Altered Regulation of β like Globin Genes by a Redesigned Erythroid Transcription Factor.

Abstract The well characterized switch during ontogeny of globin gene expression from embryonic/ fetal to adult type is a result of a complex interplay between cis and trans acting regulatory elements at the beta globin locus. Trans acting elements include tissue specific transcription factors that bind specific motifs within the beta globin gene cluster with high specificity. Erythroid Kruppel like factor (EKLF) is one such erythroid specific, zinc finger transcription factor that is critical for the activation of the beta globin promoter and for consolidating the switch from gamma to beta globin during development. The ability to willfully regulate the expression of endogenous genes using redesigned zinc finger transcription factors is an emerging field. There is tremendous appeal in utilizing the understanding of transcriptional control pathways to design tools that will elucidate molecular mechanisms and provide potential therapeutic tools. To this end we redesigned Erythroid Kruppel Like Factor (EKLF) as a transcriptional repressor. The zinc finger DNA binding domain was linked to the repressor domain from the Drosophila Engrailed protein with the prediction that this construct (ENG/ZNF) would bind the beta globin promoter and repress it. It was hypothesized that embryonic/fetal globin activation would result by a competitive mechanism. When introduced transiently into cells these transcription factors are effective in repressing the adult beta globin promoter CACCC element, the natural target for EKLF. In stable MEL clones, repression of the adult beta globin gene is accompanied by a reactivation of the endogenous embryonic globin gene. In order to study this effect in the context of a whole animal we generated transgenic mice expressing ENG/ZNF. A 271 bp region 5′of the ANK-1 gene was chosen to drive expression in transgenic mice as it provides erythroid specific expression with copy number dependence and minimal position dependence. D13.5 fetal livers were subject to RT-PCR analysis in the linear range to quantitate the ratios of BH1 to alpha globin transcripts. The 9 ENG/ZNF transgenic embryos express BH1 mRNa in a range of values that is statistically higher than in 9 control littermates (Mann Whitney U test, p value 0.02) and beta major globin mRNA at lower levels. We further studied ENG/ZNF in the developmentally plastic environment of differentiating murine embryonic stem cells. The construct was stably integrated into a targeting site upstream of the HPRT locus under the control of a tetracycline inducible promoter. The Doxycycline induction of ENG/ZNF transgene expression results in a 4 fold activation of embryonic globin at day 6 of embryoid body development; however there is no evidence of beta globin repression. Since at this stage of embryoid body development, primitive erythroid cells are 100–500 fold more abundant than definitive erythroid cells, this may reflect a differential effect of EKLF in primitive erythroid cells. To evaluate this further, we are currently performing analyses in primitive versus definitive erythroid colonies. In conclusion, our studies support the competitive model of globin switching and may contribute to the delineation of a stage specific role of EKLF. In addition, transcriptional reagents that augment gamma globin expression hold promise as novel therapeutic agents for sickle cell disease and other hemoglobinopathies.

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ERV-9 LTR Retrotransposon Modulates Globin Gene Switching

Blood ◽

10.1182/blood-2018-99-117280 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2341-2341

Author(s):

Dorothy Tuan

Keyword(s):

Gene Locus ◽

Globin Gene ◽

Human Endogenous Retrovirus ◽

Ltr Retrotransposon ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene ◽

Gamma Globin ◽

Globin Promoter

Abstract In the human beta-globin gene locus, an LTR retrotransposon derived from ERV-9 human endogenous retrovirus is located near the locus control region (LCR) far upstream of the globin genes. In transgenic mice carrying the 100 kb human globin gene locus, deleting the ERV-9 LTR by cre-loxP mediated in situ recombination inactivates transcription of beta-globin gene by ~50% and activates that of gamma-globin gene by up to 5 fold, to at least 20% the level of beta-globin mRNA in both fetal and adult erythroid cells. Chromosome-conformation-capture (3C) shows that the ERV-9 LTR preferentially loops with beta-globin gene, even in fetal erythroid cells where gamma-globin gene is predominantly transcribed. Unique in the locus, the ERV-9 LTR contains high density of CCAAT motifs that strongly bind transcription factor (TF) NF-Y critical for assembling the LTR enhancer complex but no CCNCNCCC motifs that bind master erythroid TF KLF1, while the beta-globin promoter contains two tandem CCNCNCCC motifs that bind KLF1 but a CCAAT motif that only weakly bind NF-Y. However, both the LTR and beta-globin promoter associate in vivo with high levels of NF-Y and KLF1 in ChIP assays. Protein immunoprecipitation shows that neither NF-Y nor KLF1 associates with transcription co-regulator Ldb1, which has been reported to mediate chromosome looping of the LCR with the globin genes. Thus, the Ldb1/LMO2/GATA -1, -2 complex potentially assembled by the GATA motifs distributed throughout the locus does not appear to mediate the preferential looping of ERV-9 LTR with beta-globin promoter. Through interaction of NF-Y and KLF1 mediated by the to be identified co-regulator, the ERV-9 LTR interacts with and activates beta-globin gene, and suppresses gamma-globin gene by a competitive mechanism. Deletion of the ERV-9 LTR removes the competitive advantage of beta-globin gene, thus re-activating gamma-globin gene. Our findings suggest that deleting the ERV-9 LTR from the globin locus by genome-editing could provide a potential new therapy for beta-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

