Stage-specific Activation Research Articles

Cell Type- and Developmental Stage-specific Activation of NF-κB by fMet-Leu-Phe in Myeloid Cells

Chemoattractants induce a variety of phagocytic functions including transendothelial migration, degranulation, and the generation of superoxide anions. We report here that the prototypic chemotactic peptide fMet-Leu-Phe (fMLF) stimulates the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor that is central to the regulation of proinflammatory immediate-early gene expression. In freshly prepared peripheral blood mononuclear cells, fMLF (1-100 nM) induced a kappaB binding activity that was receptor-dependent and involved the p50 and p65 subunits of the NF-kappaB/Rel family of proteins. The activation of NF-kappaB by fMLF appeared to be cell-specific and different from the activation of NF-kappaB by tumor necrosis factor-alpha (TNFalpha). Neutrophil preparations that responded to fMLF, TNFalpha, and lipopolysaccharides with interleukin-8 secretion did not show NF-kappaB activation, whereas N-formyl peptide receptor (FPR)-transfected HL-60 cells were responsive to TNFalpha but not fMLF for NF-kappaB activation. Differentiation of FPR-transfected HL-60 cells with dimethyl sulfoxide for 3-5 days conferred the capability of the cells to activate NF-kappaB in response to fMLF without a significant increase in the amount of FPR. These results identify NF-kappaB as a transcription factor that can be activated by the prototypic chemotactic peptide and demonstrate that this function is both highly regulated and dependent on signaling components specifically expressed during myeloid differentiation.

Studies on DNA-protein interactions in the upstream regulatory region of the human ɛ-globin gene promoter

The erythroid- and developmental stage-specific expression of the human ɛ-globin gene is controlled, in part, by the 5′-flanking DNA sequence of this gene. In the present study, we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ɛ-globin gene during development. Using gel mobility shift assays and DNaseI footprinting assays, a nuclear protein factor (termed ɛ- SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified. It could specifically bind to the positive control region (between -535 and -453bp) of the human ɛ-globin gene. We speculated that the ɛ-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ɛ-R1) in the nuclear extract from mouse fetal liver at d 18 of gestation, which could strongly bind to the silencer region (between -392 and -177bp) of this gene. Therefore, we speculated that the ɛ-R1 might be an erythroid- and developmental stage- specific repressor. Our data suggest that both ɛ-SSF1 and ɛ-R1 might play important roles in developmental regulation of the human ɛ-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the human ɛ-globin gene in three different cell lines.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

Tissue- and stage-specific activation of an endogenous provirus after transcription through its integration site in the opposite orientation.

Endogenous proviruses of the Moloney murine leukemia retrovirus (Mo-MuLV) are transcriptionally blocked in early embryos and in general remain silent even when the tissues have become permissive to the expression of newly integrated copies. Eventually, activation in presumably very few cells initiates rapid superinfection leading to viremia and leukemia, but the processes leading to provirus activation are unknown. Differences in the onset and development of viremia between several mouse strains carrying an endogenous Mo-MuLV (Mov lines) are attributed to a chromosomal position effect, but neither cell type nor stage of provirus activation is known for any strain. We have now monitored the appearance of viral transcripts and particles in the Mov13 strain, which carries the Mo-MuLV provirus in inverted orientation in the first intron of a collagen gene (Col1a1) with well-characterized transcriptional activity. We report obligatory tissue- and stage-specific virus activation in osteoblasts and odontoblasts. The significance of this activation pattern is indicated by the fact that of the great variety of cells expressing the wild-type collagen gene, only these two cell types can also transcribe the mutant allele including its viral insert. We propose that this transcription of the proviral genome, albeit in the opposite direction, leads to the activation of the viral promoter.

