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The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through the coactivation of coupled transcription factors, such as estrogen-related receptor (ERR), nuclear respiratory factor 1 (Nrf1), GA-binding protein-a (Gabpa), and Yin Yang 1 (YY1). We hypothesized that PGC-1α plays an essential role in the transcription of TCA cycle genes. Thus, utilizing localization peaks of PGC-1α to TCA cycle gene promoters would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for the transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful in understanding how those genes are dysregulated in pathological conditions., Competing Interests: Conflict of interest The authors declare that they have no competing interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Carnitine-acylcarnitine translocase (CACT) is a nuclear-encoded mitochondrial carrier that catalyzes the transfer of long-chain fatty acids across the inner mitochondrial membrane for β-oxidation. In this study, we conducted a structural and functional characterization of the CACT promoter to investigate the molecular mechanism underlying the transcriptional regulation of the CACT gene by n-3 PUFA, EPA and DHA. In hepatic BRL3A cells, EPA and DHA stimulate CACT mRNA and protein expression. Deletion promoter analysis using a luciferase reporter gene assay identified a n-3 PUFA response region extending from -202 to -29 bp. This region did not contain a response element for PPARα, a well-known PUFA-responsive nuclear receptor. Instead, bioinformatic analysis revealed two highly conserved GABP responsive elements within this region. Overexpression of GABPα and GABPβ subunits, but not PPARα, increased CACT promoter activity, more remarkably upon treatment with EPA and DHA. ChIP assays showed that n3-PUFA enhanced the binding of GABPα to the -202/-29 bp sequence. Furthermore, both EPA and DHA induced nuclear accumulation of GABPα. In conclusion, our findings indicate that the upregulation of CACT by n3-PUFA in hepatic cells is independent from PPARα and could be mediated by GABP activation.

In recent years, the acronym NRF2 has garnered significant attention in scientific discourse. However, this attention has occasionally led to confusion due to the existence of two distinct proteins sharing the same acronym: Nuclear Respiratory Factor 2 (NRF2), also known as GA-binding protein transcription factor subunit alpha (GABPA), and Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2 or NRF2). This confusion has been highlighted in various scientific forums, including PubPeer and anonymous reader comments, where the confusion between the two proteins has been expressed. In this article, we aim to elucidate the disparities between these two proteins. Both are transcription factors that play pivotal roles in cellular homeostasis and response to stress, with some overlapping functional aspects. Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2) is a key regulator of the antioxidant response element (ARE) pathway. It functions by binding to antioxidant response elements in the promoters of target genes, thereby orchestrating the expression of various cytoprotective enzymes and proteins involved in detoxification, redox balance, and cellular defense against oxidative stress. Conversely, Nuclear Respiratory Factor 2 (GABPA) is primarily associated with the regulation of mitochondrial biogenesis, in relation to PGC1α, and maintaining cellular energy metabolism. It is important to recognize and differentiate between these two proteins to avoid misconceptions and misinterpretations in scientific literature and discussions. Our laboratories (Arubala P Reddy and P. Hemachandra Reddy) focued on Nuclear Respiratory Factor 2 (NRF2), but not on Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2). We hope that the facts, figures, and discussions presented in this article will clarify the current controversy regarding the sizes, structural features, and functional aspects of these proteins., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms., Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays., Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20., Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mooli, Zhu, Khan, Nagati, Michealraj, Jurczak and Ramakrishnan.)

Cancer metabolism has emerged as a major target for cancer therapy, while the state of mitochondrial drugs has remained largely unexplored, partly due to an inadequate understanding of various mitochondrial functions in tumor contexts. Here, we report that HOMER3 is highly expressed in non-small cell lung cancer (NSCLC) and is closely correlated with poor prognosis. Lung cancer cells with low levels of HOMER3 are found to show significant mitochondrial dysfunction, thereby suppressing their proliferation and metastasis in vivo and in vitro. At the mechanistic level, we demonstrate that HOMER3 and platelet-activating factor acetylhydrolase 1b catalytic subunit 3 cooperate to upregulate the level of GA-binding protein subunit beta-1 (GABPB1), a key transcription factor involved in mitochondrial biogenesis, to control mitochondrial inner membrane genes and mitochondrial function. Concurrently, low levels of HOMER3 and its downstream target GABPB1 led to mitochondrial dysfunction and decreased proliferation and invasive activity of lung cancer cells, which raises the possibility that targeting mitochondrial synthesis is an important and promising therapeutic approach for NSCLC., (© 2023. The Author(s).)

Long noncoding RNA (lncRNA) (GABPB1-IT1) has been reported to be downregulated in lung cancer, while its expression and function in other cancers are unknown. In this study, the expression levels of GABPB1-IT1 in tissue samples from 62 ccRCC patients were measured by performing RT-qPCR. Potential base pairing formed between GABPB1-IT1 and miR-21 was explored using the online program IntaRNA 2.0 and further confirmed by Dual-luciferase activity assay and RNA pulldown assay. The role of GABPB1-IT1 and miR-21 in regulating the expression of PTEN was evaluated by RT-qPCR and Western blot. The role of GABPB1-IT1, miR-21, and PTEN in regulating the proliferation of Caki-2 cells was explored by CCK-8 assay. It was observed that GABPB1-IT1 was downregulated in ccRCC and predicted poor survival. GABPB1-IT1 directly interacted with miR-21, while it did not regulate the expression of each other. Moreover, upregulation of PTEN, which is a target of miR-21, was observed in ccRCC cells with overexpression of GABPB1-IT1. Overexpression of GABPB1-IT1 and PTEN decreased the proliferation rates of ccRCC cells. In addition, overexpression of GABPB1-IT1 reduced the enhancing effects of miR-21 on cell proliferation. Therefore, GABPB1-IT1 may upregulate PTEN by sponging miR-21 in ccRCC to inhibit cancer cell proliferation. Our study characterized a novel GABPB1-IT1/miR-21/PTEN axis in ccRCC., (© 2023 Wiley Periodicals LLC.)

