Library Of Transcription Factors Research Articles

Discovery and validation of PURA as a transcription target of 20(S)-protopanaxadiol: Implications for the treatment of cognitive dysfunction

Background20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for the central nervous system, especially in improving learning and memory. However, its transcriptional targets in brain tissue remain unknown. MethodsIn this study, we first used mass spectrometry-based drug affinity responsive target stability (DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcription factor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayer interferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of target genes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used to analyze the potential biological features of these target proteins. ResultsThe results showed three overlapping transcription factors between the proteomics of DARTS and transcription factor library. BLI analysis further showed that PPD had a higher direct interaction with PURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations in key amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showed that PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformatics analysis showed that these target proteins were involved in learning and memory function. ConclusionThe above-mentioned findings indicate that PURA is a transcription target of PPD in brain, and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by binding PURA, which could provide a chemical and biological basis for the study of treating cognitive impairment by targeting PURA.

Performance upgrade of a microbial explosives’ sensor strain by screening a high throughput saturation library of a transcriptional regulator

We present a methodology for a high-throughput screening (HTS) of transcription factor libraries, based on bacterial cells and GFP fluorescence. The method is demonstrated on the Escherichia coli LysR-type transcriptional regulator YhaJ, a key element in 2,4-dinitrotuluene (DNT) detection by bacterial explosives’ sensor strains. Enhancing the performance characteristics of the YhaJ transcription factor is essential for future standoff detection of buried landmines. However, conventional directed evolution methods for modifying YhaJ are limited in scope, due to the vast sequence space and the absence of efficient screening methods to select optimal transcription factor mutants. To overcome this limitation, we have constructed a focused saturation library of ca. 6.4 × 107 yhaJ variants, and have screened over 70 % of its sequence space using fluorescence-activated cell sorting (FACS). Through this screening process, we have identified YhaJ mutants exhibiting superior fluorescence responses to DNT, which were then effectively transformed into a bioluminescence-based DNT detection system. The best modified DNT reporter strain demonstrated a 7-fold lower DNT detection threshold, a 45-fold increased signal intensity, and a 40 % shorter response time compared to the parental bioreporter. The FACS-based HTS approach presented here may hold a potential for future molecular enhancement of other sensing and catalytic bioreactions.

Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein.

Transcription factors (TFs) play an important role in plant development; however, their role during viral infection largely remains unknown. The present study was designed to uncover the role transcription factors play in Cucumber mosaic virus (CMV) infection. During the screening of an Arabidopsis thaliana (Col-0) transcription factor library, using the CMV 2b protein as bait in the yeast two-hybrid system, the 2b protein interacted with Homeobox protein 27 (HB27). HB27 belongs to the zinc finger homeodomain family and is known to have a regulatory role in flower development, and responses to biotic and abiotic stress. The interaction between CMV 2b and HB27 proteins was further validated using in planta (bimolecular fluorescence complementation assay) and in vitro far-Western blotting (FWB) methods. In the bimolecular fluorescence complementation assay, these proteins reconstituted YFP fluorescence in the nucleus and the cytoplasmic region as small fluorescent dots. In FWB, positive interaction was detected using bait anti-MYC antibody on the target HB27-HA protein. During CMV infection, upregulation (~3-fold) of the HB27 transcript was observed at 14 days post-infection (dpi) in A. thaliana plants, and expression declined to the same as healthy plants at 21 dpi. To understand the role of the HB27 protein during CMV infection, virus accumulation was determined in HB27-overexpressing (HB27 OE) and knockout mutants. In HB27-overexpressing lines, infected plants developed mild symptoms, accumulating a lower virus titer at 21 dpi compared to wild-type plants. Additionally, knockout HB27 mutants had more severe symptoms and a higher viral accumulation than wild-type plants. These results indicate that HB27 plays an important role in the regulation of plant defense against plant virus infection.

