Similar DNA-binding Specificity Research Articles

PclR is a transcriptional activator of the gene that encodes the pneumococcal collagen-like protein PclA

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that shows high levels of genetic variability. The pneumococcal R6 genome harbours several gene clusters that are not present in all strains of the species. One of these clusters contains two divergent genes, pclA, which encodes a putative surface-exposed protein that contains large regions of collagen-like repeats, and spr1404 (here named pclR). PclA was shown to mediate pneumococcal adherence to host cells in vitro. In this work, we demonstrate that PclR (494 amino acids) is a transcriptional activator. It stimulates transcription of the pclA gene by binding to a specific DNA site upstream of the core promoter. In addition, we show that PclR has common features with the MgaSpn transcriptional regulator (493 amino acids), which is also encoded by the R6 genome. These proteins have high sequence similarity (60.3%), share the same organization of predicted functional domains, and generate multimeric complexes on linear double-stranded DNAs. However, on the PpclA promoter region, MgaSpn binds to a site different from the one recognized by PclR. Our results indicate that PclR and MgaSpn have similar DNA-binding properties but different DNA-binding specificities, pointing to a different regulatory role of both proteins.

Opposing Roles for the Related ETS-Family Transcription Factors Spi-B and Spi-C in Regulating B Cell Differentiation and Function.

Generation of specific antibodies during an immune response to infection or vaccination depends on the ability to rapidly and accurately select clones of antibody-secreting B lymphocytes for expansion. Antigen-specific B cell clones undergo the cell fate decision to differentiate into antibody-secreting plasma cells, memory B cells, or germinal center B cells. The E26-transformation-specific (ETS) transcription factors Spi-B and Spi-C are important regulators of B cell development and function. Spi-B is expressed throughout B cell development and is downregulated upon plasma cell differentiation. Spi-C is highly related to Spi-B and has similar DNA-binding specificity. Heterozygosity for Spic rescues B cell development and B cell proliferation defects observed in Spi-B knockout mice. In this study, we show that heterozygosity for Spic rescued defective IgG1 secondary antibody responses in Spib–/– mice. Plasma cell differentiation was accelerated in Spib–/– B cells. Gene expression, ChIP-seq, and reporter gene analysis showed that Spi-B and Spi-C differentially regulated Bach2, encoding a key regulator of plasma cell and memory B cell differentiation. These results suggest that Spi-B and Spi-C oppose the function of one another to regulate B cell differentiation and function.

In vivo Hox binding specificity revealed by systematic changes to a single cis regulatory module

Hox proteins belong to a family of transcription factors with similar DNA binding specificities that control animal differentiation along the antero-posterior body axis. Hox proteins are expressed in partially overlapping regions where each one is responsible for the formation of particular organs and structures through the regulation of specific direct downstream targets. Thus, explaining how each Hox protein can selectively control its direct targets from those of another Hox protein is fundamental to understand animal development. Here we analyse a cis regulatory module directly regulated by seven different Drosophila Hox proteins and uncover how different Hox class proteins differentially control its expression. We find that regulation by one or another Hox protein depends on the combination of three modes: Hox-cofactor dependent DNA-binding specificity; Hox-monomer binding sites; and interaction with positive and negative Hox-collaborator proteins. Additionally, we find that similar regulation can be achieved by Amphioxus orthologs, suggesting these three mechanisms are conserved from insects to chordates.

Enforcement of developmental lineage specificity by transcription factor Oct1.

Embryonic stem cells co-express Oct4 and Oct1, a related protein with similar DNA-binding specificity. To study the role of Oct1 in ESC pluripotency and transcriptional control, we constructed germline and inducible-conditional Oct1-deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal and growth but manifest defects upon differentiation. They fail to form beating cardiomyocytes, generate neurons poorly, form small, poorly differentiated teratomas, and cannot generate chimeric mice. Upon RA-mediated differentiation, Oct1-deficient cells induce lineage-appropriate developmentally poised genes poorly while lineage-inappropriate genes, including extra-embryonic genes, are aberrantly expressed. In ESCs, Oct1 co-occupies a specific set of targets with Oct4, but does not occupy differentially expressed developmental targets. Instead, Oct1 occupies these targets as cells differentiate and Oct4 declines. These results identify a dynamic interplay between Oct1 and Oct4, in particular during the critical window immediately after loss of pluripotency when cells make the earliest developmental fate decisions.