Background: The ETS transcription factor GABPA has long been thought of as an oncogenic factor and recently suggested as a target for cancer therapy due to its critical effect on telomerase activation, but the role of GABPA in clear cell renal cell carcinoma (ccRCC) is unclear. In addition, ccRCC is characterized by metabolic reprograming with aberrant accumulation of L-2-hydroxyglurate (L-2HG), an oncometabolite that has been shown to promote ccRCC development and progression by inducing DNA methylation, however, its downstream effectors remain poorly defined., Methods: siRNAs and expression vectors were used to manipulate the expression of GABPA and other factors and to determine cellular/molecular and phenotypic alterations. RNA sequencing and ChIP assays were performed to identify GABPA target genes. A human ccRCC xenograft model in mice was used to evaluate the effect of GABPA overexpression on in vivo tumorigenesis and metastasis. ccRCC cells were incubated with L-2-HG to analyze GABPA expression and methylation. We carried out immunohistochemistry on patient specimens and TCGA dataset analyses to assess the effect of GABPA on ccRCC survival., Results: GABPA depletion, although inhibiting telomerase expression, robustly enhanced proliferation, invasion and stemness of ccRCC cells, whereas GABPA overexpression exhibited opposite effects, strongly inhibiting in vivo metastasis and carcinogenesis. TGFBR2 was identified as the GABPA target gene through which GABPA governed the TGFβ signaling to dictate ccRCC phenotypes. GABPA and TGFBR2 phenocopies each other in ccRCC cells. Higher GABPA or TGFBR2 expression predicted longer survival in patients with ccRCC. Incubation of ccRCC cells with L-2-HG mimics GABPA-knockdown-mediated phenotypic alterations. L-2-HG silenced the expression of GABPA in ccRCC cells by increasing its methylation., Conclusions: GABPA acts as a tumor suppressor by stimulating TGFBR2 expression and TGFβ signaling, while L-2-HG epigenetically inhibits GABPA expression, disrupting the GABPA-TGFβ loop to drive ccRCC aggressiveness. These results exemplify how oncometabolites erase tumor suppressive function for cancer development/progression. Restoring GABPA expression using DNA methylation inhibitors or other approaches, rather than targeting it, may be a novel strategy for ccRCC therapy., (© 2022. The Author(s).)

GABPB1, known as nuclear respiratory factor 2 (Nrf2), activates the mitochondrial genes that are responsible for antioxidant action and detoxification. Two single nucleotide polymorphisms (SNPs) of GABPB1 , such as rs7181866 and rs8031031, were reported to be associated with the prevention of the increasing cancer risk caused by environmental deterioration. Between March 1 and May 1, 2018, human peripheral blood mononuclear cells (PBMCs) from a cohort of 300 volunteers working in adverse occupational environments were genotyped for the two SNPs in the present study. The SNP rs7181866 was found to be significantly greater in the male group than in the female group. Frequencies of SNP rs7181866 and bi-allele SNPs (rs7181866 + rs8031031) were significantly different between the <35-year-old group and the ≥35-year-old group. Further, multinomial logistic regression analysis of the occupational environments revealed the highest predictive frequency of SNPs for four environmental factors, of which chemical factors accounted for 15.33% rs7181866, physical factors accounted for 34.79% rs7181866 + rs8031031, physical + chemical factors accounted for 39.5% rs8031031, and unknown factors accounted for 26.5% rs7181866 + rs8031031. In conclusion, the G allele of rs7181866 was found to be significantly more susceptible than the rs8031031 allele under adverse occupational environmental factors, and physical factors such as noise, which appear to play vital roles in causing SNP mutations.

Background: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by abnormal immune cell activation. This study aimed to investigate differentially expressed long non-coding RNA (lncRNA) in peripheral blood mononuclear cells (PBMCs) in patients with pSS to identify lncRNAs that affect pSS pathogenesis., Methods: Total RNA was extrated from PBMCs of 30 patients with pSS and 15 healthy persons. Transcriptome sequencing was used to screen differentially expressed lncRNAs and mRNAs in 8 RNA samples from the discovery cohort. The differentially expressed mRNAs underwent functional enrichment analysis. A protein interaction relationship (PPI) and competitive endogenous RNA (ceRNA) network was constructed. Real-time PCR was used to validate screened lncRNAs in all 45 RNA samples.., Results: 1180 lncRNAs and 640 mRNAs were differentially expressed in pSS patients (fold change > 2 in healthy persons). The PPI network was constructed with 640 mRNAs and a ceRNA network with four key lncRNAs (GABPB1-AS1, PSMA3-AS1, LINC00847 and SNHG1). Real-time PCR revealed that GABPB1-AS1 and PSMA3-AS1 were significantly up-regulated 3.0- and 1.4-fold in the pSS group, respectively. The GABPB1-AS1 expression level was positively correlated with the percentage of B cells and IgG levels., Conclusions: GABPB1-AS1 was significently up-regulated in pSS patients, and its expression level is positively correlated with the percentage of B cells and IgG levels. GABPB1-AS1 may be involved in the pathogenesis of pSS and may be a promising biological marker., (© 2021. The Author(s).)

Background: GA-binding protein A (GABPA), a transcription factor, is broadly involved in physiological and pathological processes. Several studies have investigated the relationship between GABPA expression level and outcomes of various malignancies. However, the function and clinicopathological significance of GABPA in endometrial carcinoma (EC) remain obscure., Methods: The GABPA mRNA expression in EC tissues and adjacent nonneoplastic tissues in the TCGA database was involved in our study. The protein expression of GABPA in 107 EC tissues and 15 normal endometrial tissues was detected by immunohistochemistry., Results: The GABPA expression was significantly downregulated in EC tissues compared with its expression in normal tissues ( P < 0.001). The expression of GABPA was markedly correlated with type II EC ( P < 0.01) and grade 3 EC ( P < 0.05). A tendency has been observed that patients with low GABPA levels had relatively poorer overall survival (OS) ( P = 0.036) and disease-free survival (DFS) ( P = 0.016) than patients with high GABPA levels. The multivariate Cox proportional hazard model showed that lower expression of GABPA was an independent poor prognostic factor for OS ( P = 0.043) and DFS ( P = 0.045). Similar correlation between low expression levels of GABPA and unfavorable prognosis has also been found in type II or grade 3 EC. IHC analysis showed EC tissues had low expression of GABPA, which indicated relatively poor prognosis. Moreover, we identified that the GABPA mRNA expression was negatively correlated with its methylation level ( R = -0.2512, P < 0.001) which is one of the mechanisms for the silencing of GABPA gene., Conclusion: GABPA may act as an independent predictor of clinical prognosis and serve as a potential target gene for EC therapy., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Xiaoxue Ma et al.)

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase ( TERT ) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM., Competing Interests: Competing interest statement: The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. C.F. is a co-founder of Mirimus, Inc. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences, and Inari. J.A.D. is a Director at Johnson & Johnson and has sponsored research projects by Pfizer, Roche Biopharma, and Biogen. J.F.C. is a co-founder of Telo Therapeutics, Inc. and has ownership interests. The other authors declare no competing interests., (Copyright © 2021 the Author(s). Published by PNAS.)

