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The cAMP responsive element binding protein (CREB)-regulated transcription coactivator 3 (CRTC3) is a member of the CRTC protein family and plays an important role in energy metabolism. The aim of this study was to determine if the expression of porcine CRTC3 is related to intramuscular fat (IMF) deposition and meat quality in Heigai pigs (a local fatty breed in China) and Duroc × Landrace × Yorkshire (DLY) pigs (a lean crossbred pig widely cultured in China). In addition, the effect of ectopic expression of CRTC3 on gene expression in porcine IMF adipocytes was also examined. Our results showed that Heigai pigs had lower lean percentage, thicker back fat thickness and smaller loin muscle area than DLY pigs. Compared with DLY pigs, Heigai pigs had higher marbling scores, better meat color and higher IMF contents and triglyceride concentrations. Higher levels of oxidative metabolic enzyme and expression of the slow oxidative muscle fiber-related genes were observed in longissimus dorsi muscle and psoas major muscle (P < 0.05) from Heigai pigs. Notably, CRTC3 and adipocyte-specific marker genes were highly expressed in muscle tissues of Heigai pigs. The expression of lipolysis-related genes ATGL and HSL were lower in Heigai muscles. Moreover, forced expression of CRTC3 promoted lipid accumulation and increased the expression of PPARγ, C/EBPα, leptin and FABP4 (P < 0.05), whereas it decreased the expression of ATGL and HSL in IMF adipocytes. These results suggest that CRTC3 expression is associated with lipid accumulation and IMF deposition in pigs., (© 2020 Stichting International Foundation for Animal Genetics.)

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor. Emerging studies showed that long noncoding RNAs (lncRNAs) have crucial regulatory roles in cancer biology. In the current study, differentially expressed lncRNAs between HCC and paired non-tumor tissues were identified using microarrays. The effects of a specific differentially expressed lncRNA (termed ZEB1-AS1) on tumor progression were investigated in vitro and in vivo. We found that ZEB1-AS1 is frequently upregulated in HCC samples, especially in metastatic tumor tissues. DNA methylation analysis shows a tumor-specific ZEB1-AS1 promoter hypomethylation. Aberrant methylation is tightly correlated with overexpression of ZEB1-AS1 in HCC. Patients with ZEB1-AS1 hypomethylation or with high ZEB1-AS1 expression have poor recurrence-free survival. Functionally, ZEB1-AS1 promotes tumor growth and metastasis, acts as an oncogene in HCC. The ZEB1-AS1 gene is located in physical contiguity with ZEB1 and positively regulates the ZEB1 expression. ZEB1 inhibition partially abrogates ZEB1-AS1-induced epithelial to mesenchymal transition (EMT) and cancer metastasis. Our results provide novel insights into the function of lncRNA-driven hepatocarcinogenesis, highlight the important role of ZEB1-AS1 and ZEB1 in HCC progression, and indicate that ZEB1-AS1 may be served as a valuable prognostic biomarker for HCC.

Here we study links between aminoglycoside-induced mistranslation, protein misfolding and neuropathy. We demonstrate that aminoglycosides induce misreading in mammalian cells and assess endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways. Genome-wide transcriptome and proteome analyses revealed upregulation of genes related to protein folding and degradation. Quantitative PCR confirmed induction of UPR markers including C/EBP homologous protein, glucose-regulated protein 94, binding immunoglobulin protein and X-box binding protein-1 (XBP1) mRNA splicing, which is crucial for UPR activation. We studied the effect of a compromised UPR on aminoglycoside ototoxicity in haploinsufficient XBP1 (XBP1(+/-)) mice. Intra-tympanic aminoglycoside treatment caused high-frequency hearing loss in XBP1(+/-) mice but not in wild-type littermates. Densities of spiral ganglion cells and synaptic ribbons were decreased in gentamicin-treated XBP1(+/-) mice, while sensory cells were preserved. Co-injection of the chemical chaperone tauroursodeoxycholic acid attenuated hearing loss. These results suggest that aminoglycoside-induced ER stress and cell death in spiral ganglion neurons is mitigated by XBP1, masking aminoglycoside neurotoxicity at the organismal level.

