Phosphorylation of Serine 536 of p65(RelA) Downregulates Inflammatory Responses.

Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in inflammatory diseases and immune responses. Recently, a novel transcriptional regulatory mechanism of NF-κB involving the phosphorylation of serine 536 (534 in mice; S534) of its p65 subunit was reported; however, further research is required to elucidate the physiological role of S534 phosphorylation. Therefore, we generated S534A knock-in (KI) mice, in which the S534 of p65 was substituted with alanine. Similar to the wild-type (WT) mice, S534A KI mice developed normally. After stimulation with tumor necrosis factor α (TNFα), mouse embryonic fibroblasts (MEFs) derived from S534A KI mice exhibited increased target gene expression compared with that in the WT MEFs, which was induced by long-term binding of p65 to DNA. According to comprehensive gene expression analysis after stimulation with TNFα, the expression of genes p65ted to inflammatory and immune responses was increased, and the expression of genes p65ted to lipolysis was decreased in S534A KI MEFs. Analyses of a periodontal disease model established using WT and S534A KI mice revealed that alveolar bone resorption was enhanced in S534A KI mice owing to an increase in the number of osteoclasts, which was not attributed to the differentiation of osteoclast precursor cells but to an increased expression of interleukin-1ß and receptor activator of NF-κB ligand in the periodontal tissue. Hence, phosphorylation of S536 negatively regulates inflammatory responses in vitro and in vivo.

Transcriptional Activity of Genes Regulating T-Helper Differentiation in the Accidentally Exposed Population of the Southern Urals.

The objective of this work was to study the expression of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes responsible for the regulation of the differentiation of various T-helper subpopulations in individuals chronically exposed to radiation. The object of the study was peripheral blood cells obtained from 120 persons chronically exposed to radiation in a wide range of doses on the Techa River. The mean cumulative absorbed dose to red bone marrow in the examined exposed individuals was 742.7 ± 78.6 mGy (dose range, 73.5-3516.1 mGy); in the comparison group, 17.4 ± 2.2 mGy (dose range, 0.0-55.5 mGy). The subpopulation composition of T-helpers (Th1, Th2, and Th17) was analyzed by flow cytofluorometry. The relative mRNA content of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes was estimated by real-time PCR. The study made it possible to note a decrease in the relative number of T-helpers 2 in the populations of T-helpers of the central memory in the group of chronically exposed persons compared to the comparison group. In the population of T-helpers of the central memory, a statistically significant increase in the relative number of T-helpers 1 was shown, depending on the accumulated absorbed dose to red bone marrow. No changes in mRNA expression of the studied genes were observed. The analysis of the correlation between the expression of GATA3, MAPK8, STAT3, RORC, and TBX21 mRNA and the relative number of cells in subpopulations of T-helper types 1, 2, and 17 in the examined people did not reveal statistically significant patterns.

Comparative study on the transcriptional activities of grass carp (Ctenopharyngodon idellus) peroxisome proliferator-activated receptors induced by different fatty acids.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are part of the nuclear hormone receptor family, playing a crucial role in gene expression regulation. They serve as a connection between lipid metabolism disorders and innate immunity by being activated by fatty acids and their derivatives, facilitating signal transduction between the cell surface and nucleus. However, the specific transcriptional effects of different fatty acids (FAs) in fish are not yet fully understood. In our research, we identified and characterized PPARs in grass carp (Ctenopharyngodon idellus). The complete coding sequences of pparαa, pparαb, pparγ, pparδa, and pparδb were 1443 bp, 1404 bp, 1569 bp, 1551 bp, and 1560 bp in length, respectively. Pparα showed the highest expression in the liver, pparγ was mainly expressed in abdominal adipose tissue, and pparδ exhibited increased expression in the heart compared to other tissues. Gene localization analysis revealed that only pparδa was present in both the nucleus and cytoplasm, while the other four genes were exclusively located in the nucleus. Furthermore, our study explored the influence of various fatty acids (docosahexaenoic acid, palmitic acid, lauric acid and oleic acid at concentrations of 0, 50, 100, and 200 µM) on the transcriptional activities of different PPARs, demonstrating the diverse effects of fatty acid ligands on PPAR transcriptional activity. These results have significant implications for understanding the regulation of PPARs transcriptional activity.

