dehydrogenase (GPDH) to assess the integrity and amount of RNA and with a probe for -globin (D; numbers above the lanes represent days in DMSO). (C) In situ hybridization was performed with a 271-bp restriction fragment from the unique 3' region of HERF1 labeled with [33P]UTP. The left side shows the bright-field view of a 12-µm sagittal section from an E15 embryo stained with hematoxylin and eosin; the right side is a dark-field view of the same section hybridized with the HERF1 probe. Sense control shows no specific hybridization (data not shown).

Critical role of HERF1 in erythroid differentiation. To investigate the role of HERF1 in erythroid differentiation, we determined what effect loss of HERF1 would have on DMSO-induced MEL cell differentiation. To perform this experiment, MEL cells were first transfected by electroporation with a plasmid containing a tetracycline-regulated promoter that drives expression of a Tet-VP16 fusion protein (11, 37). In the presence of tetracycline, the Tet-VP16 fusion protein fails to bind to DNA and is thus unable to induce the transcription of either itself or any other cotransfected genes that are under the control of a tetracycline-regulated promoter. By contrast, in the absence of tetracycline, the Tet-VP16 protein binds DNA and strongly transactivates its own expression, resulting in the rapid accumulation of transcriptionally active Tet-VP16. This, in turn, results in the transcription of any other tetracycline-regulated gene (11, 37). Individual MEL cell clones that express the Tet-VP16 chimeric protein after removal of tetracycline were identified by Western blot analysis using antibodies against VP16 and were then assessed for the ability to undergo DMSO-induced differentiation, either in the presence or in the absence of tetracycline (data not shown). Several clones that maintained the ability to differentiate into terminal erythroid cells after treatment with DMSO were isolated and used in the following experiments.

View larger version (110K): [in this window] [in a new window]   FIG. 3.   Inhibition of DMSO-induced MEL cell differentiation by expression of -sense HERF1. MEL cells expressing the tetracycline-regulated Tet-VP16 fusion protein, stably transfected with either an empty vector (parental) or a tetracycline-regulated -sense HERF1-containing plasmid ( sense HERF1), were treated for 3 to 5 days with DMSO in the presence (+) or absence () of tetracycline (tet). Following the indicated treatments, cells were isolated and assessed by Northern blot analysis (A) and morphology (B). Northern blots of total RNA were sequentially hybridized with probes specific for the genes listed on the right side.

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Induction of MEL cell differentiation by enforced HERF1 expression. MEL cells expressing the tetracycline-regulated Tet-VP16 fusion protein, either with an empty plasmid vector (parental) or with a tetracycline-regulated HERF1-containing plasmid (clones 1 to 3), were grown in the presence (+) or absence () of tetracycline (tet). Following the indicated treatments, cells were isolated and analyzed by Northern blot analysis (A) and morphology (B). Northern blots were sequentially hybridized with probes specific for the genes listed on the right side.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Western blot analysis of HERF1, performed with an anti-HERF1 rabbit serum on total cell lysates prepared from Cos cells transfected with an empty vector (HERF1) or a HERF1 expression plasmid (+HERF1) (A) or the B-lineage leukemic cell line MPC11, parental MEL cells treated with the differentiation-inducing agent DMSO, and MEL cells transfected with a tetracycline (tet)-regulated sense or -sense HERF1 cDNA (B).

	DISCUSSION

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We have identified a novel gene encoding a hematopoietic specific RING finger-containing protein, termed HERF1, whose expression is markedly reduced in the absence of AML1. The expression of HERF1 during embryogenesis coincided with the initiation of definitive erythropoiesis and, in leukemic cell lines from adult mice, was restricted to the erythroid lineage, increasing over 30-fold during terminal differentiation. Importantly, we have demonstrated that inhibition of HERF1 expression blocked terminal erythroid differentiation, whereas its overexpression in erythroid cells induced -major globin expression and morphologic maturation. Taken together, these results suggest that HERF1 plays an important role in the development of mature erythroid cells.

