Bifunctional Immunoactive siRNAs as an Approach to Personalized AML Therapy

Abstract The prognosis of acute myeloid leukemia (AML) is poor due to frequent relapse after initial remission. The development of new approaches to postremission therapy for elimination of minimal residual disease remains a major scientific and clinical challenge. We strive to combine two different innovative therapeutic concepts to develop a new specific and personalized treatment for AML. siRNAs are used to knock down either a gene that drives leukemogenesis due to genetic alterations in specific cases of AML (e.g., FLT3, NPM1) or a gene that is essential for the survival of the leukemic cells (e.g., BRD4, MCL1, PLK1). By adding a triphosphate modification to the 5’ end, the siRNA molecules additionally become ligands for the cytosolic pattern recognition receptor RIG-I (retinoic acid inducible gene I). Its activation mimics viral infection and leads to the production of inflammatory cytokines and induction of apoptosis in the target cell. We expect these bifunctional molecules to result in a decrease of viable AML cells and in the induction of an immune response similar to an active immunization. This concept was successfully tested in vitro for several target genes in AML cell lines. We could demonstrate that the specific gene knockdown leads to inhibited proliferation, increased apoptosis and higher sensitivity to chemotherapeutic agents. Activation of RIG-I by triphosphate-modified RNA additionally stimulated an inflammatory response by the leukemic cells and increased the apoptosis rate. A major hurdle for all siRNA-based anti-cancer strategies is the specific delivery of the RNA into tumor cells. In vivo liposomal transfection of siRNA molecules has been used in various tumor models, but generally results in ineffective and unspecific delivery. We are testing DNA-based nanoparticles coupled with molecules that target receptors specific for or overexpressed on AML cells. By coupling bifunctional siRNA molecules to these nanoparticles, they should be efficiently and selectively transported into the cytosol of AML cells. Proof-of-concept in vivo studies in AML mouse models are in preparation. The long-term goal of this project is the development of a set of bifunctional siRNA molecules for the individualized treatment of AML. Disclosures: No relevant conflicts of interest to declare.

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Adaptation of a Xenograft AML Model to Evaluate Chemotherapeutic Efficacy In Vivo

Blood ◽

10.1182/blood.v116.21.3304.3304 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3304-3304 ◽

Cited By ~ 1

Author(s):

Mark Wunderlich ◽

Fu-Sheng Chou ◽

Mahesh Shrestha ◽

Benjamin Mizukawa ◽

James C. Mulloy

Keyword(s):

Blood Cell ◽

Treatment Period ◽

Conflicts Of Interest ◽

Residual Disease ◽

Synergistic Effects ◽

Day Treatment ◽

Chemotherapeutic Agents ◽

Mouse Strains

Abstract Abstract 3304 Although significant progress has been made in the treatment of leukemia, relapse continues to be a major problem, particularly in acute myeloid leukemia (AML). The prognosis for relapsed leukemia is poor, indicating an area for potential improvements. However, animal models to study the response of human AML to chemotherapeutics and subsequent relapse are lacking. Recently we developed an improved NOD/SCID mouse with IL2RG knockout and transgenic expression of myelo-supportive cytokines SCF, GM-CSF, and IL-3 (the NSGS mouse). This mouse is remarkable in its ability to accept human AML grafts more efficiently than all other available strains. When coupled with in vitro derived AML cells, the NSGS mouse allows for a more predictable AML model with shorter latency and smaller range of death than in other mouse strains, including NSG mice. Importantly, very low numbers of cells reliably generate fatal AML in roughly 40 days, even in non-irradiated NSGS mice, allowing for rapid experimental conclusions and reduced toxicity. With the benefits of these unique tools, we sought to develop a model system to evaluate the efficacy of chemotherapeutic agents on human AML cells in vivo. Engrafted mice received a chemotherapy regimen over a 5-day treatment period consisting of a daily dose of cytarabine with simultaneous injection of doxorubicin during the first three days. Treated mice experienced striking weight loss during the treatment period with a nadir at days 8–10 post-treatment. Mice recovered body weight within 3 weeks. Serial complete blood counts indicated a rapid transient drop in total white blood cell and neutrophil counts and a delayed transient drop in red blood cell and platelet numbers, reminiscent of the effects observed in patients undergoing chemotherapy. The drugs successfully targeted the cells of the bone marrow, as evidenced by a profound loss of cellularity in treated mice relative to controls. When mice harboring N-Ras(G12D) positive AML cells were treated at early time points post-transplant, a significant reduction of tumor burden was observed in the BM and PB, with the grafts of treated mice essentially undetectable for weeks after treatment cessation. Nevertheless, treated mice inevitably succumbed to disease, although with a significantly prolonged latency compared to mock treated mice. However, when AML cells containing the FLT3-ITD mutation were used, a shift in disease latency was not reproducibly seen. This data correlates well with patient data showing that FLT3-ITD mutant AML has a worse prognosis than AML samples with N-Ras mutations. Importantly, the reappearance of AML within weeks of treatment affords the opportunity to model drug resistance and relapse, as well as the potential synergistic effects of experimental compounds used in combination with traditional chemotherapy. Additionally, the period following treatment may allow for studies of minimal residual disease as well as the testing of potential maintenance therapies. Finally, this approach permits a detailed analysis of the critical few cancer stem cells that remain after induction therapy with the goal of identifying novel compounds capable of targeting these cells. Disclosures: No relevant conflicts of interest to declare.

