A single helix repression domain is functional across diverse eukaryotes.

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL-and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1)-can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae. Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across many eukaryotes.

Groucho co-repressor proteins regulate ß cell development and proliferation by repressing Foxa1 in the developing mouse pancreas.

Groucho-related genes (GRGs) are transcriptional co-repressors that are crucial for many developmental processes. Several essential pancreatic transcription factors are capable of interacting with GRGs; however, the in vivo role of GRG-mediated transcriptional repression in pancreas development is still not well understood. In this study, we used complex mouse genetics and transcriptomic analyses to determine that GRG3 is essential for ß cell development, and in the absence of Grg3 there is compensatory upregulation of Grg4Grg3/4 double mutant mice have severe dysregulation of the pancreas gene program with ectopic expression of canonical liver genes and Foxa1, a master regulator of the liver program. Neurod1, an essential ß cell transcription factor and predicted target of Foxa1, becomes downregulated in Grg3/4 mutants, resulting in reduced ß cell proliferation, hyperglycemia, and early lethality. These findings uncover novel functions of GRG-mediated repression during pancreas development.

OsbHLH061 links TOPLESS/TOPLESS-RELATED repressor proteins with POSITIVE REGULATOR OF IRON HOMEOSTASIS 1 to maintain iron homeostasis in rice.

As excess iron (Fe) is toxic, uptake of this essential micronutrient must be tightly controlled. Previous studies have shown that Oryza sativa (rice) POSITIVE REGULATOR OF IRON HOMEOSTASIS1 (OsPRI1) acts upstream of the iron-related transcription factor 2 (OsIRO2) and OsIRO3 to positively regulate root-to-shoot Fe translocation. However, as expression of OsPRI1 is constitutive it is unclear how the Fe-deficiency response is turned off to prevent toxicity when Fe is sufficient. The bHLH transcription factor OsbHLH061 interacts with OsPRI1, and this study used molecular, genetics, biochemical and physiological approaches to functionally characterise OsbHLH061 and how it affects Fe homeostasis. OsbHLH061 knockout or overexpression lines increase or decrease Fe accumulation in shoots respectively. Mechanistically, OsbHLH061 expression is upregulated by high Fe, and physically interacts with OsPRI1, the OsbHLH061-OsPRI1 complex recruits TOPLESS/TOPLESS-RELATED (OsTPL/TPR) co-repressors to repress OsIRO2 and OsIRO3 expression. The OsbHLH061 ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif is required for this transcriptional repression activity. These results define a functional OsTPL/TPR-OsbHLH061-OsPRI1-OsIRO2/3 module that negatively controls long-distance transport of Fe in plants for adaptation to changing Fe environments and maintain Fe homeostasis in rice.

Nuclear Functions of KaeA, a Subunit of the KEOPS Complex in Aspergillus nidulans.

Kae1 is a subunit of the highly evolutionarily conserved KEOPS/EKC complex, which is involved in universal (t6A37) tRNA modification. Several reports have discussed the participation of this complex in transcription regulation in yeast and human cells, including our previous observations of KaeA, an Aspergillus nidulans homologue of Kae1p. The aim of this project was to confirm the role of KaeA in transcription, employing high-throughput transcriptomic (RNA-Seq and ChIP-Seq) and proteomic (LC-MS) analysis. We confirmed that KaeA is a subunit of the KEOPS complex in A. nidulans. An analysis of kaeA19 and kaeA25 mutants showed that, although the (t6A37) tRNA modification is unaffected in both mutants, they reveal significantly altered transcriptomes compared to the wild type. The finding that KaeA is localized in chromatin and identifying its protein partners allows us to postulate an additional nuclear function for the protein. Our data shed light on the universal bi-functional role of this factor and proves that the activity of this protein is not limited to tRNA modification in cytoplasm, but also affects the transcriptional activity of a number of nuclear genes. Data are available via the NCBI's GEO database under identifiers GSE206830 (RNA-Seq) and GSE206874 (ChIP-Seq), and via ProteomeXchange with identifier PXD034554 (proteomic).