Abstract 2206: Pilot phase I clinical trial of RANKL inhibition and breast tissue gene expression in high-risk premenopausal women with dense breasts

Abstract Background: Identifying pathways that can be targeted to reduce breast density and breast cancer incidence is an unmet need, especially in premenopausal women. Receptor activator of nuclear factor-κB ligand (RANKL) signaling mediates the major proliferative response of mammary epithelium to progesterone and is positively associated with breast density but there are no clinical trial data on the impact of RANKL inhibition on breast tissue markers in women with dense breasts. We, therefore, determined the impact of RANKL inhibition on breast tissue gene expression in high-risk premenopausal women with dense breasts. Study design: Pilot phase I clinical trial of 9 healthy high-risk pre-menopausal women (≥35 years of age) with dense breasts performed at Washington University School of Medicine, St. Louis, MO from July 2018-December 2018. Fifty-five percent of participants had a positive family history of breast cancer in a first degree relative. Intervention: Participants were given a single dose of subcutaneous RANKL antibody, denosumab (60mg), at baseline (Day 1). On the same day, prior to the denosumab injection, participants had an ultrasound guided core needle biopsy and a blood draw. All study participants returned for repeat core needle biopsy and blood draw after 60 days (Day 60). Outcome Measures: Changes in breast tissue gene expression between Day 1 and Day 60 with a focus on immune/inflammatory and hormone markers. Gene expression was profiled using NanoString nCounter platform. We performed pathway enrichment analysis and used Cell Maker Enrichment library from EnrichR to identify differentially expressed genes. Genes with a p-value<0.05 on differential expression analysis were considered statistically significant for utilization in pathway enrichment analysis. Libraries utilized for pathway enrichment using the EnrichR platform included the ChEA transcription factor library, GO Biological Process library, and Cellular Components library. Results: Macrophage markers, including CCR5, CD86, CD300A, and CD84 and targets downstream of IRF8: (p-value =4.73e-7), neutrophil degranulation/neutrophil activation in the immune response (p-value=6.0e-21), and the secretory granule membrane pathways (p-value=2.4e-14) were downregulated in breast tissues on Day 60. Pathways involved in progesterone metabolism (p-value=0.0003), estrogen response (p-value=0.0014) and fatty acid metabolism, (p-value=0.0001) were significantly upregulated on Day 60. Specifically, genes involved in steroid hormone metabolism such as DHRS2 (p-value=3.13e-05), and AKR1B15: p-value=4.12e-05) were upregulated on Day 60. Conclusions: A single 60 mg dose of subcutaneous denosumab injection was associated with alterations in pathways involved in hormone, immune and inflammatory signaling in the breast tissues of healthy premenopausal women with dense breasts. Citation Format: Michael Waters, Kay Jayachandran, Debbie Bennet, Jin Zhang, Katherine Weilbaecher, Ian S. Hagemann, Graham A. Colditz, Catherine M. Appleton, Adetunji T. Toriola. Pilot phase I clinical trial of RANKL inhibition and breast tissue gene expression in high-risk premenopausal women with dense breasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2206.

Functional Antagonism of WRI1 and TCP20 Modulates GH3.3 Expression to Maintain Auxin Homeostasis in Roots.

Auxin is a well-studied phytohormone, vital for diverse plant developmental processes. The GH3 genes are one of the major auxin responsive genes, whose expression changes lead to modulation of plant development and auxin homeostasis. However, the transcriptional regulation of these GH3 genes remains largely unknown. WRI1 is an essential transcriptional regulator governing plant fatty acid biosynthesis. Recently, we identified that the expression of GH3.3 is increased in the roots of wri1-1 mutant. Nevertheless, in this study we found that AtWRI1 did not activate or repress the promoter of GH3.3 (proGH3.3) despite of its binding to proGH3.3. Cross-family transcription factor interactions play pivotal roles in plant gene regulatory networks. To explore the molecular mechanism by which WRI1 controls GH3.3 expression, we screened an Arabidopsis transcription factor library and identified TCP20 as a novel AtWRI1-interacting regulator. The interaction between AtWRI1 and TCP20 was further verified by several approaches. Importantly, we found that TCP20 directly regulates GH3.3 expression via binding to TCP binding element. Furthermore, AtWRI1 repressed the TCP20-mediated transactivation of proGH3.3. EMSAs demonstrated that AtWRI1 antagonized TCP20 from binding to proGH3.3. Collectively, we provide new insights that WRI1 attenuates GH3.3 expression through interaction with TCP20, highlighting a new mechanism that contributes to fine-tuning auxin homeostasis.

Cell fate engineering through TF activation.