Estrogen receptor/androgen receptor transcriptional activation of benzophenone derivatives and integrated approaches to testing and assessment (IATA) for estrogenic effects.

Estrogen receptor (ER) and androgen receptor (AR) transactivation assays for the benzophenone compounds (BPs) were performed using hERα-HeLa-9903 cells for ER and MMTV/22Rv1_GR-KO cells for AR. Results showed that some BPs, such as BP-1, BP-2, 4OH-BP, 4DHB, and 4-MBP, showed agonistic activity on ER with a higher RPCmax than 1 nM 17-ß estradiol. The other BPs (BP, BP-3, BP-6, BP-7, and BP-8) showed low RPCmax in accordance with the OECD Test guideline (TG) 455 criteria, with BP-4 as the only ER-negative. However, the potency of the BPs was at least 1000 times less than the reference chemical, 17-ß-estradiol. None of the BPs exhibited agonistic activity on AR except BP-2 which showed a small increase in activity. For further evaluation of the estrogenic effect of BPs based on the integrated approaches to testing and assessment (IATA) approach, existing data on ER binding, steroidogenesis, MCF-7 cell proliferation, and in vivo uterotrophic assays were collected and evaluated. There seemed to be a close association between the in vitro data on BPs, especially ER transcriptional activity, and the in vivo results of increased uterine weight. This case study implied that integrated approaches using in vitro data can be a useful tool for the prediction of in vivo data for estrogenic effects, without the need for additional animal toxicity tests.

SGK1 promotes the lipid accumulation via regulating the transcriptional activity of FOXO1 in bovine.

OBJECTIVES: Serum/glucocorticoid-inducible kinase 1 (SGK1) gene encodes a serine/threonine protein kinase that plays an essential role in cellular stress response and regulation of multiple metabolic processes. However, its role in bovine adipogenesis remains unknown. In this study, we aimed to clarify the role of SGK1 in bovine lipid accumulation and improvement of meat quality. METHODS: Preadipocytes were induced to differentiation to detect the temporal expression pattern of SGK1. Heart, liver, lung, spleen, kidney, muscle and fat tissues were collected to detect its tissue expression profile. Recombinant adenovirus and the lentivirus were packaged for overexpression and knockdown. Oil Red O staining, quantitative real-time PCR, Western blot analysis, Yeast two-hybrid assay, luciferase assay and RNA-seq were performed to study the regulatory mechanism of SGK1. RESULTS: SGK1 showed significantly higher expression in adipose and significantly induced expression in differentiated adipocytes. Furthermore, overexpression of SGK1 greatly promoted adipogenesis and inhibited proliferation, which could be shown by the remarkable increasement of lipid droplet, and the expression levels of adipogenic marker genes and cell cycle-related genes. Inversely, its knockdown inhibited adipogenesis and facilitated proliferation. Mechanistically, SGK1 regulates the phosphorylation and expression of two critical proteins of FoxO family, FOXO1/FOXO3. Importantly, SGK1 attenuates the transcriptional repression role of FOXO1 for PPARγ via phosphorylating the site S256, then promoting the bovine fat deposition. CONCLUSIONS: SGK1 is a required epigenetic regulatory factor for bovine preadipocyte proliferation and differentiation, which contributes to a better understanding of fat deposition and meat quality improvement in cattle.

Regulation of Oocyte mRNA Metabolism: A Key Determinant of Oocyte Developmental Competence.

The regulation of mRNA transcription and translation is uncoupled during oogenesis. The reason for this uncoupling is two-fold. Chromatin is only accessible to the transcriptional machinery during the growth phase as it condenses prior to resumption of meiosis to ensure faithful segregation of chromosomes during meiotic maturation. Thus, transcription rates are high during this time period in order to produce all of the transcripts needed for meiosis, fertilization, and embryo cleavage until the newly formed embryonic genome becomes transcriptionally active. To ensure appropriate timing of key developmental milestones including chromatin condensation, resumption of meiosis, segregation of chromosomes, and polar body extrusion, the translation of protein from transcripts synthesized during oocyte growth must be temporally regulated. This is achieved by the regulation of mRNA interaction with RNA binding proteins and shortening and lengthening of the poly(A) tail. This chapter details the essential factors that regulate the dynamic changes in mRNA synthesis, storage, translation, and degradation during oocyte growth and maturation.