The transcriptional corepressor CBFA2T3 inhibits all-trans-retinoic acid-induced myeloid gene expression and differentiation in acute myeloid leukemia.

CBFA2/RUNX1 partner transcriptional co-repressor 3 (CBFA2T3, also known as MTG16 or ETO2) is a myeloid translocation gene family protein that functions as a master transcriptional corepressor in hematopoiesis. Recently, it has been shown that CBFA2T3 maintains leukemia stem cell gene expression and promotes relapse in acute myeloid leukemia (AML). However, a role for CBFA2T3 in myeloid differentiation of AML has not been reported. Here, we show that CBFA2T3 represses retinoic acid receptor (RAR) target gene expression and inhibits all-trans-retinoic acid (ATRA)-induced myeloid differentiation of AML cells. ChIP-Seq revealed that CBFA2T3 targets the RARα/RXRα cistrome in U937 AML cells, predominantly at myeloid-specific enhancers associated with terminal differentiation. CRISPR/Cas9-mediated abrogation of CBFA2T3 resulted in spontaneous and ATRA-induced activation of myeloid-specific genes in a manner correlated with myeloid differentiation. Importantly, these effects were reversed by CBFA2T3 re-expression. Mechanistic studies showed that CBFA2T3 inhibits RAR target gene transcription by acting at an early step to regulate histone acetyltransferase recruitment, histone acetylation, and chromatin accessibility at RARα target sites, independently of the downstream, RAR-mediated steps of transcription. Finally, we validated the inhibitory effect of CBFA2T3 on RAR in multiple AML subtypes and patient samples. To our knowledge, this is the first study to show that CBFA2T3 down-regulation is both necessary and sufficient for enhancing ATRA-induced myeloid gene expression and differentiation of AML cells. Our findings suggest that CBFA2T3 can serve as a potential target for enhancing AML responsiveness to ATRA differentiation therapies.

SCARECROW-LIKE3 regulates the transcription of gibberellin-related genes by acting as a transcriptional co-repressor of GAI-ASSOCIATED FACTOR1.

KEY MESSAGE: SCL3 inhibits transcriptional activity of IDD-DELLA complex by acting as a co-repressor and repression activity is enhanced in the presence of GAF1 in a TOPLESS-independent manner. GRAS [GIBBERELLIN-INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA) and SCARECROW (SCR)] proteins are a family of plant-specific transcriptional regulators that play diverse roles in development and signaling. GRAS family DELLA proteins act as growth repressors by inhibiting gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also act as co-activators of transcription factor GAI-ASSOCIATED FACTOR1 (GAF1)/INDETERMINATE DOMAIN2 (IDD2), the GAF1-DELLA complex activating transcription of GAF1 target genes. GAF1 also interacts with TOPLESS (TPL), a transcriptional co-repressor, in the absence of DELLA, the GAF1-TPL complex repressing transcription of the target genes. SCARECROW-LIKE3 (SCL3), another member of the GRAS family, is thought to inhibit transcriptional activity of the IDD-DELLA complex through competitive interaction with IDD. Here, we also revealed that SCL3 inhibits transcriptional activation by the GAF1-DELLA complex via repression activity rather than via competitive inhibition of the GAF1-DELLA interaction. Moreover, the repression activity of SCL3 was enhanced by GAF1 in a TPL-independent manner. While the GRAS domain of DELLA has transcriptional activation activity, that of SCL3 has repression activity. SCL3 also inhibited transcriptional activity of GAF1-RGA fusion proteins. Results from the co-immunoprecipitation assays and the yeast three-hybrid assay suggested the possibility that SCL3 forms a ternary complex with GAF1 and DELLA. These findings provide important information on DELLA-regulated GA signaling and new insight into the transcriptional repression mechanism.

