Multiple ETS Factors Participate in the Transcriptional Control of TERT Mutant Promoter in Thyroid Cancers

Hotspot mutations in the TERT (telomerase reverse transcriptase) gene are key determinants of thyroid cancer progression. TERT promoter mutations (TPM) create de novo consensus binding sites for the ETS (“E26 transformation specific”) family of transcription factors. In this study, we systematically knocked down each of the 20 ETS factors expressed in thyroid tumors and screened their effects on TERT expression in seven thyroid cancer cell lines with defined TPM status. We observed that, unlike in other TPM-carrying cancers such as glioblastomas, ETS factor GABPA does not unambiguously regulate transcription from the TERT mutant promoter in thyroid specimens. In fact, multiple members of the ETS family impact TERT expression, and they typically do so in a mutation-independent manner. In addition, we observe that partial inhibition of MAPK, a central pathway in thyroid cancer transformation, is more effective at suppressing TERT transcription in the absence of TPMs. Taken together, our results show a more complex scenario of TERT regulation in thyroid cancers compared with other lineages and suggest that compensatory mechanisms by ETS and other regulators likely exist and advocate for the need for a more comprehensive understanding of the mechanisms of TERT deregulation in thyroid tumors before eventually exploring TPM-specific therapeutic strategies.

Multiple ETS Factors Participate in the Transcriptional Control of TERT Mutant Promoter in Thyroid Cancers

Hotspot mutations in the TERT (telomerase reverse transcriptase) gene are key determinants of thyroid cancer progression. TERT promoter mutations (TPM) create de novo consensus binding sites for the ETS (“E26 transforming sequence”) family of transcription factors. In this study, we systematically knocked down each of the 20 ETS factors expressed in thyroid tumors and screened their effects on TERT expression in seven thyroid cancer cell lines with defined TPM status. We observed that, unlike in other TPM-carrying cancers such as glioblastomas, ETS factor GABPA does not unambiguously regulate transcription from the TERT mutant promoter in thyroid specimens. In fact, multiple members of the ETS family impact TERT expression, and they typically do so in a mutation-independent manner. In addition, we observe that partial inhibition of MAPK, a central pathway in thyroid cancer transformation, is more effective at suppressing TERT transcription in the absence of TPMs. Taken together, our results show a more complex scenario of TERT regulation in thyroid cancers compared to other lineages, suggest that compensatory mechanisms by ETS and other regulators likely exist and advocate for the need of a more comprehensive understanding of the mechanisms of TERT deregulation in thyroid tumors before eventually exploring TPM-specific therapeutic strategies.Graphical Abstract

Model-driven promoter strength prediction based on a fine-tuned synthetic promoter library in Escherichia coli

Promoters are one of the most critical regulatory elements controlling metabolic pathways. However, in recent years, researchers have simply perfected promoter strength, but ignored the relationship between the internal sequences and promoter strength. In this context, we constructed and characterized a mutant promoter library of Ptrc through dozens of mutation-construction-screening-characterization engineering cycles. After excluding invalid mutation sites, we established a synthetic promoter library, which consisted of 3665 different variants, displaying an intensity range of more than two orders of magnitude. The strongest variant was 1.52-fold stronger than a 1 mM isopropyl-β-D-thiogalactoside driven PT7 promoter. Our synthetic promoter library exhibited superior applicability when expressing different reporters, in both plasmids and the genome. Different machine learning models were built and optimized to explore relationships between the promoter sequences and transcriptional strength. Finally, our XgBoost model exhibited optimal performance, and we utilized this approach to precisely predict the strength of artificially designed promoter sequences. Our work provides a powerful platform that enables the predictable tuning of promoters to achieve the optimal transcriptional strength.