A study in Nature Biotechnology presents a library of transcription factors that are able to induce differentiation of human induced pluripotent stem cells as a resource for cell and tissue engineering.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR4 Interacts with WRINKLED1 to Mediate Seed Oil Biosynthesis.

Cross-family transcription factor (TF) interactions play critical roles in the regulation of plant developmental and metabolic pathways. WRINKLED1 (WRI1) is a key TF governing oil biosynthesis in plants. However, little is known about WRI1-interacting factors and their roles in oil biosynthesis. We screened a TF library using Arabidopsis (Arabidopsis thaliana) WRI1 (AtWRI1) as bait in yeast two-hybrid assays and identified three TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family TFs, namely TCP4, TCP10, and TCP24, as AtWRI1-interacting partners. The physical interaction between AtWRI1 and TCPs was further validated using bimolecular fluorescence complementation assays. TCPs play important roles in various plant developmental processes; however, their involvement in fatty acid biosynthesis was not previously known. Coexpression of TCP4, but not TCP10 or TCP24, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves. Transcriptomic analysis in transgenic Arabidopsis plants with enhanced TCP4 activity engineered by expressing rTCP4 (i.e. miR319-resistant TCP4) revealed that AtWRI1 target genes were significantly repressed. TCP4 expression is strongly correlated with AtWRI1 during embryo development. A tcp4 loss-of-function mutant, the jaw-D mutant with a strong reduction of TCP4 expression, and a tcp2 tcp4 tcp10 triple mutant accumulated more seed oil than wild-type Arabidopsis. In addition, TCP4 repressed the AtWRI1-mediated transactivation of the promoters of fatty acid biosynthetic genes. Collectively, our findings suggest that TCP4 represses fatty acid biosynthetic gene expression through interaction with AtWRI1, leading to a reduction of AtWRI1-mediated seed oil accumulation.

FLOWERING HTH1 is involved in CONSTANS-mediated flowering regulation in Arabidopsis

Flowering at the right time is essential for maximum reproductive fitness. In Arabidopsis thaliana, the CONSTANS (CO) protein facilitates the transition from the vegetative phase to the reproductive phase under long-day conditions. The formation of heterodimeric complexes between CO and DNA binding domain-containing transcription factors is important for the induction of day length-dependent flowering. Here, we report a myb-like helix turn helix (HTH) transcriptional regulator family protein as a new modulator of floral transition, which we have named FLOWERING HTH1 (FHTH1). We isolated FHTH1 as a CO-interacting protein by a yeast two-hybrid screen using an Arabidopsis transcription factor library. Our analysis showed that FHTH1 presented in the nucleus and the FHTH1-CO complex was formed in the same subcellular location. We also observed the expression of a FHTH1:GUS construct in the leaf vasculature, where CO exists. Transgenic plants overexpressing FHTH1 fused with the plant-specific repression domain SRDX showed a delayed flowering phenotype in long days, resembling the phenotype of the co mutant. Our results suggest that FHTH1 may contribute to CO-mediated photoperiodic flowering regulation.

The Oxidative Stress Response in Caenorhabditis elegans Requires the GATA Transcription Factor ELT-3 and SKN-1/Nrf2.

Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the Caenorhabditis elegans Brap2 ortholog (BRAP-2) and found that it is required to prevent larval arrest in response to elevated levels of oxidative stress. Here, we report that C. elegans brap-2 mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK C. elegans ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target gst-4 in brap-2 mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of gst-4p::gfp expression. We found that ELT-3 interacts with SKN-1 to activate gst-4 transcription in vitro and that elt-3 is required for enhanced gst-4 expression in the brap-2(ok1492) mutant in vivo Furthermore, nematodes overexpressing SKN-1 required ELT-3 for life-span extension. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of SKN-1 through inhibition of p38 MAPK activity, and that the GATA transcription factor ELT-3 is required along with SKN-1 for the phase II detoxification response in C. elegans.

Identification of Novel Components of the Unfolded Protein Response in Arabidopsis.