The telomerase reverse transcriptase (TERT) has long been pursued as a direct therapeutic target in human cancer, which is currently hindered by the lack of effective specific inhibitors of TERT. The FOS/GABPB/(mutant) TERT cascade plays a critical role in the regulation of mutant TERT , in which FOS acts as a transcriptional factor for GABPB to up-regulate the expression of GABPB, which in turn activates mutant but not wild-type TERT promoter, driving TERT-promoted oncogenesis. In the present study, we demonstrated that inhibiting this cascade by targeting FOS using FOS inhibitor T-5224 suppressed mutant TERT cancer cells and tumors by inducing robust cell apoptosis; these did not occur in wild-type TERT cells and tumors. Mechanistically, among 35 apoptotic cascade-related proteins tested, the apoptosis induced in this process specifically involved the transcriptional activation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 ( TRAIL-R2 ) and inactivation of survivin, two key players in the apoptotic cascade, which normally initiate and suppress the apoptotic cascade, respectively. These findings with suppression of FOS were reproduced by direct knockdown of TERT and prevented by prior knockdown of TRAIL-R2. Further experiments demonstrated that TERT acted as a direct transcriptional factor of survivin, up-regulating its expression. Thus, this study identifies a therapeutic strategy for TERT promoter mutation-driven cancers by targeting FOS in the FOS/GABPB/(mutant) TERT cascade, circumventing the current challenge in pharmacologically directly targeting TERT itself. This study also uncovers a mechanism through which TERT controls cell apoptosis by transcriptionally regulating two key players in the apoptotic cascade., Competing Interests: The authors declare no competing interest.

Combat sports are an intermittent sport, with mixed anaerobic and aerobic energy production. Here, we investigated whether the polymorphisms that have been previously suggested as genetic markers for endurance or power phenotypes were associated with combat-sport athletic status. A total of 23 previously reported performance-related polymorphisms were examined in a cohort of 1,129 Brazilian individuals (164 combat-sport athletes and 965 controls), using a case-control association study. We found that the GA-binding protein transcription factor subunit beta 1 ( GABPβ1 ) gene (also known as nuclear respiratory factor 2; NRF2 ) was associated with athletic status, with the minor G (rs7181866) and T (rs8031031) alleles overrepresented in athletes ( P ≤ 0.003), especially among world-class athletes ( P ≤ 0.0002). These findings indicate that single-nucleotide polymorphisms (SNPs) within the GABPβ1 gene increase the likelihood of an individual being a combat-sport athlete, possibly because of a better mitochondrial response to intermittent exercises.

The transcriptional de-repression of the telomerase reverse transcriptase (TERT) gene and subsequent activation of telomerase is a prerequisite step in malignant transformation and progression. Recently, the gain-of-function mutation of the TERT promoter was identified in many types of human malignancies, and the mutated promoter acquires de novo ETS binding motifs through which the TERT transcription is activated. The ETS family transcription factors GABPA and GABPB1 have been shown to act as master drivers for the mutant TERT promoter activity. Indeed, GABPA or GABPB1 depletion leads to the down-regulation of TERT expression in the mutant TERT promoter-bearing cancer cells, and is thus proposed as targets for cancer therapy. Surprisingly, however, despite its key role in activating the mutant TERT promoter and telomerase, GABPA may itself function as a potent tumor suppressor in several malignancies. In this review, we address the collaboration between GABPA and mutant TERT promoter in cancer development, discuss selection trade-offs among different activities of GABPA in cancer evolution, and underscore the suppressive function of GABPA in cancer progression and implications in precision oncology., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Telomerase reverse transcriptase ( TERT ) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC , as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.

Context: Lysine-specific demethylase 1 (LSD1) stabilizes hypoxia-inducible factor 1α (HIF1α) to advance tumor progression, while HIF1α functions as a transcription factor to increase the expression of microRNA-146a (miR-146a)., Objective: We aim to investigate whether LSD1 affects the development of papillary thyroid carcinoma (PTC) via HIF1α and miR-146a., Design: In vitro assays were performed with Nthy-ori 3-1, BHP5-16, BCPAP, K1, and BHP2-7 cell lines. In vivo assays were conducted with established xenograft tumors in nude mice., Setting: This study was conducted at our lab., Patients and Materials: PTC tissues and corresponding adjacent normal tissues were obtained from 45 patients hospitalized in Sun Yat-Sen Memorial Hospital. Assays were conducted using Nthy-ori 3-1, BHP5-16, BCPAP, K1, and BHP2-7 cell lines, as well as 50 male BALB/c nude mice., Intervention: Cells were transfected with sh-LSD1, sh-GABPA, oe-LSD1, oe-HIF1α, miR-146a mimic, and miR-146a inhibitor. In addition, K1 cells expressing lv-oe-LSD1, lv-miR-146a inhibitor, lv-oe-LSD1 or miR-146a inhibitor were injected into the right side of the mice. LSD1 gene and protein expression patterns were analyzed in 45 clinical PTC tissue samples., Main Outcome Measure: Expression of LSD1, HIF1α, miR-146a, and GA-binding protein transcription factor alpha (GABPA), as well as their effects on PTC., Results: LSD1 was highly expressed in clinical PTC tissues. LSD1 stabilized HIF1α and inhibited the degradation of its ubiquitin proteasome. HIF1α was enriched in the promoter region of miR-146a, an upregulated miRNA in PTC. HIF1α increased miR-146a expression to promote PTC progression in vitro, which was achieved by inhibiting GABPA, a target gene of miR-146a. LSD1 upregulated miR-146a to enhance the development and metastasis of PTC in nude mice., Conclusion: Our results show that LSD1 functions as an oncogene in PTC by upregulating HIF1α and miR-146a, elucidating an understanding of undefined mechanisms associated with tumor progression in PTC., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To investigate the effects of microRNA-29a (miR-29a) on myocardial ischemia-reperfusion (I/R) injury and its specific mechanisms, we used H9C2 myocardial cells to establish a myocardial ischemia model by hypoxia/reoxygenation (H/R), and microRNA-29a inhibitor was interfered. Annexin V/propidium iodide and flow cytometry were used to detect the effects of cell death. C57 mice were used to establish were used to establish the I/R injury model, and H&E staining was used to detect pathologic damage to heart tissues. The expressions of miR-29a silent information regulator factor 2-related enzyme 1 (SIRT1) and nucleotide-binding oligomerization domain like receptor protein 3 (NLRP3), as well as pyroptosis-related proteins were determined by quantitative reverse-transcription polymerase chain reaction and Western blot analysis. The serum levels of 2-hydroxybutyrate dehydrogenase (HBDH), lactate dehydrogenase-1 (LDH), creatine kinase (CK), creatine kinase MB activity (CK-MB), IMA, and inflammatory factors in I/R rats were significantly up-regulated. In the I/R group, the expression of miR-29a was significantly up-regulated while SIRT1 was remarkably down-regulated. Dual luciferase reporter assay showed SIRT1 was a direct target of miR-29a. Inhibition of miR-29a significantly up-regulated the expression of peroxisome proliferator-activated receptor gamma coactivator-1 α /nuclear respiratory factor-2 and endothelial nitric oxide synthase while remarkably down-regulating levels of inducible nitric oxide synthase and malondialdehyde in I/R. The oxidative stress that was induced by I/R injury was also suppressed by inhibition of miR-29a. All these effects of miR-29a inhibition were reversed by small interfering SIRT1. The in vitro H/R results showed that NLRP3-caspase-1-mediated pyroptosis was activated in H/R but was significantly inhibited by the inhibition of miR-29a. Inhibition of miR-29a improved myocardial I/R injury by targeting SIRT1 through suppressing oxidative stress and NLRP3-mediated pyroptosis. SIGNIFICANCE STATEMENT: In this study, we showed for the first time that miR-29a could improve myocardial I/R injury through inhibition of pyroptosis., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Ferroptosis is a non-apoptotic, iron-dependent oxidative form of cell death that is specifically induced by erastin in RAS mutant cancer cells. Ferroptotic cell death is the result of membrane lipid peroxide damage caused by the accumulation of hydroxyl radicals derived from H 2 O 2 by the Fenton reaction. Peroxidases are key cellular antioxidant enzymes that block such damaging processes. Few studies have examined the roles of long non-coding RNAs (lncRNAs) in the regulation of cellular oxidative stress, especially in ferroptosis. Here, we demonstrated that erastin upregulated the lncRNA GABPB1-AS1, which downregulated GABPB1 protein levels by blocking GABPB1 translation, leading to the downregulation of the gene encoding Peroxiredoxin-5 (PRDX5) peroxidase and the eventual suppression of the cellular antioxidant capacity. Such effects critically inhibited the cellular antioxidant capacity and cell viability. Additionally, high expression levels of GABPB1 were correlated with poor prognosis of hepatocellular carcinoma (HCC) Patients, while high GABPB1-AS1 levels in HCC patients correlated with improved overall survival. Collectively, these data demonstrate a mechanistic link between GABPB1 and its antisense lncRNA GABPB1-AS1 in erastin-induced ferroptosis and establish GABPB1 and GABPB1-AS1 as attractive therapeutic targets for HCC.