E3 ubiquitin ligase UBR5 modulates circadian rhythm by facilitating the ubiquitination and degradation of the key clock transcription factor BMAL1.

The circadian clock is the inner rhythm of life activities and is controlled by a self-sustained and endogenous molecular clock, which maintains a ~ 24 h internal oscillation. As the core element of the circadian clock, BMAL1 is susceptible to degradation through the ubiquitin-proteasome system (UPS). Nevertheless, scant information is available regarding the UPS enzymes that intricately modulate both the stability and transcriptional activity of BMAL1, affecting the cellular circadian rhythm. In this work, we identify and validate UBR5 as a new E3 ubiquitin ligase that interacts with BMAL1 by using affinity purification, mass spectrometry, and biochemical experiments. UBR5 overexpression induced BMAL1 ubiquitination, leading to diminished stability and reduced protein level of BMAL1, thereby attenuating its transcriptional activity. Consistent with this, UBR5 knockdown increases the BMAL1 protein. Domain mapping discloses that the C-terminus of BMAL1 interacts with the N-terminal domains of UBR5. Similarly, cell-line-based experiments discover that HYD, the UBR5 homolog in Drosophila, could interact with and downregulate CYCLE, the BMAL1 homolog in Drosophila. PER2-luciferase bioluminescence real-time reporting assay in a mammalian cell line and behavioral experiments in Drosophila reveal that UBR5 or hyd knockdown significantly reduces the period of the circadian clock. Therefore, our work discovers a new ubiquitin ligase UBR5 that regulates BMAL1 stability and circadian rhythm and elucidates the underlying molecular mechanism. This work provides an additional layer of complexity to the regulatory network of the circadian clock at the post-translational modification level, offering potential insights into the modulation of the dysregulated circadian rhythm.

Alternative splicing of clock transcript mediates the response of circadian clocks to temperature changes.

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although clock (clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whether clk exhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed that clk transcripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative clk transcript, hereinafter termed clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, and in vitro experiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.

Photoregulatory protein kinases fine-tune plant photomorphogenesis by directing a bifunctional phospho-code on HY5 in Arabidopsis.

Photomorphogenesis is a light-dependent plant growth and development program. As the core regulator of photomorphogenesis, ELONGATED HYPOCOTYL 5 (HY5) is affected by dynamic changes in its transcriptional activity and protein stability; however, little is known about the mediators of these processes. Here, we identified PHOTOREGULATORY PROTEIN KINASE 1 (PPK1), which interacts with and phosphorylates HY5 in Arabidopsis, as one such mediator. The phosphorylation of HY5 by PPK1 is essential to establish high-affinity binding with B-BOX PROTEIN 24 (BBX24) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which inhibit the transcriptional activity and promote the degradation of HY5, respectively. As such, PPKs regulate not only the binding of HY5 to its target genes under light conditions but also HY5 degradation when plants are transferred from light to dark. Our data identify a PPK-mediated phospho-code on HY5 that integrates the molecular mechanisms underlying the regulation of HY5 to precisely control plant photomorphogenesis.

PDK1 promotes breast cancer progression by enhancing the stability and transcriptional activity of HIF-1α.