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5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4690 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4690-4697 ◽

Cited By ~ 74

Author(s):

J G Glauber ◽

N J Wandersee ◽

J A Little ◽

G D Ginder

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Adult Chicken ◽

Beta Globin Gene ◽

Flanking Sequences ◽

Murine Erythroleukemia ◽

Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

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Erythroleukemia (K562) cells contain a functional beta-globin gene

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2553-2555.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2553-2555

Author(s):

M Donovan-Peluso ◽

K Young ◽

C Dobkin ◽

A Bank

Keyword(s):

Hela Cells ◽

Globin Gene ◽

K562 Cells ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene ◽

Rna Transcripts

K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult beta-globin. A cloned beta-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal beta-globin gene.

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Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.7.11.4024-4029.1987 ◽

1987 ◽

Vol 7 (11) ◽

pp. 4024-4029

Author(s):

M Trudel ◽

J Magram ◽

L Bruckner ◽

F Costantini

Keyword(s):

Transgenic Mice ◽

Fusion Gene ◽

Globin Gene ◽

Regulatory Elements ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Base Pairs ◽

Beta Globin Gene ◽

Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

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A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽

10.1182/blood.v81.5.1384.1384 ◽

1993 ◽

Vol 81 (5) ◽

pp. 1384-1392 ◽

Cited By ~ 64

Author(s):

I Plavec ◽

T Papayannopoulou ◽

C Maury ◽

F Meyer

Keyword(s):

Bone Marrow Cells ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Beta Globin Gene ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia ◽

In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

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Human Beta-Globin Expression during Erythroid Cells Development in Transgenic Mice Bearing Human Beta-Globin Gene with Mutated BP1 Binding Site.

Blood ◽

10.1182/blood.v106.11.3651.3651 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3651-3651

Author(s):

Olga P. Zoueva ◽

David Bodine ◽

Griffin P. Rodgers

Keyword(s):

Transgenic Mice ◽

Binding Site ◽

Fetal Liver ◽

Globin Gene ◽

Fold Increase ◽

Mouse Embryos ◽

Mrna Levels ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene

Abstract Binding of beta protein 1 (BP1) to its site on the promoter of adult beta-globin gene has silencing effect on beta-globin transcription in vitro. To better understand the mechanism of the negative regulation of beta-globin expression by BP1 we have developed transgenic mice. Specifically, we introduced a mutated BP1 binding site into the promoter of beta-globin gene sequence of 35 kb cosmid construct. This construct containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into the single cell mouse embryos. To detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we studied the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5. In addition, we analyzed adult erythroid cells. To minimize experimental error, samples from individual animals of three transgenic lines were analyzed independently using real-time PCR assays. Levels of expression of murine alpha-globin mRNA were used as internal controls. The BP1 gene and its mouse analog Dlx4 belongs to the Distal-less family of homeobox genes, which are expressed during early development. We found that the mRNA levels of human beta-globin in transgenic mice containing mutated BP1 binding site were higher at all stages of erythroid cells development as compared with control transgenic mice bearing cosmid construct with wild type sequence of BP1 site. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our data indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice. These results may help develop the novel clinic approaches for the inhibition of the expression of abnormal beta-globin genes, such as sickle (hbs) and hbc.

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Erythroleukemia (K562) cells contain a functional beta-globin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2553 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2553-2555 ◽

Cited By ~ 10

Author(s):

M Donovan-Peluso ◽

K Young ◽

C Dobkin ◽

A Bank

Keyword(s):

Hela Cells ◽

Globin Gene ◽

K562 Cells ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene ◽

Rna Transcripts

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Improved Lentiviral Vectors for the Cure of Hemoglobinopathies

Blood ◽

10.1182/blood-2018-99-111319 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3477-3477

Author(s):

Laura Breda ◽

Valentina Ghiaccio ◽

Hanyia Zaidi ◽

Silvia Pires Lourenco ◽

Carla Casu ◽

...