A Pyrrole-Imidazole Polyamide Is Active against Enzalutamide-Resistant Prostate Cancer

The LREX' prostate cancer model is resistant to the antiandrogen enzalutamide via activation of an alternative nuclear hormone receptor, glucocorticoid receptor (GR), which has similar DNA-binding specificity to the androgen receptor (AR). Small molecules that target DNA to interfere with protein-DNA interactions may retain activity against enzalutamide-resistant prostate cancers where ligand-binding domain antagonists are ineffective. We reported previously that a pyrrole-imidazole (Py-Im) polyamide designed to bind the consensus androgen response element half-site has antitumor activity against hormone-sensitive prostate cancer. In enzalutamide-resistant LREX' cells, Py-Im polyamide interfered with both AR- and GR-driven gene expression, whereas enzalutamide interfered with only that of AR. Genomic analyses indicated immediate interference with the AR transcriptional pathway. Long-term treatment with Py-Im polyamide demonstrated a global decrease in RNA levels consistent with inhibition of transcription. The polyamide was active against two enzalutamide-resistant xenografts with minimal toxicity. Overall, our results identify Py-Im polyamide as a promising therapeutic strategy in enzalutamide-resistant prostate cancer. Cancer Res; 77(9); 2207-12. ©2017 AACR.

Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1 (Fujioka and Jaynes, 2012). Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. We localized cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function.

Cofactor Binding Evokes Latent Differences in DNA Binding Specificity between Hox Proteins

Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation

AbstractIn megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

Linking Yeast Transcription Factor Structural Class and Detailed Binding Preferences with in vivo Regulatory Functions

Cellular responses to internal and external stimuli through changes in gene expression are in part controlled by the binding of regulatory transcription factors (TFs) to specific sequences of DNA. These TFs belong to a wide variety of DNA binding domain (DBD) structural classes. Sequence specificity is diverse between structural classes, but TFs within each class often have apparently redundant sequence preferences. To understand how cells use these regulators to coordinate networks of responses, it is essential to determine how regulatory function is shared and partitioned between factors of similar and different structures and DNA binding specificities. Are certain structural classes better suited for certain functions? Are detailed differences in binding preferences among apparently similar TFs relevant to in vivo function? Using genome-wide datasets, we have described trends in biological function and regulatory mechanisms within TF structural classes in the yeast Saccharomyces cerevisiae. These trends suggest general ways in which TF function may be distributed across structural classes according to the biophysical constraints dictated by each DBD structure. Such analyses do not show, however, how specific details of an individual TF's binding specificity might affect its biological function. New data from protein binding microarrays (PBMs) provide such detailed TF binding preference information at all possible 8 base-pair DNA sequences. By combining these PBM data with in vivo binding locations measured by chromatin immunoprecipitation (ChIP-chip) experiments, we can infer the functional importance of specific types of TF binding sites (ie low and high affinity sites). This and other analyses made possible by the high resolution PBM data, when combined with observed functional trends in structural classes, will demonstrate how the cell utilizes both general and specific biophysical TF properties to accomplish cellular functions.

A general mechanism for transcription regulation by Oct1 and Oct4 in response to genotoxic and oxidative stress

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure.

Overlapping and distinct transcriptional regulator properties of the GLI1 and GLI2 oncogenes

The GLI transcription factors mediate the hedgehog signal in development and carcinogenesis. Basal cell carcinoma can be caused by overexpression of either GLI1 or GLI2. Though GLI1 and GLI2 have identical or very similar DNA binding specificities, some of their activities are overlapping, some are clearly distinct. We analyzed target gene specificities of GLI1 and constitutively active GLI2 (GLI2ΔN) by global expression profiling in an inducible, well-characterized HaCaT keratinocyte expression system. Four hundred fifty-six genes up- or downregulated at least twofold were identified. GLI target gene profiles correlated well with the biological activities of these transcription factors in hair follicles and basal cell carcinoma. Upregulation of largely overlapping sets of target genes was effected by both factors, repression occurred predominantly in response to GLI2. Also, significant quantitative differences in response to GLI1 and GLI2ΔN were found for a small number of activated genes. Since we have not detected a putative processed GLI2 repressor, these results point to specific but indirect target gene repression by GLI2ΔN via preferential activation of one or more negative regulators.