Pyruvate dehydrogenase kinase 1 (PDK1) phosphorylates the pyruvate dehydrogenase complex, which inhibits its activity. Inhibiting pyruvate dehydrogenase complex inhibits the tricarboxylic acid cycle and the reprogramming of tumor cell metabolism to glycolysis, which plays an important role in tumor progression. This study aims to elucidate how PDK1 promotes breast cancer progression. We found that PDK1 was highly expressed in breast cancer tissues, and PDK1 knockdown reduced the proliferation, migration, and tumorigenicity of breast cancer cells and inhibited the HIF-1α (hypoxia-inducible factor 1α) pathway. Further investigation showed that PDK1 promoted the protein stability of HIF-1α by reducing the level of ubiquitination of HIF-1α. The HIF-1α protein levels were dependent on PDK1 kinase activity. Furthermore, HIF-1α phosphorylation at serine 451 was detected in wild-type breast cancer cells but not in PDK1 knockout breast cancer cells. The phosphorylation of HIF-1α at Ser 451 stabilized its protein levels by inhibiting the interaction of HIF-1α with von Hippel-Lindau and prolyl hydroxylase domain. We also found that PDK1 enhanced HIF-1α transcriptional activity. In summary, PDK1 enhances HIF-1α protein stability by phosphorylating HIF-1α at Ser451 and promotes HIF-1α transcriptional activity by enhancing the binding of HIF-1α to P300. PDK1 and HIF-1α form a positive feedback loop to promote breast cancer progression.

Steroid receptor coactivator 1 promotes human hepatocellular carcinoma invasiveness through enhancing MMP-9.

SRC-1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC-1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC-1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta-catenin signalling. In this study, we uncover a previously unknown role of SRC-1 in HCC metastasis. Our findings reveal that SRC-1 promotes HCC metastasis through the augmentation of MMP-9 expression. The knockdown of SRC-1 effectively mitigated HCC cell metastasis both in vitro and in vivo by suppressing MMP-9 expression. Furthermore, we observed a positive correlation between SRC-1 mRNA levels and MMP-9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC-1 operates as a coactivator for NF-κB and AP-1, enhancing MMP-9 promoter activity in HCC cells. Higher levels of SRC-1 and MMP-9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC-1 expression, significantly decreased MMP-9 expression and inhibited HCC metastasis in both in vitro and in vivo settings. Our results demonstrated the pivotal role of SRC-1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.

Jun Dimerization Protein 2 (JDP2) Increases p53 Transactivation by Decreasing MDM2.

The AP-1 protein complex primarily consists of several proteins from the c-Fos, c-Jun, activating transcription factor (ATF), and Jun dimerization protein (JDP) families. JDP2 has been shown to interact with the cAMP response element (CRE) site present in many cis-elements of downstream target genes. JDP2 has also demonstrates important roles in cell-cycle regulation, cancer development and progression, inhibition of adipocyte differentiation, and the regulation of antibacterial immunity and bone homeostasis. JDP2 and ATF3 exhibit significant similarity in their C-terminal domains, sharing 60-65% identities. Previous studies have demonstrated that ATF3 is able to influence both the transcriptional activity and p53 stability via a p53-ATF3 interaction. While some studies have shown that JDP2 suppresses p53 transcriptional activity and in turn, p53 represses JDP2 promoter activity, the direct interaction between JDP2 and p53 and the regulatory role of JDP2 in p53 transactivation have not been explored. In the current study, we provide evidence, for the first time, that JDP2 interacts with p53 and regulates p53 transactivation. First, we demonstrated that JDP2 binds to p53 and the C-terminal domain of JDP2 is crucial for the interaction. Second, in p53-null H1299 cells, JDP2 shows a robust increase of p53 transactivation in the presence of p53 using p53 (14X)RE-Luc. Furthermore, JDP2 and ATF3 together additively enhance p53 transactivation in the presence of p53. While JDP2 can increase p53 transactivation in the presence of WT p53, JDP2 fails to enhance transactivation of hotspot mutant p53. Moreover, in CHX chase experiments, we showed that JDP2 slightly enhances p53 stability. Finally, our findings indicate that JDP2 has the ability to reverse MDM2-induced p53 repression, likely due to decreased levels of MDM2 by JDP2. In summary, our results provide evidence that JDP2 directly interacts with p53 and decreases MDM2 levels to enhance p53 transactivation, suggesting that JDP2 is a novel regulator of p53 and MDM2.

PPARγ Modulators in Lung Cancer: Molecular Mechanisms, Clinical Prospects, and Challenges.