Aims/hypothesis: Rapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells., Methods: Experiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB., Results: Rapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment., Conclusions/interpretation: This report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) α factor (LITAF) is a multiple functional molecule whose sequence is identical to the small integral membrane protein of the lysosome/late endosome. LITAF was initially identified as a transcription factor that activates transcription of proinflammatory cytokine in macrophages in response to LPS. Mutations of the LITAF gene are associated with a genetic disease, called Charcot-Marie-Tooth syndrome. Recently, we have reported that mRNA levels of LITAF and TNF superfamily member 15 (TNFSF15) are upregulated by 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK). The present study further assesses their biological functions. Thus, we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a pharmacological activator of AMPK, increases the abundance of LITAF and TNFSF15 in LNCaP and C4-2 prostate cancer cells, which is abrogated by small hairpin RNA (shRNA) or the dominant-negative mutant of AMPK α1 subunit. Our data further demonstrate that AMPK activation upregulates the transcription of LITAF. Intriguingly, silencing LITAF by shRNA enhances proliferation, anchorage-independent growth of these cancer cells and tumor growth in the xenograft model. In addition, our study reveals that LITAF mediates the effect of AMPK by binding to a specific sequence in the promoter region. Furthermore, we show that TNFSF15 remarkably inhibits the growth of prostate cancer cells and bovine aortic endothelial cells in vitro, with a more potent effect toward the latter. In conjuncture, intratumoral injection of TNFSF15 significantly reduces the size of tumors and number of blood vessels and induces changes that are characteristic of tumor cell differentiation. Therefore, our studies for the first time establish the regulatory axis of AMPK-LITAF-TNFSF15 and also suggest that LITAF may function as a tumor suppressor.

Saccharomyces cerevisiae mutants that fail to complete meiotic recombination are blocked by the RAD17/RAD24/MEC1 checkpoint signaling pathway in pachytene when early sporulation genes are expressed. Middle genes are not activated in checkpoint-arrested cells because the Ndt80 transcription factor is inhibited. We find that the pachytene checkpoint requires Sum1, a transcriptional repressor that recognizes a subset of Ndt80-binding sites. Mutants lacking Sum1 or Rad17 partially bypass the block to the nuclear divisions but do not form spores, while mutants lacking both Sum1 and Rad17 completely bypass the block and form morphologically normal spores. The level of Sum1 protein decreases as middle genes are expressed, and this decrease is blocked in checkpoint-arrested cells. These data suggest that Sum1 levels are regulated by the checkpoint and that progression of the meiotic divisions and spore differentiation can be differentially controlled by competition of the Sum1 repressor and Ndt80 activator for occupancy at key middle promoters.

NC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA-bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2beta activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA-less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a non-essential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.

Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically relevant regions of NC2 have been determined and shown to match the histone-fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2beta yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP-TFIIA-DNA complexes. Wild-type but not the mt-Toa1 can relieve NC2 effects in purified transcription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.

Meiotic development in yeast is characterized by the sequential induction of temporally distinct classes of genes. Genes that are induced at the middle stages of the pathway share a promoter element, termed the middle sporulation element (MSE), which interacts with the Ndt80 transcriptional activator. We have found that a subclass of MSEs are strong repressor sites during mitosis. SUM1 and HST1, genes previously associated with transcriptional silencing, are required for MSE-mediated repression. Sum1 binds specifically in vitro to MSEs that function as strong repressor sites in vivo. Repression by Sum1 is gene specific and does not extend to neighboring genes. These results suggest that mechanisms used to silence large regions of chromatin may also be used to regulate the expression of specific genes during development. NDT80 is regulated during mitosis by both the Sum1 and Ume6 repressors. These results suggest that progression through sporulation may be controlled by the regulated competition between the Sum1 repressor and Ndt80 activator at key MSEs.

The human myeloid nuclear differentiation antigen, MNDA, is expressed only in myelomonocytic and a subset of B lymphoid hematopoietic cells. MNDA is uniformly distributed throughout the interphase cell nucleus and associates with chromatin, but does not bind specific DNA sequences. We recently demonstrated that MNDA binds nucleolin and nucleophosmin/NPM/B23 and both of these nuclear proteins bind the ubiquitous zinc finger transcription factor YY1. Investigations of the possible effect of MNDA on the interaction between YY1 and NPM, showed that MNDA bound YY1 directly under both in vitro and in vivo conditions. The MNDA-YY1 interaction enhanced the affinity of YY1 for its target DNA and decreased its rate of dissociation. The N-terminal half (200 amino acids) of MNDA was sufficient for maximum enhancement of YY1 DNA binding and a portion of this sequence was responsible for binding YY1. MNDA participated in a ternary complex with YY1 and the YY1 target DNA element. The results show that MNDA affects the ability of YY1 to bind its target DNA sequnce and that MNDA participates in a ternary complex possibly acting as a cofactor to impart lineage specific features to YY1 function.