Changes in Chromatin Structure Support Constitutive and Developmentally Regulated Transcription of the Bone-Specific Osteocalcin Gene in Osteoblastic Cells

Transcription of the osteocalcin gene, which encodes a 10 kDa bone-specific protein, is controlled by modularly organized basal regulatory sequences and hormone-responsive enhancer elements. We have previously shown that in the ROS 17/2.8 rat osteosarcoma cell line, which continuously expresses the osteocalcin gene, key regulatory elements reside in two DNase I hypersensitive sites that are fucntionally correlated with transcriptional activity. We now report that a specific nucleosomal organization supports this constitutive expression in ROS 17/2.8 cells, and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of the osteocalcin gene during differentiation of normal diploid rat osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNAse I hypersensitive sites (-170 to -70 and -600 to -400) and a selective nucleosome positioning over the OC gene promoter are directly associated with developmental stage-specific transcriptional activation in bone-derived cells.

Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated. In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.

The Embryonic Enhancer-Binding Protein SSAP Contains a Novel DNA-Binding Domain Which Has Homology to Several RNA-Binding Proteins

Stage-specific activator protein (SSAP) is a 43-kDa polypeptide that binds to an enhancer element of the sea urchin late histone H1 gene. This enhancer element mediates the transcriptional activation of the late histone H1 gene in a temporally specific manner at the mid-blastula stage of embryogenesis. We have cloned cDNAs encoding SSAP by using polyclonal antibodies raised against purified SSAP to screen expression libraries. SSAP is unrelated to previously characterized transcription factors; however, it exhibits striking homology to a large family of proteins involved in RNA processing. The protein is a sequence-specific DNA-binding protein that recognizes both single- and double-stranded DNA. The DNA-binding domain of the protein was localized to the conserved RNA recognition motif (RRM). In addition to tandem copies of this conserved domain, SSAP contains a central domain that is rich in glutamine and glycine and a C-terminal domain that is enriched in serine, threonine, and basic amino acids. Overexpression of SSAP in sea urchin embryos by microinjection of either synthetic mRNA or an SSAP expression vector results in four- to eightfold transactivation of target reporter genes that contain the enhancer sequence. Transactivation occurs beginning only at the mid-blastula stage of development, suggesting that SSAP must be modified in a stage-specific manner in order to activate transcription. In addition, there are a number of other RRM-containing proteins that contain glutamine-rich regions which are postulated to function in the regulation of RNA processing. Instead, we suggest that SSAP is a member of a family of glutamine-rich RRM proteins which constitute a novel class of transcription factors.

Distinct negative regulatory mechanisms involved in the repression of human embryonic epsilon- and fetal G gamma-globin genes in transgenic mice

A current model for human beta-globin gene switching proposes that the stage-specific activation of embryonic and fetal globin genes requires the interaction of the beta-globin locus control region with proximal promoter elements. Subsequent repression in fetal and adult stages likely involves negative regulatory promoter elements and factors. To begin addressing these negative regulatory mechanisms, the regulation of human fetal G gamma-globin promoter fused to the SV40 T antigen gene was analyzed in transgenic mice. The results showed correct developmental expression in erythroid tissue, but lower levels of expression were also detected in non-erythroid tissue. Thus, the 5'-flanking G gamma-globin promoter sequence contains stage-specific erythroid elements but is lacking nonerythroid-specific negative elements. In contrast, the human embryonic epsilon-globin gene was only expressed in nonerythroid tissue of transgenic embryos, suggesting the presence of an erythroid-specific negative element(s). With the locus control region, complete repression of epsilon-globin RNA in fetal liver was observed in epsilon-globin genes without the previously characterized silencer, suggesting the presence of additional negative elements. Overall, this transgenic study suggests that distinct negative regulatory mechanisms function in the repression of embryonic and fetal globin genes.

Stage-specific expression of activation antigens on NK cells at uterine implantation sites in mice.