Lung cancer is one of the most lethal malignancies worldwide. Peroxisome proliferator-activated receptor gamma (PPARγ, NR1C3) is a ligand-activated transcriptional factor that governs the expression of genes involved in glucolipid metabolism, energy homeostasis, cell differentiation, and inflammation. Multiple studies have demonstrated that PPARγ activation exerts anti-tumor effects in lung cancer through regulation of lipid metabolism, induction of apoptosis, and cell cycle arrest, as well as inhibition of invasion and migration. Interestingly, PPARγ activation may have pro-tumor effects on cells of the tumor microenvironment, especially myeloid cells. Recent clinical data has substantiated the potential of PPARγ agonists as therapeutic agents for lung cancer. Additionally, PPARγ agonists also show synergistic effects with traditional chemotherapy and radiotherapy. However, the clinical application of PPARγ agonists remains limited due to the presence of adverse side effects. Thus, further research and clinical trials are necessary to comprehensively explore the actions of PPARγ in both tumor and stromal cells and to evaluate the in vivo toxicity. This review aims to consolidate the molecular mechanism of PPARγ modulators and to discuss their clinical prospects and challenges in tackling lung cancer.

The N-terminal α2 helix element is critical for the activity of the rice transcription factor MYC2.

Jasmonates (JAs) are a class of phytohormones that play a crucial role in plant growth, development, and environmental stress responses. Central to JA signaling are the MYC2-type transcription factors, as they activate the expression of JA-responsive genes. We previously used CRISPR-Cas9-based genome editing to engineer rice OsMYC2 and yielded a mutant (myc2-5) with a single amino acid (aa) deletion (75I) outside the known functional domains of the protein. This myc2-5 mutant also showed some JA-deficient phenotypes, promoting us to investigate how 75I deletion affects JA responses. The mutation is found in the α2 helix element at the N-terminal of OsMYC2. The deletion of 75I in OsMYC2 rendered plants deficient in most of the JA responses, including root growth, leaf senescence, spikelet development, and resistance to pathogens and herbivores. Biochemical assays revealed that the 75I deletion markedly reduced OsMYC2 protein accumulation, subsequently diminishing its transcriptional activity. However, the deletion did not influence the protein's subcellular localization, DNA-binding capability, or its interactions with JAZ transcriptional repressors and the Mediator complex subunit MED25. Additionally, the screening of seven other deletions in the α2 helix further reinforces the importance of this protein element. Our results highlight the significance of the α2 helix in the N-terminus for OsMYC2's functionality, primarily through modulating its protein levels. This insight expands our knowledge of JA signaling and opens new avenues for research into the yet-to-be-explored domains of the MYC2 protein, with the potential to tailor JA responses in rice and other plant species.

IKZF4/NONO-RAB11FIP3 axis promotes immune evasion in gastric cancer via facilitating PD-L1 endosome recycling.

As an immune checkpoint protein expressed by diverse cancer cells, programmed death ligand 1 (PD-L1) facilitates immune evasion by interacting with programmed cell death-1 (PD-1) on T cells. Despite the clinical benefits observed in various cancer types, strategies targeting PD-1/PD-L1 have demonstrated limited efficacy in gastric cancer (GC). Furthermore, the regulation of PD-L1, especially at post-translational modification levels, remains largely unknown. Therefore, it is crucial to elucidate the mechanisms governing PD-L1 expression to enhance anti-tumor immunity. In this study, we have identified that IKAROS family zinc finger 4 (IKZF4) and Non-POU domain-containing octamer-binding (NONO) synergistically regulate and enhance the expression of RAB11 family-interacting protein 3 (RAB11FIP3) in GC. The IKZF4/NONO-RAB11FIP3 axis facilitates the endosomal recycling of PD-L1, particularly on the cell membrane of GC cells. Moreover, overexpression of RAB11FIP3 mitigates the hypo-expression of PD-L1 protein resulting from IKZF4 or NONO deletion. Functionally, the silencing of RAB11FIP3 or IKZF4 promotes T cell proliferation, and enhances T-cell cytotoxicity towards GC cells in vitro, which further inhibits tumor immune evasion in mice via increasing the infiltration of CD8+ T cells into the tumor microenvironment (TME) to suppress GC progression. Our study suggests that the IKZF4/NONO-RAB11FIP3 axis promotes immune evasion by facilitating PD-L1 endosome recycling, thus presenting a potential therapeutic target for GC treatment.

Molecular study of the presence and transcriptional activity of HPV in semen.