All mammalian metallothionein genes studied to date have several metal responsive elements (MRE) with consensus sequences of TGCRCNC (R, purine) in their regulatory region. MRE-like sequences were also found in many other metal-related genes. To see whether there is also such a sequence at the genetic locus (13q14.3) of Wilson disease, which is a genetic disorder of copper metabolism, junction-trapping method based on the MRE sequence was used. A fragment containing MRE and MRE-like sequences from YAC 27D8 at the WND locus was successfully cloned and mapped back to the YAC by PCR. Presence of such a sequence in the copper transporter gene at the WD locus might imply that it has a possible interesting role in the regulation of WD gene expression.

The myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in developing cells of the human myelomonocytic lineage, including the end-stage monocytes/macrophages and granulocytes. Nuclear localization, lineage- and stage-specific expression, association with chromatin, and regulation by interferon alpha indicate that this protein is involved in regulating gene expression uniquely associated with the differentiation process and/or function of the monocyte/macrophage. MNDA does not bind specific DNA sequences, but rather a set of nuclear proteins that includes nucleolin (C23). Both in vitro binding assays and co-immunoprecipitation were used to demonstrate that MNDA also binds protein B23 (nucleophosmin/NPM). Three reciprocal chromosome translocations found in certain cases of leukemia/lymphoma involve fusions with the NPM/B23 gene, t(5;17) NPM-RARalpha, t(2;5) NPM-ALK, and the t(3;5) NPM-MLF1. In the current study, MNDA was not able to bind the NPM-ALK chimera originating from the t(2;5) and containing residues 1-117 of NPM. However, MNDA did bind the NPM-MLF1 product of the t(3;5) that contains the N-terminal 175 residues of NPM. The additional 58 amino acids (amino acids 117-175) of the NPM sequence that are contained in the product of the NPM-MLF1 fusion gene relative to the product of the NPM-ALK fusion appear responsible for MNDA binding. This additional NPM sequence contains a nuclear localization signal and clusters of acidic residues believed to bind nuclear localization signals of other proteins. Whereas NPM and nucleolin are primarily localized within the nucleolus, MNDA is distributed throughout the nucleus including the nucleolus, suggesting that additional interactions define overall MNDA localization.

Human myeloid cell nuclear differentiation antigen (MNDA) is a myelomonocytic lineage-specific protein that influences gene expression through interactions with other nuclear proteins and transcription factors. MNDA also self-associates and chemical cross-linking was used to demonstrate that MNDA forms a dimer. C-terminal and internal deletion mutants were used to identify two regions in the N-terminal half of MNDA essential for self-association. One region contains an imperfect leucine zipper and the second is highly enriched in basic residues. The sequences that are essential for dimerization are separated by a highly basic amphipathic alpha-helical region which was not required for dimerization.

The Oct-2 transcription factor has been shown previously to repress both the cellular tyrosine hydroxylase and the herpes simplex virus immediate-early genes in neuronal cells. Here we identify the gene encoding the neuronal nitric-oxide synthase (nNOS) as the first example of a gene activated in neuronal cells by Oct-2. The levels of the nNOS mRNA and protein are greatly reduced in neuronal cell lines in which Oct-2 levels have been reduced by an antisense method, although these cells have enhanced levels of tyrosine hydroxylase. Moreover, the nNOS gene regulatory region is activated by Oct-2 expression vectors upon cotransfection into both neuronal and non-neuronal cells, and this response is dependent upon a 20-amino acid region within the COOH-terminal activation domain of Oct-2. Of the two closely linked promoters that drive nNOS gene expression, only the downstream 5.1 promoter is activated by Oct-2, whereas the 5.2 promoter is unaffected. These effects are discussed in terms of the potential role of Oct-2 in regulating nNOS expression in the nervous system.

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in cells of the myelomonocytic lineage and regulated by interferon alpha in a cell-specific fashion. MNDA is also a member of a family of interferon-regulated genes of unknown function. In an effort to elucidate the function of MNDA, three techniques (affinity purification, coimmunoprecipitation, and protein blot assay) were used to characterize its specific protein binding activities. Microsequence analysis showed that MNDA bound the 100 kDa nucleolin protein. The identification of nucleolin was confirmed by immunoreaction with specific antibodies. MNDA contains motifs which could account for specific binding to nucleolin. Nucleolin binds other macromolecules and exhibits features consistent with roles in signal transduction, production of ribosomes, nuclear matrix structure, and regulation of transcription. The present results indicate that the function of MNDA is most likely related to interactions with other proteins. Through these associations, MNDA could contribute cell/lineage- and differentiation-specific limits to the function of ubiquitous proteins such as nucleolin. Further analysis of MNDA protein binding could be critical to elucidating the function of MNDA and could contribute to understanding the function of the products of other members of this interferon-inducible family of genes.