TaNAC69 from the NAC superfamily of transcription factors is up-regulated by abiotic stresses in wheat and recognises two consensus DNA-binding sequences.

NAC proteins are one of the largest families of plant transcription factors and have recently been implicated in diverse physiological processes. To elucidate their role in gene regulation, we determined the DNA-binding specificity of a drought- and cold-inducible NAC protein, TaNAC69 from wheat, and analysed its homologues from other species. Two consensus DNA-binding sequences (spanning 23-24 bp) of TaNAC69 were identified through binding site selection and both consisted of two half sites. Comprehensive data on the DNA-binding specificity of TaNAC69 were generated through extensive base substitution mutagenesis. TaNAC69 and its homologue in Arabidopsis, NAP, sharing 75% sequence identity in the NAC domain, exhibited similar DNA-binding specificity. TaNAC69 was able to homodimerise through its NAC domain. The NAC domain consists of five conserved subdomains. Subdomain mutation showed that a loss or reduction in TaNAC69 dimerisation capacity was accompanied with abolition or decrease in its DNA-binding activity. These data suggest that all subdomains are necessary to maintain a functional NAC domain structure required for interaction with DNA and dimerisation.

Biochemical characterization of the mammalian Cux2 protein

The CCAAT displacement protein (CDP) and cux (Cut homeobox) genes were originally identified as the human and mouse orthologs of Drosophila melanogaster cut. More recently, vertebrates were found to possess a second cut orthologs that was generated by gene duplication: Cux2. We report the initial biochemical characterization of the Cux2 protein in tissue culture and in vitro. We generated four polyclonal antibodies that were able to recognize the human and mouse Cux2 protein but displayed little or no cross-reactivity towards CDP1 and Cux1. The expression of the Cux2 protein was convincingly detected in only one among 19 neuronal cell lines: the SH-SY5Y human neuroblastoma cell line. CDP/Cux proteins contain four DNA binding domains, three Cut repeat (CR1, CR2 and CR3) and one Cut homeodomain (HD). Purified fusion proteins containing either CR1CR2, CR2CR3HD or CR3HD exhibited similar DNA binding specificities as the corresponding domains of Cux1, but their DNA binding kinetics were much more rapid. Similarly, the full-length Cux2 protein made rapid but transient interactions with DNA. We did not observe an N-terminally processed Cux2 isoform equivalent to the Cux1 p110 isoform. Whereas Cux1 can function as a repressor or activator in a promoter-specific manner, Cux2 functioned exclusively as a transcriptional repressor in NIH3T3 cells. Overall, our results suggest that the Cux1 and Cux2 proteins carry distinct biochemical functions. Cux2 is able, like Cux1, to perform the CCAAT-displacement activity. However, Cux2 is unlikely to execute transcriptional regulatory functions that require stable interaction with DNA.

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc.

USF and c-Myc are basic helix-loop-helix transcription factors with similar DNA-binding specificities, but antagonistic effects on cellular transformation. In order to determine how these opposite functions correlate with the transcriptional activities of the two factors on particular downstream targets, we investigated the roles of USF and c-Myc in expression of CDK4, a known direct target of c-Myc. Overexpression of either c-Myc or USF2, but not USF1, stimulated the expression of CDK4 promoter-driven reporter genes in the non-tumorigenic mammary epithelial MCF-10A cells. Dominant-negative mutants specific to either Myc or USF family proteins inhibited reporter gene activity as well as endogenous CDK4 expression, demonstrating involvement of both USF and Myc in CDK4 transcriptional control. In contrast, in two different breast cancer cell lines where USF is transcriptionally inactive and c-Myc is overexpressed, CDK4 promoter activity was no longer responsive to either transcription factor. Accordingly, chromatin immunoprecipitation revealed significantly lower levels of both USF and c-Myc bound to the endogenous CDK4 promoter in breast cancer cells than in MCF-10A cells, with a concomitant decrease in associated histone H3 acetylation. These results suggest that a major switch in the transcriptional control of CDK4 occurs during breast carcinogenesis, with likely alteration of cell cycle regulation.