PURPOSE: Human Papillomavirus (HPV) in semen represents a controversial topic. Recent evidence suggests a correlation with poor semen quality, but its detection is still unstandardized in this biological fluid. Thus, the aims of this study were to verify the ability of nested PCR to reveal HPV-DNA in semen; to evaluate association of seminal HPV with sperm parameters and risk factors for infection; to investigate the rate of HPV-DNA positivity in patients with and without risk factors; to assess HPV transcriptional activity. METHODS: We enrolled sexually active men and collected clinical and anamnestic data during andrological and sexually transmitted infections (STIs) evaluation. For each patient, we performed semen analysis and nested PCR to detect HPV-DNA in semen. In positive semen samples, we proceeded with genotyping and RNA quantification to detect HPV transcriptional activity. RESULTS: We enrolled 185 men (36.0 ± 8.3 years), of which 85 with (Group A) and 100 without HPV risk factors (Group B). Nested PCR was able to reveal HPV-DNA in semen, discovering a prevalence of 8.6% (11.8% in Group A and 6% in Group B, respectively). We observed no correlation between sperm quality and seminal HPV. Genital warts and previous anogenital infection were significantly associated with the risk of HPV positivity in semen. Moreover, no viral transcriptional activity was detected in positive semen samples. CONCLUSIONS: Our study suggests that searching for seminal HPV could be important in patients both with and without risk factors, especially in assisted reproduction where the risk of injecting sperm carrying HPV-DNA is possible.

CCR5 promoter polymorphisms associated with nonsmall cell lung cancer.

C-C chemokine receptor 5 (CCR5) plays a crucial role in the regulation of immune cell activation and migration as well as the progression of many cancers. We performed an in silico analysis using public data resources and found that the lung cancer patients with higher CCR5 expression had a notably better overall survival than those with lower CCR5 expression patients and CCR5 expression level is positive correlated with the infiltration of immune cells, such as B, CD8+ T and CD4+ T cells, in both lung adenocarcinoma and lung squamous cell cancer. In the present study, we investigated the association between the promoter polymorphism of CCR5 and nonsmall cell lung cancer (NSCLC). A case-control study of 449 NSCLC patients and 516 controls of Chinese Han population was conducted, along with polymorphism detection using a sequencing method. A dual-luciferase reporter assay system was used to analyse the transcriptional activity of CCR5 promoter variations. Our results showed that the frequency of rs1799987-AA was significantly higher in the NSCLC group than in the controls in recessive model (p = .007, OR = 1.66 95% confidence interval [CI]: 1.14-2.40, adjusted by sex and age); the G allele showed a significant associated with NSCLC in dominant model (p = .003, OR = 1.64, 95%CI: 1.18-2.28, adjusted by sex and age). Compared with haplotype H1 rs2227010-rs2734648-rs1799987-rs1799988-rs1800023-rs1800024: A-T-G-T-G-C, haplotype H5: A-G-G-T-G-C increased the risk of NSCLC by over 10-fold (p < .0001, OR = 16.09, 95%CI: 5.37-48.20, adjusted by sex and age) and notably depressed the transcriptional activity of the CCR5 promoter in 293T, A549, H1299 and HeLa cells. In conclusion, CCR5 promoter polymorphisms are significantly associated with NSCLC by affecting the transcriptional activity of the CCR5 promoter.

The presence of a G-quadruplex prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast Saccharomyces cerevisiae.

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

Discordance between chromatin accessibility and transcriptional activity during the human primed-to-naïve pluripotency transition process.

Naïve pluripotent state can be obtained by several strategies from various types of cells, in which the cell fate roadmap as well as key biological events involved in the journey have been described in detail. Here, we carefully explored the chromatin accessibility dynamics during the primed-to-naïve transition by adopting a dual fluorescent reporter system and the assay for transposase-accessible chromatin (ATAC)-seq. Our results revealed critical chromatin remodeling events and highlight the discordance between chromatin accessibility and transcriptional activity. We further demonstrate that the differential epigenetic modifications and transcription factor (TF) activities may play a critical role in regulating gene expression, and account for the observed variations in gene expression despite similar chromatin landscapes.