Keyword(s):

Clinical Trials ◽

Gene Transfer ◽

Copy Number ◽

Research Funding ◽

Globin Gene ◽

Lentiviral Vectors ◽

Beta Globin ◽

Beta Globin Gene ◽

Vector Copy Number ◽

Globin Promoter

Abstract Given that both Sickle Cell Disease (SCD) and beta-thalassemia (BT) are caused by mutations in the beta-globin gene, several lentivirus-based gene addition therapies have been developed. Results from recent trials indicate that the vectors used are safe; however, their efficacy inversely correlates with the severity of patients' hemoglobinopathy. The severity of the mutations (non-beta0 vs beta0) largely influences the outcome of the gene transfer. In fact, the data indicate that a relatively low number of integrations (in the range of 1-2 copies per genome) or vector copy number (VCN) is sufficient to cure patients whose mutations are categorized as non-beta0 and express relative high levels of endogenous hemoglobins (adult hemoglobin, HbA, and/or fetal hemoglobin, HbF). In contrast, the same level of VCN alleviates the transfusion regimen of patients with beta0 mutations, but it does not cure them. In addition, the lentiviruses currently used in clinical trials were engineered by different groups and to date no one has directly compared them side by side. In light of these limitations, here we describe a study that supplies a platform for rapid screening of lentiviral vectors expressing curative hemoglobin, based on the correlation between VCN and the increase in HbA levels. We also compared newly generated lentiviral vectors to vectors currently used in clinical trials. Our ultimate goal is to generate a new vector that can increase the yield of beta globin expressed per VCN in patients' cells. Using CRISPR-Cas9 we modified the erythroid Hudep-2 cell line (Kurita et al, 2013) to generate a clonal cell line, named Hudep #M13, which, upon differentiation, produces a hemoglobin variant (HbMut) that can be discriminated from that produced by the lentiviruses (HbA). In parallel, we immortalized erythroid progenitor cells isolated from a SCD donor (SCD #13), using the HPV16-E6/E7 expression system, which was introduced into the cells by lentiviral transduction. Using Hudep #M13, we compared the correlation between gene transfer and the production of HbA for 5 novel lentiviral vectors, indicated as ALS16-20. Our new vectors include the Ankyrin insulator in the 3' LTR (Breda et al 2012), the full beta-globin gene (including the native introns), the full 3' enhancer region, a combination of different portions of the beta-globin promoter, as well as modifications and inclusion of novel genomic elements from the locus control region (LCR). Our ALS- constructs were then compared to lentiviral vectors currently utilized in clinical trials. These constructs were reproduced based on information available from the literature (Negre et al, 2015; Miccio et al, 2008; and Boulad et al, 2014) and indicated as CV-1, CV-2, and CV-3, respectively. All these vectors contain the beta-globin gene with deletions in intron 2, different portions of the beta-globin promoter and/or 3' enhancer region, and different elements and sizes of the hypersensitive sites (HS) of the LCR. In Hudep #M13, linear regression analysis of the ratio of HbA to vector copy number (VCN) for each treatment, indicates that ALS17 and ALS20 yield roughly 40, 157 and 84% more HbA per copy than CV-1, CV-2 and CV-3, respectively. Similar increment in HbA% were confirmed on primary and immortalized (SCD #13) SCD erythroblasts derived CD34+ cells isolated from patients' blood. In these specimens, ALS20 maintained a 40% HbA increase compared to CV-1, when exploring a range of VCN from 0 to 3 with a linear mixed effects model. To assess the ability of these constructs to increase hemoglobin content in vivo, we are performing murine bone marrow transplants using thalassemic hematopoietic stem cells treated with CV1 and our two most powerful vectors. Based on most recently reported data (Thompson et al, 2018), 1 copy of the vector we reproduced as CV-1, makes on average 6.8g/dL of HbA. Hence, 1 copy of our best vector has the potential to make up to 9.5g/dL HbA. This could lead to a much greater clinical impact for patient with hemoglobinopathies, especially those who require higher Hb production to become transfusion independent, like patients with the beta0 genotype. The completion of these studies will provide not only a comparative analysis of our new best vector to those already in clinical trial, but also a way to predict how much therapeutic hemoglobin per vector copy number will be produced in the clinical setting. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Kwiatkowski:bluebird bio: Consultancy, Honoraria, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; Novartis: Research Funding; Apopharma: Research Funding; Terumo: Research Funding. Rivella:Disc Medicine: Consultancy; Protagonist: Consultancy; Ionis: Consultancy; Meira GTX: Membership on an entity's Board of Directors or advisory committees.