GA binding protein (GABP) is a ubiquitously expressed transcription factor that regulates the development of multiple cell types, including osteoblast, hematopoietic stem cells, B cells and T cells. However, so little is known about its biological function in the development of central nervous system. In this report, we show that GABP is highly expressed in neural stem/progenitor cells (NSPCs) and down-regulated in neurons, and that GABPβ1 is required for the proper proliferation of NSPCs. Knockdown of GABPα resulted in an elevated expression level of GABPβ1, and GABPβ1 down-regulation significantly decreased the proliferation of NSPCs, whereas GABPβ2 knockdown did not result in any changes in the proliferation of NSPCs. We observed that there was nearly a 21-fold increase of the GABPβ1S mRNA level in GABPβ1L KO NSPCs compared to WT cells, and knocking down of GABPβ1S in GABPβ1L KO NSPCs could further reduce their proliferation potential. We also found that knockdown of GABPβ1 promoted neuronal and astrocytic differentiation of NSPCs. Finally, we identified dozens of downstream target genes of GABPβ1, which are closely associated with the cell proliferation and differentiation. Collectively, our results suggest that both GABPβ1L and GABPβ1S play an essential role in regulating the proper proliferation and differentiation of NSPCs., (Copyright © 2019. Published by Elsevier B.V.)

GABPB1, known as nuclear respiratory factor 2 (Nrf2), activates mitochondrial genes that are responsible for oxidative phosphorylation. Earlier studies on GABPB1 reported that two single nucleotide polymorphisms (SNPs) such as rs7181866 and rs8031031, to be associated with increased endurance in athletes. In the present study, a cohort of 302 South Indians, including normoglycemic healthy controls, T2DM with and without obesity were genotyped for the two SNPs by PCR-RFLP method and correlated with serum adipokines. The 'G' allele of rs7181866 was found to be associated with obesity whereas rs8031031 didn't show any significant association with obese individuals. The increased levels of adipokines such as Leptin, IL-6 and TNF-α and decreased adiponectin were found among obese-T2DM, when compared to non-obese T2DM subjects. Further, Factor analysis on metabolic components revealed four factors which accounts for 71.5% for non-obese control and 88.3% for obese T2DM of variance. The bias-corrected and accelerated bootstrap analysis revealed GG genotype to have significant positive and negative correlation with both TNF-α and adiponectin. In conclusion, the G allele of (rs7181866 A/G) was found to be significantly associated with risk for obesity among T2DM subjects., (Copyright © 2019. Published by Elsevier B.V.)

Mitotic bookmarking constitutes a mechanism for transmitting transcriptional patterns through cell division. Bookmarking factors, comprising a subset of transcription factors (TFs), and multiple histone modifications retained in mitotic chromatin facilitate reactivation of transcription in the early G1 phase. However, the specific TFs that act as bookmarking factors remain largely unknown. Previously, we identified the "early G1 genes" and screened TFs that were predicted to bind to the upstream region of these genes, then identified GA-binding protein transcription factor alpha subunit (GABPA) and Sp1 transcription factor (SP1) as candidate bookmarking factors. Here we show that GABPA and multiple histone acetylation marks such as H3K9/14AC, H3K27AC, and H4K5AC are maintained at specific genomic sites in mitosis. During the M/G1 transition, the levels of these histone acetylations at the upstream regions of genes bound by GABPA in mitosis are decreased. Upon depletion of GABPA, levels of histone acetylation, especially H4K5AC, at several gene regions are increased, along with transcriptional induction at 1 h after release. Therefore, we proposed that GABPA cooperates with the states of histone acetylation to act as a novel bookmarking factor which, may negatively regulate transcription during the early G1 phase.

The ETS family transcription factor GABPA is suggested as an oncogenic element, which is further supported by the recent reporting of it as the sole ETS member to activate the mutant TERT promoter in thyroid carcinomas (TC). However, it remains unclear how GABPA contributes to TC pathogenesis. The present study is designed to address this issue. TERT expression was significantly diminished in TERT promoter-mutated TC cells upon GABPA inhibition. Surprisingly, GABPA depletion led to robustly increased cellular invasion independently of TERT promoter mutations and TERT expression. DICER1, a component of the microRNA machinery, was identified as a downstream effector of GABPA. GABPA facilitated Dicer1 transcription while its depletion reduced Dicer1 expression. The mutation of the GABPA binding site in the DICER1 promoter led to diminished basal levels of DICER1 promoter activity and abolishment of GABPA-stimulated promoter activity as well. The forced DICER1 expression abrogated the invasiveness of GABPA-depleted TC cells. Consistently, the analyses of 93 patients with papillary thyroid carcinoma (PTC) revealed a positive correlation between GABPA and DICER1 expression. GABPA expression was negatively associated with TERT expression and promoter mutations, in contrast to published observations in cancer cell lines. Lower GABPA expression was associated with distant metastasis and shorter overall/disease-free survival in PTC patients. Similar results were obtained for PTC cases in the TCGA dataset. In addition, a positive correlation between GABPA and DICER1 expression was seen in multiple types of malignancies. Taken together, despite its stimulatory effect on the mutant TERT promoter and telomerase activation, GABPA may itself act as a tumor suppressor rather than an oncogenic factor to inhibit invasion/metastasis in TCs and be a useful predictor for patient outcomes.

