Zinc Finger DNA-binding Protein Research Articles

Method of Detecting and Targeting Mutations in Cancer

While there are many differences between tumor and non-tumor cells, the basic underlying distinction is in the DNA. Tumor cells harbor mutations, at least some of which are not present in non-tumor cells. Thus, a method of directly targeting cells containing specific mutations has potential for detection or treatment of cancer without the toxicity associated with more indirect approaches. Also, as mutations are a necessary component of malignancy, such a method is potentially applicable to all tumors. I propose a method by which several recently developed techniques can be utilized in a novel way to accomplish the goal of directly targeting mutations for cancer detection and therapy. The model can be summarized as follows: (1) Determine potential target mutations present in tumor cells but not non-tumor cells. (2) Construct molecules that will bind to DNA at the sites of mutation, but will not bind to DNA in normal cells. And, as a consequence of the molecules binding to the mutation, the cells will be destroyed. (3) Deliver these molecules to all cells (or at least all tumor cells). I hypothesize that such molecules can now be constructed using sequence-specific DNA binding proteins (such as customized zinc-finger DNA binding proteins) fused to transcriptional activator domains (such as VP16) and reporter or toxin genes. The necessary genes can be linked to the DNA binding proteins utilizing a recently described method based on expressed protein ligation.

Method of Detecting and Targeting Mutations in Cancer

AbstractWhile there are many differences between tumor and non-tumor cells, the basic underlying distinction is in the DNA. Tumor cells harbor mutations, at least some of which are not present in non-tumor cells. Thus, a method of directly targeting cells containing specific mutations has potential for detection or treatment of cancer without the toxicity associated with more indirect approaches. Also, as mutations are a necessary component of malignancy, such a method is potentially applicable to all tumors. I propose a method by which several recently developed techniques can be utilized in a novel way to accomplish the goal of directly targeting mutations for cancer detection and therapy. The model can be summarized as follows: (1) Determine potential target mutations present in tumor cells but not non-tumor cells. (2) Construct molecules that will bind to DNA at the sites of mutation, but will not bind to DNA in normal cells. And, as a consequence of the molecules binding to the mutation, the cells will be destroyed. (3) Deliver these molecules to all cells (or at least all tumor cells). I hypothesize that such molecules can now be constructed using sequence-specific DNA binding proteins (such as customized zinc-finger DNA binding proteins) fused to transcriptional activator domains (such as VP16) and reporter or toxin genes. The necessary genes can be linked to the DNA binding proteins utilizing a recently described method based on expressed protein ligation.

Predominant Interaction of Both Ikaros and Helios with the NuRD Complex in Immature Thymocytes

Ikaros is the founding member of a small family of C2H2 zinc-finger DNA-binding proteins that carry out critical functions during lymphocyte development. Although interactions between Ikaros and various proteins have been reported, Ikaros-containing complexes have not been purified to determine their composition and identify the predominant interacting partners. In this study, a tandem affinity purification-mass spectrometry strategy was developed for the isolation of complexes formed by Ikaros and by Helios, a T-cell-restricted member of the Ikaros family that remains largely uncharacterized. This strategy, which appears to be well suited for general use in mammalian cells, relies on an N-terminal polypeptide containing a double FLAG epitope, followed by a tobacco etch virus protease cleavage site and calmodulin binding peptide. In extracts from a murine thymocyte line, Ikaros and Helios associated under moderate stringency conditions only with other members of the Ikaros family. However, under low stringency conditions, both tagged proteins assembled into higher molecular weight complexes. Mass spectrometry revealed that both proteins associated predominantly with subunits of NuRD, an ATP-dependent nucleosome remodeling complex implicated in transcriptional repression and activation and previously reported to associate with Ikaros. Further analysis of the affinity-purified Ikaros revealed that several serines and threonines are phosphorylated in the thymocyte line, with apparent changes upon thymocyte maturation. These results support the hypothesis that the NuRD complex makes major contributions to the functions of both Ikaros and Helios and that the activities of these proteins may be regulated in part by changes in phosphorylation.

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

Artificial endonucleases consisting of a FokI cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites. Here we describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.