The differentiation of the cellular components of the uterine decidua, in particular the life history of NK cells, is poorly understood. With the use of two mAbs that recognize stage-specific activation Ags on NK cells, we investigated the development of NK cells known as granulated metrial gland (GMG) cells. Immunohistochemical analysis demonstrated that mAb 3C2, but not mAb 4H12, recognized numerous cells throughout the uterine decidua basalis during early gestation. Isolated (panned) 3C2+ decidual cells from day 7 of pregnancy co-expressed the NK1.1 Ag, displayed NK cytolytic activity, and proliferated in IL-2-containing media. A small percentage of those cells expressed the GMG-associated Ag 4H12. Immunohistochemical analysis of serial sections at midgestation demonstrated that most of the 3C2+ NK cells co-expressed 4H12 Ag. During the later part of pregnancy, however, 3C2 expression in the decidua was down-regulated, and the cells expressed high levels of 4H12 Ag. When 3C2+ NK cells were isolated from cell suspensions of decidua from 7-day pregnant mice, and cultured in IL-2-containing medium, the cells developed the large and granular morphology characteristics of GMG cells, and acquired 4H12 Ag. These results demonstrate that 4H12+ GMG cells differentiate from 3C2+, NK1.1+, cytolytic precursors that reside in the decidua during early gestation.

A novel developmental regulatory motif required for stage-specific activation of the epsilon-globin gene and nuclear factor binding in embryonic erythroid cells.

Members of the human beta-globin gene family are expressed at discrete stages of development and therefore provide an important model system for examining mechanisms of temporal gene regulation. We have previously shown that expression of the embryonic beta-like globin gene (epsilon) is mediated by a complex array of positive and negative upstream control elements. Correct developmental stage- and tissue-specific gene expression is conferred by synergistic interactions between a positive regulatory element (termed epsilon-PRE II) which is active only in embryonic erythroid cells and at least two other regulatory domains upstream of the epsilon-globin gene promoter. A nuclear factor highly enriched in cultured embryonic erythroid cells and in mouse embryonic yolk sac binds to a novel, evolutionarily conserved sequence within epsilon-PRE II. We show here that binding of this factor to the conserved element within epsilon-PRE II is critical for transcriptional activity. Point mutations that interfere with protein binding to epsilon-PRE II abolish transcriptional activation of the constitutive epsilon-globin promoter. Adult erythroid nuclei (from cultured cells or adult mouse liver) also contain a factor that binds to this region, but the complex formed migrates more rapidly during nondenaturing electrophoresis, suggesting either that distinct proteins bind to epsilon-PRE II or that a single protein is differentially modified in these cells in a way that modulates its activity. Several lines of evidence suggest that the binding factors in embryonic and adult erythroid cells are distinguished by posttranscriptional differences.

A Novel Developmental Regulatory Motif Required for Stage-Specific Activation of the ɛ-Globin Gene and Nuclear Factor Binding in Embryonic Erythroid Cells

Members of the human beta-globin gene family are expressed at discrete stages of development and therefore provide an important model system for examining mechanisms of temporal gene regulation. We have previously shown that expression of the embryonic beta-like globin gene (epsilon) is mediated by a complex array of positive and negative upstream control elements. Correct developmental stage- and tissue-specific gene expression is conferred by synergistic interactions between a positive regulatory element (termed epsilon-PRE II) which is active only in embryonic erythroid cells and at least two other regulatory domains upstream of the epsilon-globin gene promoter. A nuclear factor highly enriched in cultured embryonic erythroid cells and in mouse embryonic yolk sac binds to a novel, evolutionarily conserved sequence within epsilon-PRE II. We show here that binding of this factor to the conserved element within epsilon-PRE II is critical for transcriptional activity. Point mutations that interfere with protein binding to epsilon-PRE II abolish transcriptional activation of the constitutive epsilon-globin promoter. Adult erythroid nuclei (from cultured cells or adult mouse liver) also contain a factor that binds to this region, but the complex formed migrates more rapidly during nondenaturing electrophoresis, suggesting either that distinct proteins bind to epsilon-PRE II or that a single protein is differentially modified in these cells in a way that modulates its activity. Several lines of evidence suggest that the binding factors in embryonic and adult erythroid cells are distinguished by posttranscriptional differences.