Unfavorable environmental and developmental conditions may cause disturbances in protein folding in the endoplasmic reticulum (ER) that are recognized and counteracted by components of the Unfolded Protein Response (UPR) signaling pathways. The early cellular responses include transcriptional changes to increase the folding and processing capacity of the ER. In this study, we systematically screened a collection of inducible transgenic Arabidopsis plants expressing a library of transcription factors for resistance toward UPR-inducing chemicals. We identified 23 candidate genes that may function as novel regulators of the UPR and of which only three genes (bZIP10, TBF1, and NF-YB3) were previously associated with the UPR. The putative role of identified candidate genes in the UPR signaling is supported by favorable expression patterns in both developmental and stress transcriptional analyses. We demonstrated that WRKY75 is a genuine regulator of the ER-stress cellular responses as its expression was found to be directly responding to ER stress-inducing chemicals. In addition, transgenic Arabidopsis plants expressing WRKY75 showed resistance toward salt stress, connecting abiotic and ER-stress responses.

Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5′-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5′-TGTAAGCCCTAACA-3′) upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.

The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles.

Over the past two decades, Zea mays (maize) has been established as a model system for the study of indirect plant defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles, mainly composed of sesquiterpenes, to attract the natural enemies of the herbivores. This is associated with a swift transcriptional induction of terpene synthases such as TPS10; however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure. Here, by exploiting the finding that the maize TPS10 promoter retained its full responsiveness to herbivory in Arabidopsis, we identified the region from -300 to -200 of the TPS10 promoter as both necessary and sufficient for its herbivore inducibility through 5' deletion mapping. A high-throughput screening of an Arabidopsis transcription factor library using this promoter region as the bait identified seven AP2/ERF family transcription factors. Among their close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Meanwhile, EREB58 was also responsive to Jasmonate. In vivo and in vitro assays indicated that EREB58 promotes TPS10 expression by directly binding to the GCC-box within the region from -300 to -200 of the TPS10 promoter. Transgenic maize plants overexpressing EREB58 constitutively over-accumulate TPS10 transcript, and also (E)-β-farnesene and (E)-α-bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles was abolished in EREB58-RNAi transgenic lines. In sum, these results demonstrate that EREB58 is a positive regulator of sesquiterpene production by directly promoting TPS10 expression.

An overproduction of astellolides induced by genetic disruption of chromatin-remodeling factors in Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae is an important industrial mold. Recent genomic analysis indicated that A. oryzae has a large number of biosynthetic genes for secondary metabolites (SMs), but many of the SMs they produce have not been identified. For better understanding of SMs production by A. oryzae, we screened a gene-disruption library of transcription factors including chromatin-remodeling factors and found two gene disruptions that show similarly altered SM production profiles. One is a homolog of Aspergillus nidulans cclA, a component of the histone 3 lysine 4 (H3K4) methyltransferase complex of proteins associated with Set1 complex, and the other, sppA, is an ortholog of Saccharomyces cerevisiae SPP1, another component of a complex of proteins associated with Set1 complex. The cclA and sppA disruptions in A. oryzae are deficient in trimethylation of H3K4. Furthermore, one of the SMs that increased in the cclA disruptant was identified as astellolide F (14-deacetyl astellolide B). These data indicate that both cclA and sppA affect production of SMs including astellolides by affecting the methylation status of H3K4 in A. oryzae.

A special member of the rice SRO family, OsSRO1c, mediates responses to multiple abiotic stresses through interaction with various transcription factors

SIMILAR TO RCD ONE (SRO) is a plant-specific gene family involved in development and abiotic stress responses. SRO proteins are characterized by containing poly (ADP-ribose) polymerase catalytic (PARP) and C-terminal RCD1-SRO-TAF4 domains, and can be classified into two groups and five subgroups on the basis of their PARP domain. Expression analysis of rice SRO genes in response to various abiotic stresses showed that OsSRO1c, a rice SRO gene which functions downstream of the stress-responsive transcription factor SNAC1, is the major stress-responsive gene in the rice SRO family. The ossro1c-1 mutant showed resistance not only to chloroplastic oxidative stress, but also to apoplastic oxidative stress. However, the ossro1c-1 mutant and artificial microRNA-OsSRO1c transgenic rice were significantly impaired in cold tolerance. When compared with the well-characterized Arabidopsis SRO protein radical-induced cell death 1 (RCD1), OsSRO1c has considerable variation in the protein sequence, and the two genes exhibit different expression profiles under abiotic stresses. Furthermore, ossro1c-1 and rcd1 showed different responses to multiple abiotic stresses. By screening an Arabidopsis transcription factor library, 29 transcription factors interacted with OsSRO1c in yeast, but only two of these transcription factors were reported to interact with RCD1, which may partly explain the different responses of the two mutants under various stresses. The data presented in this report provide important clues for further elucidating the molecular and biochemical mechanisms of OsSRO1c in mediating responses to multiple abiotic stresses.