Myelin transcription factor 1 (Myt1) expression in demyelinated lesions of rodent and human CNS

Myelin transcription factor 1 (Myt1) is a zinc-finger DNA binding protein that influences developing oligodendrocyte progenitor (OP) cell proliferation, differentiation, and myelin gene transcription in vitro. The potential of Myt1 to play a role in OP responses leading to remyelination was examined using murine hepatitis virus strain A59 (MHV) to induce spinal cord demyelination and potential relevance to human pathology was evaluated in multiple sclerosis (MS) lesions. In MHV-infected mice, the density of Myt1 expressing cells markedly increased in lesioned areas of spinal cord white matter. Myt1 expressing cells proliferated most extensively during active demyelination and subsequently accumulated to maximal levels during early remyelination. Cells with nuclear Myt1 immunoreactivity were mainly OP cells, identified by co-localization with platelet-derived growth factor alpha receptor, with additional phenotypes being either oligodendrocytes or neural stem cells, identified by CC1 antigen and Musashi1, respectively. The density of OP cells expressing Myt1 was significantly increased in white matter of MHV-infected mice during demyelination and early remyelination then as remyelination advanced the values returned to levels comparable to PBS-injected control mice. In MHV lesions, Myt1 was not expressed in astrocytes, lymphocytes, or macrophage/microglial cells. MS lesions demonstrated increased Myt1 expression in both the periplaque white matter adjacent to lesions and within early remyelinating lesions. These results suggesta potential role for Myt1 in the regeneration of oligodendrocyte lineage cells in response to demyelination.

Chemical Biology

Chemical Biology

Maximal Activity of the Luteinizing Hormoneβ-Subunit Gene Requires β-Catenin

GnRH regulates expression of LHB via transcriptional regulation of early growth response 1 (EGR1), an immediate early gene that encodes a zinc-finger DNA-binding protein. EGR1 interacts functionally with the orphan nuclear receptor steroidogenic factor 1 (SF1) and pituitary homeobox 1, a member of the paired-like homeodomain family. The functional synergism of this tripartite interaction defines the maximal level of LHB transcription that can occur in response to GnRH. Results presented herein provide new evidence that the interaction between SF1 and EGR1 also requires beta-catenin, a transcriptional coactivator and member of the canonical Wnt signaling pathway. For instance, targeted reduction of beta-catenin attenuates activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of beta-catenin, or its targeted reduction by small interfering RNA, modulates activity of both SF1 and EGR1 as well as their functional interaction. beta-Catenin coimmunoprecipitates with SF1. Moreover, an SF1 mutant that lacks a beta-catenin binding domain has compromised transcriptional activity and fails to interact synergistically with EGR1. Finally, GnRH promotes beta-catenin colocalization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together, these data suggest that beta-catenin binds to SF1 and that this interaction is required for subsequent functional interaction with EGR1. Thus, these data identify beta-catenin as a new and required member of the basal transcriptional complex that allows the LHB promoter to achieve maximal activity in response to GnRH.

In vitro selection of zinc finger DNA-binding proteins through ribosome display

DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however, crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique.

Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 beta-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences.

Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics.

973. SEquence-Enabled Reassembly of β- Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 β-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic D...

1000. Down-Regulating the Epithelial Glycoprotein-2 Promoter Utilizing Engineered Zinc Finger Protein Transcription Factors

Top of pageAbstract Engineered zinc finger protein transcription factors (ZFP-TFs) are emerging as novel powerful tools to regulate endogenous gene expression. Zinc finger proteins consist of modular building blocks, with each domain recognizing 3-4 base pairs. Stitching these modular domains together results in the recognition of an extended DNA sequence, increasing the specificity of the target sequence. Specific amino acid positions within each domain recognize the DNA. Altering the amino acid residues at these positions therefore results in the recognition of different base pairs. A wide variety of effector domains can be attached to the DNA-binding zinc finger protein, for example to activate or inhibit gene transcription. In this study we utilized engineered ZFP-TFs to specifically inhibit the expression of the epithelial glycoprotein-2 (EGP-2). EGP-2 is expressed in the most prevalent types of carcinomas, including breast, ovarian, and colon cancer. Recently, others have demonstrated that down-regulation of EGP-2 using siRNA or ODNs resulted in a decrease in the migration, invasion, and proliferation potential of tumor cells. Therefore, inhibiting the expression of EGP-2 can provide a novel anti-cancer treatment for a broad range of tumor types. Down-regulating EGP-2 expression by targeting the mRNA requires the targeting of many molecules. Direct down-regulation of the EGP-2 expression at the DNA level using engineered ZFP-TFs can therefore be more effective. We engineered a zinc finger protein that specifically bound to a 9 base-pair target sequence in the EGP-2 promoter sequence. After attaching a gene-expression inhibition domain to the DNA-binding ZFP, co-transfection of EGP-2-positive cells with a plasmid expressing the engineered ZFP-TF and a plasmid containing the luciferase gene under the control of the EGP-2 promoter resulted in 60% inhibition of luciferase expression. To further increase the specificity for binding to the EGP-2 promoter, we engineered three different ZFP-TFs targeting 18 base pair sequences within the EGP-2 promoter. Co-transfection experiments demonstrated that the engineered ZFP-TFs caused up to 80% down-regulation of the EGP-2 promoter while no down-regulation of a non-target promoter was observed. Combining inhibition at the DNA level with inhibition at the RNA level using siRNA might even further enhance the down-regulation of EGP-2 promoter activity. In this study we have demonstrated that ZFP-TFs can be engineered to down-regulate the EGP-2 promoter, generating a novel therapeutic for the treatment of a broad variety of tumor types.