Changes in the Expression of Many Ets Family Transcription Factors and of Potential Target Genes in Normal Mammary Tissue and Tumors

Interfering with Ets transcription factor function reverses multiple aspects of the transformed phenotype of mouse or human tumor cells. However, the unknown number of individual Ets factors expressed in any cellular context and the similar DNA binding specificities of Ets family members complicates the identification of those that mediate transformation. By utilizing quantitative PCR assays for 25 mouse Ets factors, we analyzed the expression of essentially the entire Ets family in normal mammary tissue, mammary-related cell lines, and mammary tumors. In normal mammary tissue, 24 Ets factors were expressed. Even clonal derived cell lines expressed 14-20 Ets members. The most abundant Ets factor mRNAs measured in normal mammary tissue were Elk4, Elf1, and Ets2. Subtractive analysis of mammary tissue identified which Ets factors were predominantly expressed in the myeloid/lymphoid or epithelial cell compartments. Comparison of Ets factor expression in normal mammary tissue and mammary tumors identified significantly elevated expression of Pse/PDEF, Ese2/Elf5, Ese3/Ehf, TEL/Etv6, and Elf2/NERF in mammary tumors and confirmed previously reported alterations in expression of Ese1/Elf3 and the PEA3 subfamily. Expression of 13 Ets target genes, implicated in various aspects of tumor progression, was also analyzed. Altered expression of particular Ets target genes was significantly correlated with particular Ets factors (e.g. maspin and Ese2), suggesting specific in vivo regulatory roles. Together, this comprehensive analysis revealed unexpectedly diverse Ets family gene expression, characterized novel Ets factor changes in mammary tumors, and implicated specific Ets factors in the regulation of multiple genes involved in mammary tumor progression.

Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase.

The Mpk1 MAP kinase of the Saccharomyces cerevisiae cell wall integrity signalling pathway phosphorylates and activates the Rlm1 transcription factor in response to cell wall stress. Rlm1 is related to mammalian MEF2 isoforms, and shares a similar DNA-binding specificity. Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis. We report here the transcriptional induction by agents of cell wall stress of a set of lacZ reporter plasmids derived from several Rlm1-responsive genes. Analysis of substitution mutations at putative Mpk1 phosphorylation sites within Rlm1 revealed that Ser427 and Thr439 are important for its stress-induced transcriptional activation of these reporter plasmids. Assessment of Rlm1 activation potency when fused to a heterologous DNA-binding domain showed that the identified seryl and threonyl residues are necessary for the Rlm1 transcriptional activation function independently of its DNA binding. We also demonstrate that a MAP kinase docking site, shown recently to mediate activation of MEF2A and MEF2C, is conserved in Rlm1 and is required for its ability to mediate transcriptional activation in response to agents that induce cell wall stress. Finally, intracellular localization analyses show that Rlm1 resides in the nucleus regardless of its activation and phosphorylation status. Together these observations support the inference that Mpk1 regulates the Rlm1 transcriptional activation function by phosphorylation of Ser427 and Thr439.

A molecular basis for differential developmental anomalies in Axenfeld-Rieger syndrome.

Pitx2, a bicoid-like homeodomain transcription factor and Dlx2 are two transcriptional markers observed during early tooth development. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 30-fold in Chinese hamster ovary cells. Mutations in PITX2 associated with Axenfeld-Rieger syndrome (ARS) provided the first link of this homeodomain transcription factor to tooth development. We are investigating the molecular basis of developmental anomalies associated with human PITX2 mutations. A phenotypically less severe ARS mutant (without tooth anomalies), PITX2 R84W, has a similar DNA binding specificity compared to wild-type PITX2 and transactivates the Dlx2 promoter. This mutation is associated with iris hypoplasia (IH); in contrast a Rieger syndrome mutation, PITX2 T68P, which presents clinically with the full spectrum of developmental anomalies (including tooth anomalies), is unable to transactivate the Dlx2 promoter. Since Dlx2 expression is required for tooth and craniofacial development the lack of tooth anomalies in the patient with IH may be due to the residual activity of this mutant in activating the Dlx2 promoter. We demonstrate that PITX2 phosphorylation increases PITX2 and PITX2 R84W DNA binding. The PITX2 T68P ARS mutation occurs at a protein kinase C phosphorylation site in the homeodomain. Surprisingly, phosphorylation of PITX2 T68P is increased compared to wild-type PITX2 but has little effect on its DNA binding activity. Altogether these data suggest a molecular mechanism for tooth development involving Dlx2 gene expression in ARS patients.