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Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Blood ◽

10.1182/blood.v120.21.1882.1882 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1882-1882 ◽

Cited By ~ 1

Author(s):

Charlotte Victoria Cox ◽

Paraskevi Diamanti ◽

Allison Blair

Keyword(s):

Functional Capacity ◽

Conflicts Of Interest ◽

Residual Disease ◽

Survival Rates ◽

In Vivo Studies ◽

Nsg Mice ◽

Early Treatment Response ◽

Current Therapies

Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study

Nanomaterials ◽

10.3390/nano8121061 ◽

2018 ◽

Vol 8 (12) ◽

pp. 1061 ◽

Cited By ~ 7

Author(s):

María Martínez-Negro ◽

Laura Blanco-Fernández ◽

Paolo Tentori ◽

Lourdes Pérez ◽

Aurora Pinazo ◽

...

Keyword(s):

Biochemical Study ◽

Cationic Lipid ◽

Green Fluorescence Protein ◽

In Vivo Studies ◽

Interleukin 12 ◽

Specific Gene ◽

Gene Therapies ◽

Biological Studies

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120–290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

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Abstract W P93: MiR-122 Improves Stroke Outcomes after Middle Cerebral Artery Occlusion in Rats

Stroke ◽

10.1161/str.46.suppl_1.wp93 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

DaZhi Liu ◽

Glen C Jickling ◽

Bradley P Ander ◽

Heather Hull ◽

Xinhua Zhan ◽

...

Keyword(s):

Middle Cerebral Artery ◽

Cerebral Artery ◽

Messenger Rna ◽

Target Genes ◽

Conflicts Of Interest ◽

Neurological Impairment ◽

Artery Occlusion ◽

Neuroprotective Effects ◽

Genomic Studies

MicroRNA (miRNA) are recently discovered small (~22 nucleotides), non-coding RNA that regulate translation of messenger RNA (mRNA) to protein. Though there are only hundreds of miRNAs, each of them can potentially regulate hundreds of target genes, via base-pairing with complementary sequences in mRNA. This provides one approach that targets a single miRNA to have effects on multiple genes. Our previous genomic studies have demonstrated that miR-122 decreased significantly in blood of experimental strokes produced by middle cerebral artery (MCA) occlusion in rats as well as in blood of patients with ischemic strokes. Therefore, we hypothesized that elevating blood miR-122 has the potential for treating stroke. Using the newly developed in vivo polyethylene glycol-liposome based miRNA transfection system and rat suture MCAO occlusion model, we show that injection of chemically modified mimic miR-122 (600ug/rat, i.v.) through tail vein immediately after MCAO occlusion significantly decreases the neurological impairment and significantly attenuates brain infarct volumes. Ongoing studies are identifying the target genes that are associated with the neuroprotective effects of miR-122 following stroke. Acknowledgements: This study was supported by NIH grant R01NS066845 (FRS). There were no conflicts of interest.

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Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development

Development ◽

10.1242/dev.128.18.3405 ◽

2001 ◽

Vol 128 (18) ◽

pp. 3405-3413 ◽

Cited By ~ 4

Author(s):

Adi Inbal ◽

Naomi Halachmi ◽

Charna Dibner ◽

Dale Frank ◽

Adi Salzberg

Keyword(s):

Transcriptional Activity ◽

Target Genes ◽

Genetic Evidence ◽

In Vivo Studies ◽

Loss Of Function ◽

Native Form ◽

Downstream Target ◽

Activating Function

Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.

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One Hundred Faces of Geraniol

Molecules ◽

10.3390/molecules25143303 ◽

2020 ◽

Vol 25 (14) ◽

pp. 3303 ◽

Cited By ~ 2

Author(s):

Wanda Mączka ◽

Katarzyna Wińska ◽

Małgorzata Grabarczyk

Keyword(s):

Antibacterial Activity ◽

Biological Activities ◽

Chemotherapeutic Agents ◽

In Vivo Studies ◽

Respiratory Pathogens ◽

Important Ingredient ◽

Household Products ◽

Fragrance Compound

Geraniol is a monoterpenic alcohol with a pleasant rose-like aroma, known as an important ingredient in many essential oils, and is used commercially as a fragrance compound in cosmetic and household products. However, geraniol has a number of biological activities, such as antioxidant and anti-inflammatory properties. In addition, numerous in vitro and in vivo studies have shown the activity of geraniol against prostate, bowel, liver, kidney and skin cancer. It can induce apoptosis and increase the expression of proapoptotic proteins. The synergy of this with other drugs may further increase the range of chemotherapeutic agents. The antibacterial activity of this compound was also observed on respiratory pathogens, skin and food-derived strains. This review discusses some of the most important uses of geraniol.