Oxidative stress generated during inflammation is associated with a wide range of pathologies. Resveratrol (RESV) displays anti-inflammatory and antioxidant activities, being a candidate for the development of adjuvant therapies for several inflammatory diseases. Despite this potential, the cellular responses induced by RESV are not well known. In this work, transcriptomic analysis was performed following lipopolysaccharide (LPS) stimulation of monocyte cultures in the presence of RESV. Induction of an inflammatory response was observed after LPS treatment and the addition of RESV led to decreases in expression of the inflammatory mediators, tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1), without cytotoxicity. RNA sequencing revealed 823 upregulated and 2098 downregulated genes (cutoff ≥2.0 or ≤-2.0) after RESV treatment. Gene ontology analysis showed that the upregulated genes were associated with metabolic processes and the cell cycle, consistent with normal cell growth and differentiation under an inflammatory stimulus. The downregulated genes were associated with inflammatory responses, gene expression, and protein modification. The prediction of master regulators using the iRegulon tool showed nuclear respiratory factor 1 (NRF1) and GA-binding protein alpha subunit (GABPA) as the main regulators of the downregulated genes. Using immunoprecipitation and protein expression assays, we observed that RESV was able to decrease protein acetylation patterns, such as acetylated apurinic/apyrimidinic endonuclease-1/reduction-oxidation factor 1 (APE1/Ref-1), and increase histone methylation. In addition, reductions in p65 (nuclear factor-kappa B (NF-κB) subunit) and lysine-specific histone demethylase-1 (LSD1) expression were observed. In conclusion, our data indicate that treatment with RESV caused significant changes in protein acetylation and methylation patterns, suggesting the induction of deacetylase and reduction of demethylase activities that mainly affect regulatory cascades mediated by NF-кB and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. NRF1 and GABPA seem to be the main regulators of the transcriptional profile observed after RESV treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPβ1L (β1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting β1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of β1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPβ1L in enabling immortality in TERT promoter mutant glioblastoma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. In the present study, bioinformatics tools were systematically used to investigate the potential upstream effector involved in the progression of ccRCC. Using the Gene Expression Omnibus database and Library of Integrated Network-based Cellular Signatures L1000 platform, it was identified that GA-binding protein subunit β1 (GABPB1) was a potential effector gene. GABPB1 is a transcription factor subunit and its function in ccRCC is unclear. Elevated expression of GABPB1 mRNA in ccRCC was also observed in other clinical datasets from the Oncomine database. Following reverse transcription-quantitative polymerase chain reaction and western blot analysis, the ccRCC 786-O and A498 cell lines showed higher expression levels of GABPB1 than HK-2, a normal kidney cell line. Knockdown of GABPB1 in the 786-O and A498 cells significantly decreased the ability to form colonies by inducing the expression of p21Waf/Cip1. SurvExpress database analysis indicated that a higher expression of GABPB1 was associated with poor survival outcome in patients with renal cancer. These findings imply that GABPB1 serves an important role in the progression of ccRCC.

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.

Scope: Non-alcoholic fatty liver disease (NAFLD) is a common disease that is concomitant with obesity, resulting in increased mortality. To date, the efficiency of NAFLD treatment still needs to be improved. Therefore, we aimed to evaluate the effect of Lactobacillus mali APS1, which was isolated from sugary kefir, on hepatic steatosis in rats fed a high-fat diet (HFD)., Methods and Results: Sprague Dawley rats were fed a control diet, a HFD with saline, and a HFD with APS1 intervention by gavage daily for 12 weeks. The results showed that APS1 significantly reduced body weight and body weight gain in HFD-fed rats. APS1 reduced hepatic lipid accumulation by regulating SIRT-1/PGC-1α/SREBP-1 expression. Moreover, APS1 increased hepatic antioxidant activity by modulating Nrf-2/HO-1 expression. Notably, APS1 manipulated the gut microbiota, resulting in increasing proportions of the phylum Bacteroidetes/Firmicutes and reducing the abundance of specific NAFLD-associated bacteria., Conclusion: These results suggested that APS1 ameliorated hepatic steatosis by modulating lipid metabolism and antioxidant activity via manipulating specific NAFLD-associated gut microbiota in vivo., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The unique oncogene duet of coexisting BRAF V600E and TERT promoter mutations are widely found to be a robust genetic background promoting human cancer aggressiveness, but the mechanism is unclear. Here, we demonstrate that the BRAF V600E/MAP kinase pathway phosphorylates and activates FOS, which in turn acts as a transcription factor to bind and activate the GABPB promoter, increasing GABPB expression and driving formation of GABPA-GABPB complex; the latter selectively binds and activates mutant TERT promoter, upregulating TERT expression. Elevated TERT functions as a strong oncoprotein, robustly promoting aggressive behaviors of cancer cells and tumor development. We thus identify a molecular mechanism for the activation of mutant TERT by the BRAF V600E/MAP kinase pathway, in which FOS as a transcriptional factor of GABPB promoter plays a key role in functionally bridging the two oncogenes in cooperatively promoting oncogenesis, providing important cancer biological and clinical implications.