TERTpromoter mutation determines apoptotic and therapeutic responses ofBRAF-mutant cancers to BRAF and MEK inhibitors: Achilles Heel

Combination use of BRAF V600E inhibitor dabrafenib and MEK inhibitor trametinib has become a standard treatment for human cancers harboring BRAF V600E. Its anticancer efficacies vary, however, with dramatic efficacy in some patients and drug resistance/tumor recurrence in others, which is poorly understood. Using thyroid cancer, melanoma, and colon cancer cell models, we showed that dabrafenib and trametinib induced robust apoptosis of cancer cells harboring both BRAF V600E andTERT promoter mutations but had little proapoptotic effect in cells harboring only BRAF V600E. Correspondingly, the inhibitors nearly completely abolished the growth of in vivo tumors harboring both mutations but had little effect on tumors harboring only BRAF V600E. Upon drug withdrawal, tumors harboring both mutations remained hardly measurable but tumors harboring only BRAF V600E regrew rapidly. BRAF V600E/MAP kinase pathway is known to robustly activate mutant promoter of TERT, a strong apoptosis suppressor. Thus, for survival, cancer cells harboring both mutations may have evolved to rely on BRAF V600E-promoted and high-TERT expression-mediated suppression of apoptosis. As such, inhibition of BRAF/MEK can trigger strong apoptosis-induced cell death and hence tumor abolishment. This does not happen in cells harboring only BRAF V600E as they have not developed reliance on TERT-mediated suppression of apoptosis due to the lack of mutant promoter-driven high-TERT expression.TERTpromoter mutation governs BRAF-mutant cancer cells’ apoptotic and hence therapeutic responses to BRAF/MEK inhibitors. Thus, the genetic duet ofBRAFV600E andTERT promoter mutation represents an Achilles Heel for effective therapeutic targeting and response prediction in cancer.

Identification and characterization of sequence signatures in the Bacillus subtilis promoter Pylb for tuning promoter strength

Objective To thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity. Result The sequences flanking the − 35 and − 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the − 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA). Conclusion Our work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.

Interallelic interaction and gene regulation in budding yeast

In Drosophila, homologous chromosome pairing leads to “transvection,” in which the enhancer of a gene can regulate the allelic transcription in trans. Interallelic interactions were also observed in vegetative diploid budding yeast, but their functional significance is unknown. Here, we show that a GAL1 reporter can interact with its homologous allele and affect its expression. By ectopically inserting two allelic reporters, one driven by wild-type GAL1 promoter (WT GAL1pr) and the other by a mutant promoter with delayed response to galactose induction, we found that the two reporters physically associate, and the WT GAL1pr triggers synchronized firing of the defective promoter and accelerates its activation without affecting its steady-state expression level. This interaction and the transregulatory effect disappear when the same reporters are located at nonallelic sites. We further demonstrated that the activator Gal4 is essential for the interallelic interaction, and the transregulation requires fully activated WT GAL1pr transcription. The mechanism of this phenomenon was further discussed. Taken together, our data revealed the existence of interallelic gene regulation in yeast, which serves as a starting point for understanding long-distance gene regulation in this genetically tractable system.

Development of a novel T-vector supporting efficient green-white screening

T-vectors play an important role in cloning of polymerase chain reaction products. In the present study, a novel pUEG-T vector was developed using enhanced green fluorescent protein (EGFP) as an indicator. To improve EGFP-based green-white screening, the lipoprotein mutant promoter, a strong constitutive promoter, was utilized to control the expression of egfp gene. Two other efficient expression elements, the ColE1 replication origin of pUC18 and the expression cassette of pET-28a, were also integrated into pUEG-T vector. Expression analysis demonstrated the efficient accumulation of active EGFPs in Escherichia coli DH5α cells carrying the T-vector precursor pUEG. In T-A cloning using pUEG-T vector, white colonies containing foreign DNA and green colonies having no insertion could be handily distinguished under normal white light, without any chemical inducer or chromogenic substrate. Furthermore, no false positive was observed in any of the tested white colonies. This proves that pUEG-T is an inexpensive, convenient and efficient T-vector.