Discovery of LAMP-2A as potential biomarkers for glioblastoma development by modulating apoptosis through N-CoR degradation.

BACKGROUND: Lysosome-associated membrane protein type 2A (LAMP-2A) is the key component of chaperone-mediated autophagy (CMA), a cargo-selective lysosomal degradation pathway. Aberrant LAMP-2A expression and CMA activation have been demonstrated in various human malignancies. The study focusing on the intrinsic role of LAMP-2A and CMA in glioblastoma (GBM), and downstream mechanism could provide valuable insight into the pathogenesis and novel therapeutic modality of GBM. METHODS: The levels of LAMP-2A, nuclear receptor co-repressor (N-CoR), unfolded protein response (UPR) and apoptosis were examined in clinical samples. LAMP-2A siRNA and shRNA were constructed to manipulate CMA activation. The role of CMA and downstream mechanism through degradation of N-CoR and arresting UPR mediated apoptosis were explored in GBM cells and nude mouse xenograft model. RESULTS: Elevated LAMP-2A and associated decreased N-CoR expression were observed in GBM as compared with peritumoral region and low-grade glioma. Inhibited UPR and apoptosis were observed in GBM with high LAMP-2A expression. In vitro study demonstrated co-localization and interaction between LAMP-2A and N-CoR. LAMP-2A silencing up-regulated N-CoR and aroused UPR pathway, leading to apoptosis, while N-CoR silencing led to an opposite result. In vivo study further confirmed that LAMP-2A inhibition arrested tumor growth by promoting apoptosis. CONCLUSIONS: Our results demonstrated the central role of CMA in mediating N-CoR degradation and protecting GBM cells against UPR and apoptosis, and provided evidence of LAMP-2A as potential biomarker. Further research focusing on CMA with other tumorigenic process is needed and selective modulators of LAMP-2A remain to be investigated to provide a novel therapeutic strategy for GBM. Video Abstract.

Transcriptional regulators involved in responses to volatile organic compounds in plants.

Field studies have shown that plants growing next to herbivore-infested plants acquire higher resistance to herbivore damage. This increased resistance is partly due to regulation of plant gene expression by volatile organic compounds (VOCs) released by plants that sense environmental challenges such as herbivores. The molecular basis for VOC sensing in plants, however, is poorly understood. Here, we report the identification of TOPLESS-like proteins (TPLs) that have VOC-binding activity and are involved in VOC sensing in tobacco. While screening for volatiles that induce stress-responsive gene expression in tobacco BY-2 cells and tobacco plants, we found that some sesquiterpenes induce the expression of stress-responsive genes. These results provided evidence that plants sense these VOCs and motivated us to analyze the mechanisms underlying volatile sensing using tobacco as a model system. Using a pulldown assay with caryophyllene derivative-linked beads, we identified TPLs as transcriptional co-repressors that bind volatile caryophyllene analogs. Overexpression of TPLs in cultured BY-2 cells or tobacco leaves reduced caryophyllene-induced gene expression, indicating that TPLs are involved in the responses to caryophyllene analogs in tobacco. We propose that unlike animals, which use membrane receptors for sensing odorants, a transcriptional co-repressor plays a role in sensing and mediating VOC signals in plant cells.

AGL15 Controls the Embryogenic Reprogramming of Somatic Cells in Arabidopsis through the Histone Acetylation-Mediated Repression of the miRNA Biogenesis Genes.