Abstract 19007: Lineage Reprogramming of Mouse Fibroblasts to Induced Cardiac Progenitor Cells by Defined Factors

Several studies have reported reprogramming of mouse fibroblasts to induced cardiomyocytes (iCMs). However, reprogramming to cardiac progenitor cells (CPCs), which may be more favorable for cell therapy because of their proliferative properties and multipotency, remains to be accomplished. This study aims to reprogram mouse fibroblasts to induced CPCs (iCPCs) by transcription factor (TF)-mediated reprogramming. We have cloned a library of 25 genes including cardiac-related TFs and chromatin remodeling agents into a novel, inducible piggyBac transposon vector. Fibroblasts were derived from a Nkx 2.5- YFP cardiac progenitor reporter mouse model. We first confirmed efficient transcription/translation of our piggyBac TF library. Next, as a control experiment neonatal cardiac fibroblasts were transfected with Gata4, Mef2c, Tbx5 or Gata4, Mef2c, Tbx5, Hand2 (published factors) and monitored for appearance of beating cells. Spontaneously beating, YFP- iCMs were observed only after application of doxycycline (25 days) for both combinations of factors, demonstrating a robust reprogramming system. We then transfected neonatal cardiac fibroblasts with entire library of 25 factors and observed YFP+ cells on doxycycline treatment. The TF pool was subsequently narrowed down to 11 essential factors. On transfection with 11 factors, YFP+ cells were first observed 4 days after doxycycline treatment. By day 20 these YFP+ cells developed into two-dimensional, highly proliferative YFP+ colonies of cells that lost their parental fibroblast morphology and exhibited a high nucleus to cytoplasmic ratio. Similar reprogramming was later accomplished using adult cardiac and adult lung fibroblasts. Untransfected/iPSC factors transfected control fibroblasts yielded no beating or YFP+ cells. qPCR analysis of these YFP+ colonies revealed an upregulation of CPC markers including Mesp1, Mef2c, Irx4 accompanied with down regulation of fibroblast specific genes such as Fsp1, consistent with reprogramming towards an iCPC state. These results support the ability of inducible piggBac-based TFs to reprogram fibroblasts to iCPCs. Current experiments are focused on optimizing culture conditions to maintain iCPCs in a proliferative state and defining their potency.

Abstract LB-208: Small interfering RNA library screen to identify therapeutic transcription factor.

Abstract Post-genomic analyses of major transcription factor families, in both malignant and non-malignant cell types, have resulted in a broader revision of understanding the biology of transcription factor families. A number of oncogenic transcription factors, such as activator protein (AP)-1, estrogen receptor(ER) and signal transducer and activator of transcription (STAT)-3/STAT-5, are overactivated in human carcinomas. Up to now, three main groups of transcription factors are known to be important in human cancer. Transcription factor-directed anticancer drug development has focused on membrane or cytosolic targeting of molecules acting as ligand receptors. Recent technologies, such as small interfering RNA (siRNA), have shifted transcription factor targeting toward a more sophisticated rationale to elucidate the mechanisms that govern gene expression by analyzing the functions of proteins commonly over-expressed in transformed and malignant cells. In this study, we've applied a cell spot microarray (CSMA) method which provides reproducible multi-parametric phenotypic readouts for large-scale gene knockdown genetic screens. A human transcription factor siRNA library including 350 transcription factor genes which were significantly overexpressed in human breast cancer was screened against 6 breast cancer cell lines including (Triple negative breast cancer, HER2 positive ER positive) SUM149, BT20, HCC1569, SKBR3, MDAMB361 and MCF7 for measuring cell proliferation using EdU incorporation, cell apoptosis with c-PARP staining. We ranked the candidate gene list by the change of the rate of EdU or level of c-PARP or the combination of both. Our data showed that the silencing of LHX1, BCL6, GABPA or SMAD1 increased the c-PARP levels in most of cell lines. The Knockdown of ZNF215, NFATC1, HOXD4 or ZNF169 reduced the levels of EdU staining. Selected siRNAs were then tested on more different breast cancer cell lines to confirm the spectrum of activity. In the near future, based on this pilot screen, a complete siRNA library of transcription factors including 1000 gene list will be tested on our panel of 50 breast cancer cell lines. Our screen will provide comprehensive information on the central role of transcription factor in breast cancer, and will lead to the identity of novel therapeutics selectively against transcription factors. Citation Format: Zhi Hu, Shenda Gu, Joe Gray, Juha Rantala. Small interfering RNA library screen to identify therapeutic transcription factor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-208. doi:10.1158/1538-7445.AM2013-LB-208