Dual Mechanisms for Repression of the Monomeric Orphan Receptor Liver Receptor Homologous Protein-1 by the Orphan Small Heterodimer Partner

The orphan nuclear hormone receptor liver receptor homologous protein-1 (LRH-1; NR5A2, also known as FTF), an unusual receptor that binds DNA as a monomer, is an essential regulator of expression of a rate-limiting enzyme in bile acid formation, cholesterol 7-alpha-hydroxylase. In a classic negative feedback loop that is a crucial component of the complex regulation of cholesterol metabolism, cholesterol 7-alpha-hydroxylase expression is decreased when bile acid levels are high. This repression is thought to be based on the bile acid-dependent induction of expression of the orphan receptor small heterodimer partner (SHP) NR0B2, which inhibits the activity of LRH-1. We have explored the molecular basis for this important regulatory effect by characterizing the mechanisms by which mouse and human SHP inhibit LRH-1-mediated transactivation. Both SHP proteins specifically interact with the AF-2 transactivation domain of LRH-1 both in vivo and in vitro. This domain is a common target for coactivator interaction, and the SHP proteins can compete with p160 coactivators for binding to LRH-1. In addition to the N-terminal receptor interaction domain, SHP includes a C-terminal domain with autonomous repression function. Neither a deletion nor a point mutation specifically affecting this domain blocked the ability to interact with LRH-1 to compete for coactivator binding or to repress LRH-1 transactivation. However, the relative ability of these mutants to inhibit LRH-1-mediated transactivation was markedly decreased. We conclude that the proposed central role of SHP in cholesterol metabolism is based on a two-step mechanism that is dependent on both coactivator competition and direct transcriptional repression.

Suppression of the Ewing's sarcoma phenotype by FLI1/ERF repressor hybrids.

Fusion of the 5' half of the Ewing's sarcoma (ES) gene EWS with the DNA-binding domain of several transcription factors has been detected in many human tumors. The t(11;22)(q24;q12) chromosomal translocation is specifically linked to ES and primitive neuroectodermal tumors and results, in the majority of cases, in the fusion of the amino terminus of the EWS gene to the carboxyl-terminal DNA-binding domain of the FLI1 gene. The chimeric protein has been shown to be oncogenic, a potent transcriptional activator, and necessary for the maintenance of the Ewing's phenotype, making it an attractive target for gene therapy. In this study, we demonstrate that the ES transformed phenotype can be suppressed by chimeric transcriptional repressors containing the DNA-binding domain of FLI1 and the regulatory and repressor domain of ERF, a transcription suppressor and member of the ets gene family. The hybrid repressor is expressed at levels comparable with EWS/FLI1, does not affect EWS/FLI1 expression, and exhibits similar DNA-binding specificity but suppresses transcriptional activity. The FLI1/ERF repressor, like the wild-type ERF, is regulated by mitogen-activated protein kinase-dependent subcellular localization. Our data suggest that transformation by EWS/FLI1 may partially be due to activation of specific EWS/FLI1-regulated genes involved in cell proliferation.

Interaction between major nitrogen regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is required for their synergistic activation of gene expression in Neurospora crassa.

In Neurospora crassa, the major nitrogen regulatory protein, NIT2, a member of the GATA family of transcription factors, controls positively the expression of numerous genes which specify nitrogen catabolic enzymes. Expression of the highly regulated structural gene nit-3, which encodes nitrate reductase, is dependent upon a synergistic interaction of NIT2 with a pathway-specific control protein, NIT4, a member of the GAL4 family of fungal regulatory factors. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter, but, in addition, we show that a direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of the nit-3 structural gene. Neurospora possesses at least five different GATA factors which control different areas of cellular function, but which have a similar DNA binding specificity. Significantly, only NIT2, of the several Neurospora GATA factors examined, interacts with NIT4. We propose that protein-protein interactions of the individual GATA factors with additional pathway-specific regulatory factors determine each of their specific regulatory functions.