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PPAR, PTEN, and the Fight against Cancer

PPAR Research ◽

10.1155/2008/932632 ◽

2008 ◽

Vol 2008 ◽

pp. 1-6 ◽

Cited By ~ 18

Author(s):

Rosemary E. Teresi ◽

Kristin A. Waite

Keyword(s):

Tumor Suppressor ◽

Hormone Receptors ◽

Susceptibility Gene ◽

Genetic Alterations ◽

Cellular Growth ◽

In Vivo Studies ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor

Peroxisome proliferator-activated receptor gamma (PPAR) is a ligand-activated transcription factor, which belongs to the family of nuclear hormone receptors. Recent in vitro studies have shown that PPAR can regulate the transcription ofphosphatase and tensin homolog on chromosometen(PTEN), a known tumor suppressor.PTENis a susceptibility gene for a number of disorders, including breast and thyroid cancer. Activation of PPAR through agonists increases functional PTEN protein levels that subsequently induces apoptosis and inhibits cellular growth, which suggests that PPAR may be a tumor suppressor. Indeed, several in vivo studies have demonstrated that genetic alterations of PPAR can promote tumor progression. These results are supported by observations of the beneficial effects of PPAR agonists in the in vivo cancer setting. These studies signify the importance of PPAR andPTEN's interaction in cancer prevention.

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Maximal killing of lymphoma cells by DNA damage–inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim

Blood ◽

10.1182/blood-2010-04-280818 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5256-5267 ◽

Cited By ~ 65

Author(s):

Lina Happo ◽

Mark S. Cragg ◽

Belinda Phipson ◽

Jon M. Haga ◽

Elisa S. Jansen ◽

...

Keyword(s):

Dna Damage ◽

Drug Resistance ◽

Target Genes ◽

B Cell Lymphoma ◽

Chemotherapeutic Agents ◽

Lymphoid Cells ◽

Lymphoma Cells ◽

P53 Target Genes

Abstract DNA-damaging chemotherapy is the backbone of cancer treatment, although it is not clear how such treatments kill tumor cells. In nontransformed lymphoid cells, the combined loss of 2 proapoptotic p53 target genes, Puma and Noxa, induces as much resistance to DNA damage as loss of p53 itself. In Eμ-Myc lymphomas, however, lack of both Puma and Noxa resulted in no greater drug resistance than lack of Puma alone. A third B-cell lymphoma-2 homology domain (BH)3-only gene, Bim, although not a direct p53 target, was up-regulated in Eμ-Myc lymphomas incurring DNA damage, and knockdown of Bim levels markedly increased the drug resistance of Eμ-Myc/Puma−/−Noxa−/− lymphomas both in vitro and in vivo. Remarkably, c-MYC–driven lymphoma cell lines from Noxa−/−Puma−/−Bim−/− mice were as resistant as those lacking p53. Thus, the combinatorial action of Puma, Noxa, and Bim is critical for optimal apoptotic responses of lymphoma cells to 2 commonly used DNA-damaging chemotherapeutic agents, identifying Bim as an additional biomarker for treatment outcome in the clinic.

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Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819992116 ◽

2019 ◽

pp. 201819992 ◽

Cited By ~ 5

Author(s):

Florence Borot ◽

Hui Wang ◽

Yan Ma ◽

Toghrul Jafarov ◽

Azra Raza ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Residual Disease ◽

Immune Therapy ◽

Leukemic Cells ◽

Sequencing Analysis ◽

Hematopoietic Stem ◽

Genetic Ablation ◽

Targeted Immunotherapy

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn’t impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

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The TEA Transcription Factor Tec1 Confers Promoter-Specific Gene Regulation by Ste12-Dependent and -Independent Mechanisms

Eukaryotic Cell ◽

10.1128/ec.00251-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 514-531 ◽

Cited By ~ 20

Author(s):

Barbara Heise ◽

Julia van der Felden ◽

Sandra Kern ◽

Mario Malcher ◽

Stefan Brückner ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcriptional Activation ◽

Binding Sites ◽

Target Genes ◽

Specific Gene ◽

Dependent Manner ◽

A Genome ◽

Regulated Gene Expression

ABSTRACT In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the FLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.

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