Induction of Stable Drug Resistance in Human Breast Cancer Cells Using a Combinatorial Zinc Finger Transcription Factor Library

Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust tool for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to fulvestrant, a clinically important anti-endocrine therapeutic agent. From a diverse collection of nearly 400,000 different ZF-TFs, we isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor-positive breast cancer cell lines. Comparative gene expression profile analysis of the six different ZF-TF-transduced breast cancer cell lines revealed five distinct clusters of differentially expressed genes. One cluster was shared among all 6 ZF-TF-transduced cell lines and therefore constituted a common fulvestrant-resistant gene expression signature. Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents. Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes. Finally, we also demonstrated that the common fulvestrant-resistant signature is associated with poor prognosis by interrogating five independent, publicly available human breast cancer gene expression datasets. Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

Engineering butanol-tolerance with artificial transcription factor libraries

Engineering butanol-tolerance with artificial transcription factor libraries

Engineering butanol‐tolerance in escherichia coli with artificial transcription factor libraries

Escherichia coli has been explored as a host for butanol production because of its many advantages such as a fast growth and easy genetic manipulation. Butanol toxicity, however, is a major concern in the biobutanol production with E. coli. In particular, E. coli growth is severely inhibited by butanol, being almost completely stopped by 1% (vol/vol) butanol. Here we developed a new method to increase the butanol-tolerance of E. coli with artificial transcription factor (ATF) libraries which consist of zinc finger (ZF) DNA-binding proteins and an E. coli cyclic AMP receptor protein (CRP). Using these ATFs, we selected a butanol-tolerant E. coli which can tolerate up to 1.5% (vol/vol) butanol, with a concomitant increase in heat resistance. We also identified genes of E. coli that are associated with the butanol-tolerance. These results show that E. coli can be engineered as a promising host for high-yield butanol production.

Tpl2/AP-1 Enhances Murine Gammaherpesvirus 68 Lytic Replication

How cellular factors regulate gammaherpesvirus lytic replication is not well understood. Here, through functional screening of a cellular kinase expression library, we identified mitogen-activated protein kinase kinase kinase 8 (MAP3K8/Tpl2) as a positive regulator of murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) lytic gene expression and replication. Tpl2 enhances MHV-68 lytic replication by upregulating lytic gene expression and promoter activities of viral lytic genes, including RTA and open reading frame 57 (ORF57). By screening a cellular transcription factor library, we identified the Fos AP-1 transcription factor as a downstream factor that is both necessary and sufficient for mediating the enhancement of MHV-68 lytic replication by Tpl2. In addition, Tpl2 stimulates the promoter activities of key viral lytic genes, including RTA and ORF57, in an AP-1-dependent manner. We identified an AP-1-responsive element on the MHV-68 RTA promoter as the cis element mediating the upregulation of RTA promoter activity by Tpl2. MHV-68 lytic infection upregulates Fos expression, AP-1 activity, and RTA promoter activity in a Tpl2-dependent manner. We constructed a mutant MHV-68 virus that abolished this AP-1-responsive element. This mutant virus exhibited attenuated lytic replication kinetics, indicative of a critical role of this AP-1-responsive element during lytic replication. Moreover, Tpl2 knockdown inhibited the lytic replication of wild-type MHV-68 (MHV-68-WT) but not that of the MHV-68 mutant virus, indicating that endogenous Tpl2 promotes efficient virus lytic replication through AP-1-dependent upregulation of RTA expression. In summary, through tandem functional screens, we identified the Tpl2/AP-1 signaling transduction pathway as a positive regulator of MHV-68 lytic replication.