The embryogenic transition of somatic cells requires an extensive reprogramming of the cell transcriptome. Relevantly, the extensive modulation of the genes that have a regulatory function, in particular the genes encoding the transcription factors (TFs) and miRNAs, have been indicated as controlling somatic embryogenesis (SE) that is induced in vitro in the somatic cells of plants. Identifying the regulatory relationships between the TFs and miRNAs during SE induction is of central importance for understanding the complex regulatory interplay that fine-tunes a cell transcriptome during the embryogenic transition. Hence, here, we analysed the regulatory relationships between AGL15 (AGAMOUS-LIKE 15) TF and miR156 in an embryogenic culture of Arabidopsis. Both AGL15 and miR156 control SE induction and AGL15 has been reported to target the MIR156 genes in planta. The results showed that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of the MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1 (DICER-LIKE1), SERRATE and HEN1 (HUA-ENHANCER1). To repress the miRNA biogenesis genes AGL15 seems to co-operate with the TOPLESS co-repressors (TPL and TPR1-4), which are components of the SIN3/HDAC silencing complex. The impact of TSA (trichostatin A), an inhibitor of the HDAC histone deacetylases, on the expression of the miRNA biogenesis genes together with the ChIP results implies that histone deacetylation is involved in the AGL15-mediated repression of miRNA processing. The results indicate that HDAC6 and HDAC19 histone deacetylases might co-operate with AGL15 in silencing the complex that controls the abundance of miR156 during embryogenic induction. This study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1.

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes. The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.

Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex.

Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells.

Phenotypic variation reveals sites of evolutionary constraint in the androgenic signaling pathway.

Steroid hormone systems play an important role in shaping the evolution of vertebrate sexual traits, but several aspects of this relationship remain unclear. For example, we currently know little about how steroid signaling complexes are adapted to accommodate the emergence of behavior in response to sexual selection. We use downy woodpeckers (Dryobates pubescens) to evaluate how the machinery underlying androgen action can evolve to accommodate this bird's main territorial signal, the drum. We focus specifically on modifications to androgenic mechanisms in the primary neck muscle that actuates the hammering movements underlying this signal. Of the signaling components we examine, we find that levels of circulating testosterone (T) and androgen receptor (AR) expression are consistently increased in a way that likely enhances androgenic regulation of drumming. By contrast, the expression of nuclear receptor co-factors-the 'molecular rheostats' of steroid action-show no such relationship in our analyses. If anything, co-factors are expressed in directions that would presumably hinder androgenic regulation of the drum. These findings therefore collectively point to T levels and AR as the more evolutionarily labile components of the androgenic system, in that they are likely more apt to change over time to support sexual selection for territorial signaling in woodpeckers. Yet the signaling elements that fine-tune AR's functional effects on the genome-namely the receptor's transcriptional co-factors-do not change in such a manner, and thus may be under tighter evolutionary constraint.

Transducin ß-like 1, X-linked and nuclear receptor co-repressor cooperatively augment the ligand-independent stimulation of TRH and TSHß gene promoters by thyroid hormone receptors.

Mutations in TBL1X, a component of the nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptor co-repressor complexes, have recently been implicated in isolated central hypothyroidism (CeH). However, the mechanisms by which TBL1X mutations affect negative feedback regulation in the hypothalamus-pituitary-thyroid axis remain unclear. N-CoR was previously reported to paradoxically enhance the ligand-independent stimulation of TRH and TSHß gene promoters by thyroid hormone receptors (TR) in cell culture systems. We herein investigated whether TBL1X affects the unliganded TR-mediated stimulation of the promoter activities of genes negatively regulated by T3 in cooperation with N-CoR. In a hypothalamic neuronal cell line, the unliganded TR-mediated stimulation of the TRH gene promoter was significantly enhanced by co-transfected TBL1X, and the co-transfection of TBL1X with N-CoR further enhanced promoter activity. In contrast, the knockdown of endogenous Tbl1x using short interfering RNA significantly attenuated the N-CoR-mediated enhancement of promoter activity in the presence of unliganded TR. The co-transfection of N365Y or Y458C, TBL1X mutants identified in CeH patients, showed impaired co-activation with N-CoR for the ligand-independent stimulation of the TRH promoter by TR. In the absence of T3, similar or impaired enhancement of the TSHß gene promoter by the wild type or TBL1X mutants, respectively, was observed in the presence of co-transfected TR and N-CoR in CV-1 cells. These results suggest that TBL1X is needed for the full activation of TRH and TSHß gene promoters by unliganded TR. Mutations in TBL1X may cause CeH due to the impaired up-regulation of TRH and/or TSHß gene transcription despite low T3 levels.