Cell-free Selection of Zinc Finger DNA-binding Proteins Using In Vitro Compartmentalization

We have exploited emulsion-based in vitro compartmentalization (IVC) to devise a method for the selection of zinc finger proteins (ZFPs) on the basis of their DNA-binding specificity. A library of ZFPs fused to a C-terminal peptide tag is encoded by a set of DNA cassettes that are prepared wholly in vitro. In addition to the ZFP gene, each DNA cassette also carries a given DNA target binding site sequence for which one wishes to isolate ZFP binders. An aliquot of the library is added to bacterial S30 extract and emulsified in mineral oil so that most of the aqueous droplets contain, on average, no more than one gene. If an intra-compartmentally expressed ZFP binds specifically to its encoding DNA via the target binding site, the complex can be purified by affinity capture via the peptide tag after breaking the emulsion, thus rescuing the gene. We present proof-of-principle for this IVC selection method by selecting a specific high-affinity ZFP gene from a high background of a related gene. We also propose that high-affinity ZFPs can be used as genotype-phenotype linkages to enable selection of other proteins using IVC.

KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic β-like globin genes in vivo

AbstractThe Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the β-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the β-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and βh1-globin but not ζ-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult βmaj- and βmin-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic ϵ-globin gene but not the adult β-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death. (Blood. 2005;106: 2566-2571)

Mint Represses Transactivation of the Type II Collagen Gene Enhancer through Interaction with αA-crystallin-binding Protein 1

Collagen type II is an extracellular matrix protein important for cartilage and bone formation, and its expression is controlled by multiple cis- and trans-acting elements, including the zinc finger transcription factor alpha A-crystallin-binding protein 1 (CRYBP1). Here we show that MSX2-interacting nuclear target protein (MINT), a conserved transcriptional repressor, associates with CRYBP1 and negatively regulates the transactivation of the collagen type II gene (Col2a1) enhancer. We identified CRYBP1 as a binding partner of MINT by screening a mouse embryonic cDNA library using the yeast two-hybrid system. We demonstrated that the C terminus of MINT interacts with the C terminus of CRYBP1 using the mammalian cell two-hybrid assay, glutathione S-transferase pull-down, and co-immunoprecipitation analyses. Furthermore, MINT and CRYBP1 form a complex on the Col2a1 enhancer, as shown by chromatin immunoprecipitation and gel shift assays. In the presence of CRYBP1, overexpression of MINT or its C-terminal fragment in cells repressed a reporter construct driven by the Col2a1 enhancer elements. This transcription repression is dependent on histone deacetylase, the main co-repressor recruited by MINT. The present study shows that MINT is involved in CRYBP1-mediated Col2a1 gene repression and may play a role in regulation of cartilage development.