The HERF1 protein contains an N-terminal tripartite RBCC domain and a C-terminal rfp region. The RING finger domain, identified in well over 80 different proteins to date, defines a gene family that encodes proteins with widely varying functions (reviewed in references 2 and 34). These proteins include integral components of peroxisomes, transcriptional coregulators, modulators of the signaling capacity of the tumor necrosis factor receptor, members of the polycomb group of homeotic gene repressors, regulators of the cell cycle, proto-oncogenes, and tumor suppressor genes. The RING finger domain is characterized by a cysteine-rich motif of the general structure C₃HC₄ and binds two atoms of zinc, with each zinc atom ligated tetrahedrally by either four cysteines or three cysteines and a histidine. Although similar in structure to other zinc-binding motifs, the RING finger does not appear to mediate DNA binding but alternatively mediates protein-protein interactions that are critical to the function of these proteins.

Although our data do not address the mechanism through which HERF1 mediates its biologic activity, several possibilities are suggested from a comparison to the known activities of a number of closely related family members. The best-characterized members of this subfamily are proteins that contain the tripartite RBCC domain but lack the rfp motif; they include TIF1 (19), TIF1 (10, 18, 26), and PML (7, 16). Each member of this subfamily is a nuclear protein that functions as a critical component of intracellular regulatory pathways, often as an integral element of multisubunit protein complexes. Recent studies suggest that these RBCC domain-containing proteins either directly or indirectly regulate gene transcription. For example, TIF1 functions as a ligand-dependent coactivator for members of the retinoic acid family of transcription factors (19), whereas TIF1 functions as a transcriptional repressor in conjunction with members of the Krüppel family of transcription factors (10, 18, 26). PML functions as a nuclear protein in distinct subnuclear organelles referred to as PML nuclear bodies or PML oncogenic domains (4, 8, 9, 46). Expression of PML is essential for retinoic acid-mediated signaling during normal myeloid cell differentiation (44) and also has a direct growth suppressive function possibly due to its ability to induce apoptosis (13, 32, 45). Thus, the presence of the RBCC domain in HERF1 suggests that this gene product functions as part of a pathway that controls erythroid differentiation.

In addition to the RBCC domain, HERF1 contains a C-terminal 170-amino-acid rfp domain. The presence of this domain may provide further insight into the potential mechanism of action of HERF1. In addition to AFP and RFP, whose functions remain unknown, several other RBCC proteins contain an rfp domain: MID1, which is mutated in Opitz syndrome, an inherited human multiorgan disorder primarily affecting midline structures (31); three Xenopus nuclear proteins (xnf7, XL43, and XL75), which are involved in early development (3, 30); and the amphibian PwA33 protein, which binds to nascent transcripts on lampbrush chromosome loops in oocytes (1). The rfp domain is also found associated with an immunoglobulin domain in butyrophilin, a secreted protein found in milk (13). Although the biologic functions of each of the RBCC and rfp domain-containing proteins remain to be defined, studies on butyrophilin suggest that the rfp domain functions as a protein-binding domain, interacting with regulatory ligands. Thus, the rfp domain in HERF1 may function as a regulatory domain that controls the intrinsic activity of the protein. Taken together, the data from studies of other RBCC and rfp domain-containing proteins suggest that HERF1 functions as an integral component of a multisubunit protein complex that is required for the maturation of erythroid cells. Although we can only speculate on the function of this complex, likely possibilities include a direct role in the regulation of transcriptional signaling pathways and a mechanistic role in the morphologic changes that occur during terminal maturation of erythroid cells such as nuclear condensation and enucleation. Ultimately, determining the mechanistic function of HERF1 will require a direct assessment of the biologic consequences that result from its loss during murine development, as well as the identification of its subcellular location and the proteins with which it interacts.

Although HERF1 was cloned as a potential downstream target of AML1/CBF, our data do not address whether it is a direct transcriptional target or an element far downstream of this transcriptional cascade. Since the loss of AML1 results in a complete absence of definitive hematopoietic cells, HERF1 may have been differentially expressed in this system simply because of the absence of definitive erythroid cells. Supporting this notion are the observations that in differentiating MEL cells the level of AML1 decreases while HERF1 levels increase (11a). However, determining whether AML1 plays an essential role in the basal expression of HERF1 will require the characterization of the HERF1 promoter/enhancer regulatory sequences.

In summary, we have identified a novel RBCC and rfp domain-containing gene, HERF1, which appears to regulate critical steps required for the normal maturation of erythroid cells. Little is presently known about the regulatory pathways involved in the terminal maturation of cells of the erythroid lineage. Elucidation of the biochemical mechanism through which HERF1 functions should provide important insights into this process.