The complexity of TRIM28 contribution to cancer.

Since the first discovery in 1996, the engagement of TRIM28 in distinct aspects of cellular biology has been extensively studied resulting in identification of a complex nature of TRIM28 protein. In this review, we summarize core biological functions of TRIM28 that emerge from TRIM28 multi-domain structure and possessed enzymatic activities. Moreover, we will discuss whether the complexity of TRIM28 engagement in cancer biology makes TRIM28 a possible candidate for targeted anti-cancer therapy. Briefly, we will demonstrate the role of TRIM28 in regulation of target gene transcription, response to DNA damage, downregulation of p53 activity, stimulation of epithelial-to-mesenchymal transition, stemness sustainability, induction of autophagy and regulation of retrotransposition, to provide the answer whether TRIM28 functions as a stimulator or inhibitor of tumorigenesis. To date, number of studies demonstrate significant upregulation of TRIM28 expression in cancer tissues which correlates with worse overall patient survival, suggesting that TRIM28 supports cancer progression. Here, we present distinct aspects of TRIM28 involvement in regulation of cancer cell homeostasis which collectively imply pro-tumorigenic character of TRIM28. Thorough analyses are further needed to verify whether TRIM28 possess the potential to become a new anti-cancer target.

Oculo-facio-cardio-dental syndrome with craniosynostosis, temporal hypertrichosis, and deafness.

We report the case of a 7-month-old girl with atypical oculo-facio-cardio-dental syndrome (OFCD). A novel de novo pathogenic mutation in the BCL6 interacting co-repressor gene (BCOR) (c.4540C>T; p.Arg1514*), was identified on the X chromosome. This case expands the phenotype of OFCD as it is the first report of a case presenting with craniosynostois, temporal hypertrichosis, supraorbital grooving, and underdevelopment of the midface.

Nuclear co-repressor (NCoR) is required to maintain insulin sensitivity in C2 C12 myotubes.

Nuclear co-repressor (NCoR) regulates peripheral insulin sensitivity; however, its role in modulating insulin sensitivity in skeletal muscle remains elusive. Present study investigated protein expression and effect of NCoR on insulin sensitivity in murine skeletal muscle cell line C2 C12 . Myotubes as compared to myoblasts of C2 C12 cells were found to be more sensitive in response to insulin as increase in insulin-stimulated phosphorylation of AKT at serine 473 residue (pAKTS473 ) was significantly higher in myotubes. Incidentally, reduced protein level of NCoR coincided with differentiation of myoblasts into myotubes of C2 C12 cells. However, insulin stimulation per se failed to affect protein level of NCoR either in myoblasts or myotubes of C2 C12 cells. To assess the role of NCoR on insulin sensitivity, NCoR was transiently knocked down using siRNA in myotubes of C2 C12 . In fact, transient silencing of NCoR led to significant reduction in insulin-stimulated pAKTS473 and impaired glucose uptake. This observation is in contrast to published studies where NCoR has been reported to negatively regulate insulin signaling cascade. Furthermore, transient silencing of NCoR failed to improve insulin sensitivity in chronic hyperinsulinemia-induced insulin-resistant model of C2 C12 cells. Importantly, inhibition of lysosomal protein degradation pathway using ammonium chloride restored protein level of NCoR but failed to increase glucose uptake in serum-starved C2 C12 myotubes. Collectively, data from present study show differential protein level of NCoR under different cell state (myoblast and myotubes) of C2 C12 cells and NCoR proves to be vital for maintaining insulin sensitivity in C2 C12 myotubes.