Human Telomere Disease Due to Disruption of the CCAAT Box of the TERC Promoter

Abstract 2405 Telomeres, the structures capping the ends of linear chromosomes, consist of hundreds to thousands of TTAGGG repeats. To prevent critical telomere shortening, highly proliferative cells express telomerase (encoded by TERT), a reverse transcriptase that adds TTAGGG repeats to telomeres, using TERC as its RNA template. Mutations in TERT (resulting in amino acid changes and reduced enzymatic activity), in TERC (resulting in changes in RNA structure and impaired binding to telomerase), or in other components of the telomerase complex lead to accelerated telomere attrition. Clinically, the telomeropathies can manifest as the bone marrow (BM) failure syndromes dyskeratosis congenita and aplastic anemia; acute myeloid leukemia; hepatic nodular regenerative hyperplasia and cirrhosis; and familial pulmonary fibrosis. Sequencing strategies for the diagnosis of telomere disease are based on screening exons and their flanking regions of telomerase genes for mutations. Clearly pathogenic mutations in the promoter region of TERT or TERC have not been described. Here, we report a mutation in the CCAAT box of the TERC promoter region in a severely affected pedigree, leading to telomere disease. The index patient, a 39-year old Caucasian female with a family history of BM failure and leukemia, was diagnosed with aplastic anemia. Her peripheral blood telomere length was extremely short in comparison to age-matched healthy controls by quantitative PCR (patient, 5.4 kb; expected for age, 8.2 kb; 2.9 standard deviations below the age-matched average). Telomere lengths of her sister and her affected nephew also were very short, while an unaffected nephew had normal telomere length. No mutations were found in TERT, the coding region of TERC, or exon 6a of TINF2. However, the proband, her sister, her affected nephew, but not the unaffected nephew, carried a heterozygous −58 C>G in the CCAAT box (position −58 to −54) of the TERC promoter region. The mutation was not present in 378 healthy subjects. Gel shift and supershift assays with HeLa nuclear extract and anti-NF-YA antibody displayed binding and supershift for the wild-type but not for the −58C>G mutant biotinylated probe. Reporter gene experiments in HEK293T cells transfected with wild-type or mutant promoter showed a 9-fold decrease in luciferase activity in the mutant promoter as compared to the wild-type. These results indicate that the CCAAT-box disruption leads to reduced TERC levels, lower telomerase activity, and ultimately short telomeres, as observed in the present pedigree. The CCAAT box is an important and highly conserved promoter element that is frequently present in promoter regions of RNA polymerase II transcribed genes. In vitro, the CCAAT box in the TERC promoter has been shown to be critical for maintaining basal transcription of TERC by binding NF-Y. In vivo, mutations have been described in the CCAAT boxes of the Aγ and Gγ gene promoters, leading to hereditary persistence of fetal haemoglobin, a benign condition that benefits sickle cell anemia and β-thalassemia patients. We here provide the first instance of a mutation in a CCAAT box etiologic in human disease and the first example of a TERC promoter mutation producing a telomeropathy. Pathogenic mutations in telomerase genes may be located in promoter and likely also more distant regulatory sequences. Disclosures: No relevant conflicts of interest to declare.

Functional characterization of a 13-bp deletion (c.-1522_-1510del13) in the promoter of the von Willebrand factor gene in type 1 von Willebrand disease

We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5′ of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.

The Sigma Factor AlgU Plays a Key Role in Formation of Robust Biofilms by Nonmucoid Pseudomonas aeruginosa

ABSTRACT The extracytoplasmic function sigma factor AlgU of Pseudomonas aeruginosa is responsible for alginate overproduction, leading to mucoidy and chronic infections of cystic fibrosis patients. We investigated here the role of AlgU in the formation of nonmucoid biofilms. The algU mutant of P. aeruginosa PAO1 (PAOU) showed a dramatic impairment in biofilm formation under dynamic conditions. PAOU was defective both in cell attachment to glass and in development of robust, shear-resistant biofilms. This was explained by an impaired production of extracellular matrix, specifically of the exopolysaccharide Psl, as revealed by microscopy and enzyme-linked immunosorbent assay. Complementing the algU mutation with a plasmid-borne algU gene restored wild-type phenotypes. Compared with that in PAO1, expression of the psl operon was reduced in the PAOU strain, and the biofilm formation ability of this strain was partially restored by inducing the transcription of the psl operon. Furthermore, expression of the lectin-encoding lecA and lecB genes was reduced in the PAOU strain. In agreement with the requirement of LecB for type IV pilus biogenesis, PAOU displayed impaired twitching motility. Collectively, these genetic downregulation events explain the biofilm formation defect of the PAOU mutant. Promoter mapping indicated that AlgU is probably not directly responsible for transcription of the psl operon and the lec genes, but AlgU is involved in the expression of the ppyR gene, whose product was reported to positively control psl expression. Expressing the ppyR gene in PAOU partially restored the formation of robust biofilms.