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A Novel C to A Substitution in the CCAAT Box of beta Globin Gene.

Blood ◽

10.1182/blood.v108.11.3810.3810 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3810-3810

Author(s):

Chiara Refaldi ◽

Elena Di Pierro ◽

Maria C. Mocellini ◽

Maria D. Cappellini

Keyword(s):

Transcription Factor ◽

Globin Gene ◽

Tata Box ◽

Sequencing Analysis ◽

Beta Globin ◽

Specific Transcription Factor ◽

Beta Globin Gene ◽

Factor C ◽

Ccaat Box

Abstract The promoter of the human beta-globin gene contains three positive cis-acting elements required for maximal transcription: the CACCC box located between −86 and −90, the CCAAT box located between −72 and − 76 and the TATA box located between −28 and −31 relative to the start site of transcription. Naturally occurring mutations within the TATA and the CACCC box regions have been recorded in patients with beta+ thalassemia. Mutations within the TATA box disrupt assembly of the basal transcription complex, while mutations at the CACCC box prevent binding of an erythroid-specific transcription factor EKLF. Surprisingly, no mutations have so far been identified in the highly conserved element CCAAT box and the transcription factors responsible for the regulatory activity of the CCAAT site in vivo have been less intensively studied. We report a novel mutation −76 C>A (HBB c. −126) detected by sequencing analysis of beta globin gene in a Italian beta+ thalassemic patient. The transversion C>A hits the first nucleotide in the CCAAT box of the beta globin gene. The carrier, a male 44 years old, shows a mild hypochromic and microcytic anaemia with reduced mean corpuscular volume and mean corpuscular haemoglobin (MCV 75 fl, MCH 25 pg) and Hb A2 level slightly increased (3.9%). Recently, studies in vitro in gel-shift and reporter assays, investigating the transcriptional activity of human beta globin CCAAT box, have identified five factors: NF-Y (CP1) a ubiquitous CCAAT box binding complex, GATA-1 an erythroid-specific transcription factor, C/EBPbeta, C/EBPgamma and C/EBPdelta members of CCAAT/enhancer-binding protein family involved in hemapoietic regulation. This represents the first report of a natural mutation of the human beta-globin CCAAT box and confirms its functional significance for in vivo transcription.

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Activation of codependent transcription factors is required for transcriptional induction of the vgf gene by nerve growth factor and Ras.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.4621 ◽

1996 ◽

Vol 16 (9) ◽

pp. 4621-4631 ◽

Cited By ~ 23

Author(s):

G D'Arcangelo ◽

R Habas ◽

S Wang ◽

S Halegoua ◽

S R Salton

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Nerve Growth Factor ◽

Growth Factor ◽

Pc12 Cells ◽

Mutational Analysis ◽

Globin Gene ◽

Beta Globin ◽

Nerve Growth ◽

Transcriptional Induction

Nerve growth factor (NGF) treatment of PC12 cells leads to the elaboration of a neuronal phenotype, including the induction of neuronally expressed genes such as vgf. To study vgf transcription, we have created chimeric vgf/beta-globin genes in which vgf promoter sequences drive the expression of the beta-globin reporter gene or of a chimeric beta-globin gene fused to 3' untranslated vgf gene sequences. We have found that the level of inducibility of the latter construct by NGF resembles that of the endogenous vgf gene. Using transient transfection of the chimeric reporter genes into PC12 cells, into PC12 subclones expressing activated or dominantly interfering mutant Ras proteins, and into PC12 variants expressing specific NGF receptor/Trk mutants, we show that transcriptional regulation of the vgf promoter by NGF is mediated through a Ras-dependent signaling pathway. By mutational analysis of the vgf promoter, we have identified three promoter elements involved in mediating transcriptional induction by NGF and Ras. In addition to the cyclic AMP-responsive element (CRE), which binds to ATF-1, ATF-2, and CRE-binding protein in PC12 nuclear extracts, a novel CCAAT element and its binding proteins were identified, which, like the CRE, is necessary but not sufficient for the Ras-dependent induction of the vgf gene by NGF. We also identify a G(S)G element unusually located between the TATA box and transcriptional start site, which binds the NGF- and Ras-induced transcription factor, NGFI-A, and amplifies the transcriptional response. Integrating data from studies of vgf promoter regulation and NGF signal transduction, we present a model for vgf gene induction in which transcriptional activation is achieved through the persistent, direct activation of multiple interacting transcription factors binding to CRE and CCAAT elements, coordinated with the delayed transcription factor action at a G(S)G element resulting from the induced expression of NGFI-A.

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