The TIM23 protein is a key component of the mitochondrial import machinery in yeast and mammals. TIM23 is the channel-forming subunit of the translocase of the inner mitochondrial membrane (TIM23) complex, which mediates preprotein translocation across the mitochondrial inner membrane. In this paper, we aimed to characterize the promoter region of the highly similar human TIM23 orthologs: TIMM23 and TIMM23B. Bioinformatic analysis revealed putative sites for the GA-binding protein (GABP) and the recombination signal binding protein for immunoglobulin kappa J (RBPJ) transcription factors in both promoters. Luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments showed three functional sites for GABP and one functional site for RBPJ in both promoters. Moreover, silencing of GABPA, the gene encoding the DNA-binding subunit of the GABP transcription factor, resulted in reduced expression of TIMM23 and TIMM23B. Our results show an essential role of GABP in activating TIMM23 expression. More broadly, they suggest that physiological signals involved in activating mitochondrial biogenesis and oxidative function also enhance the transcription but not the protein level of TIMM23, which is essential for maintaining mitochondrial function and homeostasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The potential therapeutic effects of agonistic analogs of growth hormone-releasing hormone (GHRH) and their mechanism of action were investigated in diabetic retinopathy (DR). Streptozotocin-induced diabetic rats (STZ-rats) were treated with 15 μg/kg GHRH agonist, MR-409, or GHRH antagonist, MIA-602. At the end of treatment, morphological and biochemical analyses assessed the effects of these compounds on retinal neurovascular injury induced by hyperglycemia. The expression levels of GHRH and its receptor (GHRH-R) measured by qPCR and Western blotting were significantly down-regulated in retinas of STZ-rats and in human diabetic retinas (postmortem) compared with their respective controls. Treatment of STZ-rats with the GHRH agonist, MR-409, prevented retinal morphological alteration induced by hyperglycemia, particularly preserving survival of retinal ganglion cells. The reverse, using the GHRH antagonist, MIA-602, resulted in worsening of retinal morphology and a significant alteration of the outer retinal layer. Explaining these results, we have found that MR-409 exerted antioxidant and anti-inflammatory effects in retinas of the treated rats, as shown by up-regulation of NRF-2-dependent gene expression and down-regulation of proinflammatory cytokines and adhesion molecules. MR-409 also significantly down-regulated the expression of vascular endothelial growth factor while increasing that of pigment epithelium-derived factor in diabetic retinas. These effects correlated with decreased vascular permeability. In summary, our findings suggest a neurovascular protective effect of GHRH analogs during the early stage of diabetic retinopathy through their antioxidant and anti-inflammatory properties., Competing Interests: Conflict of interest statement: N.L.B. owns equity in Biscayne Pharmaceuticals. A.V.S. is a coinventor on the patent for GHRH agonist, assigned to the University of Miami and the Veterans Affairs Medical Center, Miami, FL. However, the investigation of the effects of GHRH agonist MR-409 was an academic endeavor without any commercial interests. The other authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Ets-related transcription factor GA-binding protein alpha (GABPα), which is encoded by Gabpa, is expressed in a variety of cell types and is involved in cellular functions such as cell cycle regulation, apoptosis, and differentiation. Here, we generated Gabpa conditional knockout embryonic stem cells (ESCs) and characterized its cellular phenotypes. Disruption of Gabpa revealed that the proliferation of Gabpa-null ESCs was drastically repressed and cells started to die within 2 days. The repressed proliferation of Gabpa-null ESCs was recovered by artificially forced expression of GABPα. Expression analysis showed that p53 mRNA levels were comparable; however, p53 target genes, including Cdkn1a/p21, Mdm2, and Gadd45a, were upregulated and cell cycle-related genes, including Cyclin D1/D2 and Cyclin E1/E2, were downregulated in Gabpa-null ESCs. Interestingly, p53 and cleaved Caspase3 expressions were enhanced in the cells and reduced proliferation as well as cell death of Gabpa-null ESCs were rescued by either transfection of p53 RNAi or treatment of the p53 inhibitor pifithrin-α. These results suggest that GABPα inhibits the accumulation of p53 and is involved in the proliferation and survival of ESCs. Stem Cells 2017;35:2229-2238., (© 2017 AlphaMed Press.)

Peripheral T cells are maintained in the absence of vigorous stimuli, and respond to antigenic stimulation by initiating cell cycle progression and functional differentiation. Here we show that depletion of the Ets family transcription factor GA-binding protein (GABP) in T cells impairs T-cell homeostasis. In addition, GABP is critically required for antigen-stimulated T-cell responses in vitro and in vivo. Transcriptome and genome-wide GABP-binding site analyses identify GABP direct targets encoding proteins involved in cellular redox balance and DNA replication, including the Mcm replicative helicases. These findings show that GABP has a nonredundant role in the control of T-cell homeostasis and immunity.

Associations between the γ-aminobutyric acid type-A receptors (GABAA) and alcohol dependence risk have been reported, although the receptor subunit driving the association is unclear. Recent work in mice has highlighted a possible role for variants in the Gabr β1 subunit (Gabrβ1) in alcohol dependence risk, although this gene does not contain any common nonsynonymous variants in humans. However, the GABAA receptor is a heteropentamer so multiple potential variants within the gene complex could generate the alcohol dependence phenotype. The association between GABRβ1 variants and alcohol dependence risk was explored in a British and Irish population of alcohol-dependent cases (n=450) and ancestrally-matched controls screened to exclude current or historical alcohol misuse (n=555). Twelve common single nucleotide polymorphisms (SNPs) and a rare nonsynonymous variant, rs41311286, were directly genotyped; imputation was then performed across the whole gene. No allelic association was observed between alcohol dependence risk and any of the directly genotyped or imputed SNPs. However, post-hoc testing for genotypic association identified five common intronic SNPs that showed modest evidence for association after correction for multiple testing; two, rs76112682 and rs141719901, were in complete linkage disequilibrium [Pcorrected=0.02, odds ratio (95% confidence interval)=5.9 (1.7-2.06)]. These findings provide limited support for an association between GABRβ1 and the risk for developing alcohol dependence; further testing in expanded cohorts may be warranted.

Although single-nucleotide somatic mutations in the proximal promoter of the human telomerase reverse transcriptase (TERT) gene create novel consensus sequences for transcription factors that enhance TERT expression, the precise mechanism of how telomerase is reactivated in cancer cells remains poorly understood. In this issue, Akıncılar and colleagues identify a potential mechanism of TERT reactivation that is mediated by a novel long-range chromatin interaction between the TERT promoter on chromosome 5p and a 300-kb upstream region. This permits recruitment of the transcription factor GABPA in mutant TERT promoters but not in wild-type promoters. Cancer Discov; 6(11); 1212-4. ©2016 AACRSee related article by Akıncılar and colleagues, p. 1276., (©2016 American Association for Cancer Research.)