A new human cell line for the molecular dissection of the requirement for interleukin-1 in the production of interleukin-2 by immature T cells

An immature human T cell line, PER-117, can be induced to secrete interleukin-2 (IL-2). In contrast to mature T cells or the Jurkat cell line, PER-117 cells require interleukin-1 (IL-1) for optimal IL-2 secretion, in addition to calcium ionophore and phorbol 12-myristate 12-acetate (PMA). These requirements mirror the conditions reported to be optimal for normal immature T cell receptor (TCR) negative thymocytes. IL-1 did not substitute for either of the other signals required for IL-2 production, i.e., calcium ionophore or PMA, suggesting that IL-1 activates pathways different from those elicited by the two other stimuli. For optimal effect, IL-1 needed to be provided at the same time as the two other signals. Significantly, a signal provided by a low concentration of PMA (not leading to IL-2 induction by itself in the presence of calcium ionophore) was necessary. The studies reported here provide the first evidence that three signals are required for optimal IL-2 production in a human immature T cell line. PER-117 cells produce substantial levels of IL-2 and thus provide a model to study stage-specific signal transduction and transcriptional activation of the IL-2 gene in IL-1 responsive immature thymocytes.

Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

A retinoic acid receptor expressed in the early development of Xenopus laevis.

We have isolated cDNAs coding for a putative retinoic acid receptor (RAR) of the gamma-type from a Xenopus laevis neurula cDNA library. By transient cotransfection of COS cells with an expression vector and a reporter plasmid, this cDNA is shown to direct the synthesis of a retinoic acid-dependent transcription factor. In embryos of X. laevis, transcription of the corresponding gene is greatly enhanced during gastrulation and early neurulation. Two distinct areas with high abundance of RAR gamma mRNA are located at the anterior and at the posterior end of the neurula. The two maxima have emerged by the end of gastrulation and they become more pronounced during neurulation. At tailbud and early tadpole stages, the RAR transcripts are found mainly in the head mesenchyme and in the tailbud. The expression of this RAR is region-specific but not germ-layer-specific. The strong and stage-specific activation of zygotic transcription of this RAR gene, and the specific localization of the mRNA are consistent with the temporal and spatial pattern of retinoic acid sensitivity of X. laevis embryos. Therefore it is likely that the gene product mediates the effects of endogenous and of exogenous retinoic acid on early embryogenesis of Xenopus. The significance of these findings for the specification of the anteroposterior axis is discussed.

The Regulation of γ‐Globin Gene Expression

In summary, our analysis indicates that important sequences for the proper initiation of fetal gene transcription in fetal cells are located in the gamma-globin [sequence: see text] promoter. These sequences are sufficient for tissue-specific expression but not induction in K562 cells. Sequences in the gamma-globin IVS-2 and the beta-globin 3' enhancer increase gamma beta and gamma-Neo transcripts when cells containing these genes undergo erythroid maturation as measured by induction with hemin. The mechanism by which these sequences exert their effect remains to be elucidated. [see text] Multiple protein factors bind to both the gamma promoter and the beta 3' enhancer. Both of these regions contain binding sites for the erythroid-specific factor NFE-1 and the octamer binding factor OTF-1. In the gamma upstream region, there may be a competition between OTF-1 binding and NFE-1 binding that affects gamma gene regulation. Our results indicate that the beta 3' enhancer interacts with the gamma gene promoter to permit increased gamma gene expression. We have developed a model for globin gene switching that takes into consideration the effect of cis-acting sequences on globin gene transcription. A similar model of hemoglobin switching in chickens has been proposed by Choi and Engel. In our model, competition for the beta-globin 3' enhancer is involved in stage-specific transcriptional activation of gamma-globin genes in fetal cells and beta-globin genes in adult cells. In adult cells the protein-protein interactions between adult cell-specific factors interacting with the beta-globin promoter and erythroid-specific factors interacting with the beta 3' enhancer would activate transcription of the beta-globin gene. In fetal cells protein-protein interactions between fetal cell-specific factors interacting with the gamma-globin promoter and erythroid-specific factors interacting with the beta 3' enhancer would activate the transcription of the gamma-globin genes.