Gli1 is important for medulloblastoma formation in Ptc1+/− mice

Germline mutations in the human homolog of the patched1 (PTCH1) are associated with basal cell nevus carcinoma syndrome (BCNS or Gorlin syndrome), which is characterized by developmental anomalies, radiation hypersensitivity and a predisposition to medulloblastomas and skin tumors. Patched1 (Ptc1) functions as a receptor for Sonic hedgehog (Shh) in a wide range of biological processes. Binding of Shh to Ptc1 results in activation of Smoothened (Smo), which in turn stimulates expression of downstream target genes including Ptc1 and Gli1. Gli1 is a member of a family of DNA-binding zinc-finger proteins, including Gli2 and Gli3, that function in transcription control. Here, we report that inactivation of both Gli1 alleles in Ptc1+/- mice significantly reduces spontaneous medulloblastoma formation. Therefore, Gli1 is not only a marker of pathway activation but also plays a functional role in medulloblastoma formation. Interestingly, Gli2 levels were elevated in medulloblastoma cells but not in normal granule neuron precursors during cerebellar development in mice lacking Gli1. In cultured fibroblasts, Gli1 was more potent than Gli2 at inducing cell transformation. These results demonstrate that Gli1 plays a central role in medulloblastoma formation in Ptc1+/- mice and that Gli2 may also contribute to oncogenesis.

Zinc finger domain of Snail functions as a nuclear localization signal for importin β‐mediated nuclear import pathway

Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid nuclear protein with an amino-terminal basic amino acid-rich domain (SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). A series of fusion proteins composed of green fluorescent protein (GFP) and portions of the Snail protein were generated, and their subcellular localization was examined. Fusion of the four zinc fingers to GFP led to the targeting of GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. Using an in vitro transport system, the nuclear import of Snail was reconstituted by importin (karyopherin) beta in the presence of Ran and NTF2. We further demonstrated that Snail binds directly to importin beta in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a nuclear localization signal and Snail can be transported into the nucleus in an importin beta-mediated manner.

Ikaros Induces Quiescence and T-Cell Differentiation in a Leukemia Cell Line

Ikaros is a hematopoietic cell-specific zinc finger DNA binding protein that plays an important role in lymphocyte development. Genetic disruption of Ikaros results in T-cell transformation. Ikaros null mice develop leukemia with 100% penetrance. It has been hypothesized that Ikaros controls gene expression through its association with chromatin remodeling complexes. The development of leukemia in Ikaros null mice suggests that Ikaros has the characteristics of a tumor suppressor gene. In this report, we show that the introduction of Ikaros into an established mouse Ikaros null T leukemia cell line leads to growth arrest at the G0/G1 stage of the cell cycle. This arrest is associated with up-regulation of the cell cycle-dependent kinase inhibitor p27kip1, the induction of expression of T-cell differentiation markers, and a global and specific increase in histone H3 acetylation status. These studies provide strong evidence that Ikaros possesses the properties of a bona fide tumor suppressor gene for the T-cell lineage and offer insight into the mechanism of Ikaros's tumor suppressive activity.

The Epstein-Barr Virus Replication Protein BBLF2/3 Provides an Origin-Tethering Function through Interaction with the Zinc Finger DNA Binding Protein ZBRK1 and the KAP-1 Corepressor

Herpesviruses encode a set of core proteins essential for lytic replication of their genomes. Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3. BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized. To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein. This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5. The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors. Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays. A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay. ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA. Replication of an oriLyt plasmid and a variant oriLyt (DeltaZBRK1) plasmid was examined in lytically induced D98-HR1 cells. Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication. These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex. The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.

The early gene Broad is involved in the ecdysteroid hierarchy governing vitellogenesis of the mosquito Aedes aegypti

The broad (br) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these three isoforms correlated well with those of yolk protein precursor (YPP) genes. These BR isoforms were activated by 20E in fat bodies cultured in vitro and behaved as early genes, with a self-repressive autoregulatory loop that can be blocked by the protein inhibitor, cyclohexamide. Multiple binding sites for all four BR isoforms were present in the 5'-regulatory region of the major YPP gene, vitellogenin (Vg). Effects of BR isoforms on the expression of Vg have been demonstrated by cell transfection analysis. In particular, BR isoforms by themselves had no effects on the Vg promoter. However, Z1 and Z4 each repressed Aedes aegypti ecdysone receptor (EcR)/Ultraspiracle (USP)-mediated 20E activation of the Vg promoter, while Z2 enhanced activation of the Vg promoter by AaEcR/AaUSP in the presence of 20E. Z3 had no obvious effect in the same experiment. These results suggested that BR isoforms are essential for proper activation and termination of the Vg gene in response to 20E. Overall, our study implicated br in the regulation of mosquito vitellogenesis.