The present study was carried out to investigate the molecular mechanism of arsenic-induced mitochondrial oxidative damage and its relation to biogenesis in rat brain. Chronic sodium arsenite (25 ppm, orally) administration for 12 weeks decreased mitochondrial complexes activities and mRNA expression of selective complexes subunits. The expression of mitochondrial biogenesis regulator PGC-1α, and its downstream targets NRF-1, NRF-2 and Tfam were decreased significantly both at mRNA and protein levels suggesting impaired biogenesis following chronic arsenic-exposure. In addition to this, protein expression analysis also revealed activation of Bax and caspase-3, leading to translocation of cytochrome c from mitochondria to cytosol suggesting induction of apoptotic pathway under oxidative stress. This was further confirmed by electron microscopy study which depicted morphological changes in mitochondria in terms of altered nuclear and mitochondrial shape and chromatin condensation in arsenic-treated rats. The immunohistochemical studies showed both nuclear and cytosolic localization of NRF-1 and NRF-2 in arsenic-exposed rat brain further suggesting regulatory role of these transcription factors under arsenic neurotoxicity. The results of present study indicate that arsenic-induced mitochondrial oxidative damage is associated with decreased mitochondrial biogenesis in rat brain that may present as important target to reveal the mechanism for arsenic-induced neurotoxicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In previous studies we demonstrated that exposure to selenomethionine (SeMet) causes developmental toxicities in zebrafish (Danio rerio). The objectives of this study were to establish a dose-response relationship for developmental toxicities in zebrafish after embryo microinjection of Se (8, 16 or 32 μg/g dry mass of eggs) in the form of SeMet, and to investigate potential underlying mechanism(s) of SeMet-induced developmental toxicities. A dose-dependent increase in frequencies of mortality and total deformities, and reduced hatchability were observed in zebrafish exposed to excess Se via embryo microinjection. The egg Se concentration causing 20% mortality was then used to investigate transcript abundance of proteins involved in antioxidant protection and methylation. Excess Se exposure modified gene expression of oxidant-responsive transcription factors (nuclear factor erythroid 2-related factor nrf2a and nrf2b), and enzymes involved in cellular methylation (methionine adenosyltransferase mat1a and mat2ab) in zebrafish larvae. Notably, excess Se exposure up-regulated transcript abundance of aryl hydrocarbon receptor 2 (ahr2), a signalling pathway involved in the toxicity of dioxin-related compounds. Our findings suggest that oxidative stress or modification of methylation, or a combination of these mechanisms, might be responsible for Se-induced developmental toxicities in fishes.

Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure.

A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Maintenance of hematopoietic stem cells and their potential to give rise to progenitors of differentiated lymphoid and myeloid cells are accomplished by a network of regulatory processes. As a part of this network, the heteromeric transcription factor GA-binding protein (GABP) plays a crucial role in self-renewal of murine hematopoietic and leukemic stem cells. Here, we report the consequences of functional impairment of GABP in human hematopoietic and in leukemic stem/progenitor cells. Ectopic overexpression of a dominant-negative acting GABP mutant led to impaired myeloid differentiation of CD34(+) hematopoietic stem/progenitor cells obtained from healthy donors. Moreover, drastically reduced clonogenic capacity of leukemic stem/progenitor cells isolated from bone marrow aspirates of chronic myeloid leukemia (CML) patients underlines the importance of GABP on stem/progenitor cell maintenance and confirms the relevance of GABP for human myelopoiesis in healthy and diseased states., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Aberrant telomerase reactivation in differentiated cells represents a major event in oncogenic transformation. Recurrent somatic mutations in the human telomerase reverse transcriptase (TERT) promoter region, predominantly localized to two nucleotide positions, are highly prevalent in many cancer types. Both mutations create novel consensus E26 transformation-specific (ETS) motifs and are associated with increased TERT expression. Here, we perform an unbiased proteome-wide survey of transcription factor binding at TERT promoter mutations in melanoma. We observe ELF1 binding at both mutations in vitro and we show that increased recruitment of GABP is enabled by the spatial architecture of native and novel ETS motifs in the TERT promoter region. We characterize the dynamics of competitive binding between ELF1 and GABP and provide evidence for ELF1 exclusion by transcriptionally active GABP. This study thus provides an important description of proteome-wide, mutation-specific binding at the recurrent, oncogenic TERT promoter mutations., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers.

Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression., (© 2015 Stern et al.; Published by Cold Spring Harbor Laboratory Press.)

In Philadelphia-positive chronic myeloid leukemia (CML), imatinib resistance frequently emerges because of point mutations in the ABL1 kinase domain, but may also be the consequence of uncontrolled upstream signaling. Recently, the heteromeric transcription factor GA-binding protein (GABP) was found to promote CML-like myeloproliferative disease in mice. In a cohort of 70 CML patients, we found that expression of the GABP α subunit (GABPα) is positively correlated to the BCR-ABL1/ABL1 ratio. Moreover, significantly higher GABPα expression was detected in blast crisis than in chronic phase CML after performing data mining on 91 CML patients. In functional studies, imatinib sensitivity is enhanced after GABPα knockdown in tyrosine kinase inhibitors (TKI)-sensitive K-562, as well as by overexpression of a deletion mutant in TKI-resistant NALM-1 cells. Moreover, in K-562 cells, GABP-dependent expression variations of PRKD2 and RAC2, relevant signaling mediators in CML, were observed. Notably, protein kinase D2 (Prkd2) was reported to be a GABP target gene in mice. In line with this, we detected a positive correlation between GABPA and PRKD2 expression in primary human CML, indicating that the effects of GABP are mediated by PRKD2. These findings illustrate an important role for GABP in disease development and imatinib sensitivity in human CML., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Nuclear respiratory factor‑2α (NRF‑2α) is an important transcription factor that regulates mitochondrial oxidative phosphorylation and regeneration. NRF‑2α regulates mitochondrial transcription factors (mTF)A and B, and mitochondrial DNA by indirectly regulating the mitochondrial respiratory enzyme chain subunit. In addition, NRF‑2α is involved in the mitochondrial energy metabolism. Peroxisome proliferator‑activated receptor γ coactivator 1α (PGC‑1α), is an important transcription coactivator of NRF‑2α. Adenosine monophosphate‑activated protein kinase (AMPK) is considered an important effector in the regulation of the energy metabolism balance of nervous system microenvironments. However, the signaling mechanism underlying the energy coupling of PGC‑1α and NRF‑2α in visual cortical neurons remains to be elucidated. The present study used a primary culture system of rat visual cortical neurons in order to investigate whether AMPK is involved in the regulation of NRF‑2α and PGC‑1α expression in cortical neurons. The results of the present study indicated that KCl depolarization rapidly activated AMPK, and significantly increased the expression levels of PGC‑1α, NRF‑2α and mtTFA, as well as adenosine triphosphate production in cultured neurons. Similarly, the AMPK agonists 5‑aminoimidazole‑4‑carboxamide riboside and resveratrol significantly increased the mRNA expression levels of PGC‑1α and NRF‑2α in cultured neurons. These responses were blocked by compound C, an AMPK inhibitor. In conclusion, AMPK is an important transcriptional regulator of the neuronal excitation response, and exerts its regulatory effects via the PGC‑1α/NRF‑2α signaling pathway.