Distinct, developmental stage-specific activation mechanisms of trypanosome VSG genes

The metacyclic form of African trypanosomes is the first to express genes for the Variant Surface Glycoprotein (VSG) and it uses an unusually predictable subset of the VSG gene repertoire. We have developed a model system for the analysis of metacyclic VSG (M-VSG) gene expression and have used this to demonstrate that, for two M-VSG genes, different modes of expression operate in the insect and mammalian phases of the life-cycle. In metacyclic-derived clones, these genes are expressed in situ, whereas they are routinely activated by duplication in bloodstream trypanosomes. The expression loci for both M-VSG genes studied are structurally simple and we present a model, based on this, for the maintenance of a separate M-VSG repertoire and expression system.

The beta-globin stage selector element factor is erythroid-specific promoter/enhancer binding protein NF-E4.

The analysis of transcriptional regulatory proteins is often hampered because such factors are present in cells in only sparing abundance. Although direct biochemical purification has been successfully applied to the analysis of many of these factors, such methods are labor intensive and expensive. We have developed an alternative strategy to identify and characterize such trans-acting factors and have used it to analyze the proteins that interact with the chicken adult beta-globin gene enhancer and promoter. The methodology involves (1) a sensitive 'reverse' radioimmunoassay used for the identification of antibodies to sequence-specific DNA-binding proteins, and (2) a monoclonal antibody-based DNase I footprint selection technique, which unambiguously identifies proteins responsible for particular footprints. Because this methodology relies on the isolation of antibodies to sequence-specific DNA-binding proteins, it should be of general utility in studying any trans-acting regulatory factor for which a specific DNA-binding sequence can be identified. In the present analysis, we report the identification of a 65-kD protein that is present only in mature definitive (adult) chicken erythroid cells. We show that this protein (termed NF-E4) binds to closely related sequences present in both the beta-globin promoter and enhancer. Biochemical analysis of extracts prepared from both nonerythroid and a variety of erythroid cell types suggests that NF-E4 is the trans-acting factor that confers definitive erythrocyte stage-specific transcriptional activation to the adult beta-globin gene.

Developmental appearance of transcription factors that regulate liver-specific expression of the aldolase B gene.

The region from -202 to -1 of the rat aldolase B (AldB) promoter directs tissue-specific transcription in vitro. Deletion of the half of this promoter distal to its origin reduced its ability for tissue-specific expression. Two protein-binding sites in this distal element have been identified and characterized. One bound a factor named AIF-A, which is probably identical to previously characterized liver-specific factors HNF-1 and APF. The other, containing a CCAAT motif, bound a factor named AIF-B. These two factors were considerably enriched in liver cells compared with other cells not expressing AldB. In the liver, these two factors increased prior to the activation of the AldB gene during development. Liver-specific in vitro transcription assays indicated that the binding of both factors to their target sequences was required for AldB transcription. These results suggest that AIF-A and AIF-B are positively acting transcription factors that regulate tissue-specific and development stage-specific activation of the AldB gene.

Developmental appearance of transcription factors that regulate liver-specific expression of the aldolase B gene.

The region from -202 to -1 of the rat aldolase B (AldB) promoter directs tissue-specific transcription in vitro. Deletion of the half of this promoter distal to its origin reduced its ability for tissue-specific expression. Two protein-binding sites in this distal element have been identified and characterized. One bound a factor named AIF-A, which is probably identical to previously characterized liver-specific factors HNF-1 and APF. The other, containing a CCAAT motif, bound a factor named AIF-B. These two factors were considerably enriched in liver cells compared with other cells not expressing AldB. In the liver, these two factors increased prior to the activation of the AldB gene during development. Liver-specific in vitro transcription assays indicated that the binding of both factors to their target sequences was required for AldB transcription. These results suggest that AIF-A and AIF-B are positively acting transcription factors that regulate tissue-specific and development stage-specific activation of the AldB gene.