Perspective on unraveling the versatility of 'co-repressor' complexes.

A multitude of post-translational modifications take place on histones, one of the best studied being acetylation on lysine residues, which is generally associated with gene activation. During the last decades, several so-called co-repressor protein complexes that carry out the reverse process, histone deacetylation, have been identified and characterized, such as the Sin3, N-CoR/SMRT and NuRD complexes. Although a repressive role for these complexes in regulating gene expression is well established, accumulating evidence also points to a role in gene activation. Here, we argue that integration of various state-of-the-art technologies, addressing different aspects of transcriptional regulation, is essential to unravel this apparent biological versatility of 'co-repressor' complexes.

Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types.

Transcriptional repressors function primarily by recruiting co-repressors, which are accessory proteins that antagonize transcription by modifying chromatin structure. Although a repressor could function by recruiting just a single co-repressor, many can recruit more than one, with Drosophila Brinker (Brk) recruiting the co-repressors CtBP and Groucho (Gro), in addition to possessing a third repression domain, 3R. Previous studies indicated that Gro is sufficient for Brk to repress targets in the wing, questioning why it should need to recruit CtBP, a short-range co-repressor, when Gro is known to be able to function over longer distances. To resolve this we have used genomic engineering to generate a series of brk mutants that are unable to recruit Gro, CtBP and/or have 3R deleted. These reveal that although the recruitment of Gro is necessary and can be sufficient for Brk to make an almost morphologically wild-type fly, it is insufficient during oogenesis, where Brk must utilize CtBP and 3R to pattern the egg shell appropriately. Gro insufficiency during oogenesis can be explained by its downregulation in Brk-expressing cells through phosphorylation downstream of EGFR signaling.

Regulation of carotenogenesis in the red yeast Xanthophyllomyces dendrorhous: the role of the transcriptional co-repressor complex Cyc8-Tup1 involved in catabolic repression.

BACKGROUND: The yeast Xanthophyllomyces dendrorhous produces carotenoids of commercial interest, including astaxanthin and ß-carotene. Although carotenogenesis in this yeast and the expression profiles of the genes controlling this pathway are known, the mechanisms regulating this process remain poorly understood. Several studies have demonstrated that glucose represses carotenogenesis in X. dendrorhous, suggesting that this pathway could be regulated by catabolic repression. Catabolic repression is a highly conserved regulatory mechanism in eukaryotes and has been widely studied in Saccharomyces cerevisiae. Glucose-dependent repression is mainly observed at the transcriptional level and depends on the DNA-binding regulator Mig1, which recruits the co-repressor complex Cyc8-Tup1, which then represses the expression of target genes. In this work, we studied the regulation of carotenogenesis by catabolic repression in X. dendrorhous, focusing on the role of the co-repressor complex Cyc8-Tup1. RESULTS: The X. dendrorhous CYC8 and TUP1 genes were identified, and their functions were demonstrated by heterologous complementation in S. cerevisiae. In addition, cyc8 - and tup1 - mutant strains of X. dendrorhous were obtained, and both mutations were shown to prevent the glucose-dependent repression of carotenogenesis in X. dendrorhous, increasing the carotenoid production in both mutant strains. Furthermore, the effects of glucose on the transcript levels of genes involved in carotenogenesis differed between the mutant strains and wild-type X. dendrorhous, particularly for genes involved in the synthesis of carotenoid precursors, such as HMGR, idi and FPS. Additionally, transcriptomic analyses showed that cyc8 - and tup1 - mutations affected the expression of over 250 genes in X. dendrorhous. CONCLUSIONS: The CYC8 and TUP1 genes are functional in X. dendrorhous, and their gene products are involved in catabolic repression and carotenogenesis regulation. This study presents the first report involving the participation of Cyc8 and Tup1 in carotenogenesis regulation in yeast.

Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24µM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18µM) and 3-chloro- (IC50=0.17µM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.