The heteromeric transcription factor GA-binding protein (GABP) consists of two subunits, the alpha subunit (GABPA) carrying the DNA-binding ETS domain, and the beta subunit (GABPB1) harbouring the transcriptional activation domain. GABP is involved in haematopoietic stem cell maintenance and differentiation of myeloid and lymphoid lineages in mice. To elucidate the molecular function of GABP in human haematopoiesis, the present study addressed effects of ectopic overexpression of GABP focussing on the myeloid compartment. Combined overexpression of GABPA and GABPB1 caused a proliferation block in cell lines and drastically reduced the colony-forming capacity of murine lineage-negative cells. Impaired proliferation resulted from perturbed cellular cycling and induction of myeloid differentiation shown by surface markers and myelomonocytic morphology of U937 cells. Depending on the dosage and functional integrity of GABP, ITGAM expression was induced. ITGAM encodes CD11b, the alpha subunit of integrin Mac-1, whose beta subunit, ITGB2/CD18, was already described to be regulated by GABP. Finally, Shield1-dependent proteotuning, luciferase reporter assays and chromatin immunoprecipitation showed that GABP activates the ITGAM/CD11b promoter via three binding sites close to the translational start site. In conclusion, the present study supports the crucial role of GABP in myeloid cell differentiation and identified ITGAM/CD11b as a novel GABP target gene., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study aims to examine the association between proliferator-activated receptor γ (PGC)-gene family-related single nucleotide polymorphisms (SNPs) and elite endurance runners' status in a Chinese cohort, and to gain insights into the functionality of a subset of SNPs. Genotype distributions of 133 SNPs in PPARGC1A, PPARGC1B, PPRC1, TFAM, TFB1M, TFB2M, NRF1, GABPA, GABPB1, ERRα, and SIRT1 genes were compared between 235 elite Chinese (Han) endurance runners (127 women) and 504 healthy non-athletic controls (237 women). Luciferase gene reporter activity was determined in 20 SNPs. After adjusting for multiple comparisons (in which threshold P-value was set at 0.00041), no significant differences were found in allele/genotype frequencies between athletes and controls (when both sexes were analyzed either together or separately). The lowest P-value was found in PPARGC1A rs4697425 (P = 0.001 for the comparison of allele frequencies between elite female endurance runners and their gender-matched controls). However, no association (all P > 0.05) was observed for this SNP in a replication cohort from Poland (194 endurance athletes and 190 controls). Using functional genomics tool, the following SNPs were found to have functional significance: PPARGC1A rs6821591, rs12650562, rs12374310, rs4697425, rs13113110, and rs4452416; PPARGC1B rs251466 and rs17110586; and PPRC1 rs17114388 (all P < 0.001). This study found no significant association between PGC-related SNPs and elite endurance athlete status in the Chinese population, despite some SNPs showing potential functional significance and the strong biological rationale to hypothesize that this gene pathway is a candidate to influence endurance exercise capacity., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Ever since we developed mitochondria to generate ATP, eukaryotes required intimate mito-nuclear communication. In addition, since reactive oxygen species are a cost of mitochondrial oxidative phosphorylation, this demands safeguards as protection from these harmful byproducts. Here we identified a critical transcriptional integrator which eukaryotes share to orchestrate both nutrient-induced mitochondrial energy metabolism and stress-induced nuclear responses, thereby maintaining carbon-nitrogen balance, and preserving life span and reproductive capacity. Inhibition of nutrient-induced expression of CAPER arrests nutrient-dependent cell proliferation and ATP generation and induces autophagy-mediated vacuolization. Nutrient signaling to CAPER induces mitochondrial transcription and glucose-dependent mitochondrial respiration via coactivation of nuclear receptor ERR-α-mediated Gabpa transcription. CAPER is also a coactivator for NF-κB that directly regulates c-Myc to coordinate nuclear transcriptome responses to mitochondrial stress. Finally, CAPER is responsible for anaplerotic carbon flux into TCA cycles from glycolysis, amino acids and fatty acids in order to maintain cellular energy metabolism to counter mitochondrial stress. Collectively, our studies reveal CAPER as an evolutionarily conserved 'master' regulatory mechanism by which eukaryotic cells control vital homeostasis for both ATP and antioxidants via CAPER-dependent coordinated control of nuclear and mitochondrial transcriptomic programs and their metabolisms. These CAPER dependent bioenergetic programs are highly conserved, as we demonstrated that they are essential to preserving life span and reproductive capacity in human cells-and even in C. elegans.

The trisomy of human chromosome 21 (Hsa21), which causes Down syndrome (DS), is the most common viable human aneuploidy. In contrast to trisomy, the complete monosomy (M21) of Hsa21 is lethal, and only partial monosomy or mosaic monosomy of Hsa21 is seen. Both conditions lead to variable physiological abnormalities with constant intellectual disability, locomotor deficits, and altered muscle tone. To search for dosage-sensitive genes involved in DS and M21 phenotypes, we created two new mouse models: the Ts3Yah carrying a tandem duplication and the Ms3Yah carrying a deletion of the Hspa13-App interval syntenic with 21q11.2-q21.3. Here we report that the trisomy and the monosomy of this region alter locomotion, muscle strength, mass, and energetic balance. The expression profiling of skeletal muscles revealed global changes in the regulation of genes implicated in energetic metabolism, mitochondrial activity, and biogenesis. These genes are downregulated in Ts3Yah mice and upregulated in Ms3Yah mice. The shift in skeletal muscle metabolism correlates with a change in mitochondrial proliferation without an alteration in the respiratory function. However, the reactive oxygen species (ROS) production from mitochondrial complex I decreased in Ms3Yah mice, while the membrane permeability of Ts3Yah mitochondria slightly increased. Thus, we demonstrated how the Hspa13-App interval controls metabolic and mitochondrial phenotypes in muscles certainly as a consequence of change in dose of Gabpa, Nrip1, and Atp5j. Our results indicate that the copy number variation in the Hspa13-App region has a peripheral impact on locomotor activity by altering muscle function.

Mitochondria are membrane-bound cytoplasmic organelles that serve as the major source of ATP production in eukaryotic cells. GABP (also known as nuclear respiratory factor 2) is a nuclear E26 transformation-specific transcription factor (ETS) that binds and activates mitochondrial genes that are required for electron transport and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding component of this transcription factor complex, from mouse embryonic fibroblasts (MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene expression. Gabpα loss modestly reduced mitochondrial mass, ATP production, oxygen consumption, and mitochondrial protein synthesis but did not alter mitochondrial morphology, membrane potential, apoptosis, or the expression of several genes that were previously reported to be GABP targets. However, the expression of Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for mitochondrial protein translation, was markedly reduced in Gabpα-null MEFs. We conclude that Gabp regulates Tfb1m expression and plays an essential, nonredundant role in mitochondrial biogenesis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)