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-1β and receptor activator of NF-κB ligand in the periodontal tissue. Hence, phosphorylation of S536 negatively regulates inflammatory responses in vitro and in vivo.

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 (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-β 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-β-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.

OBJECTIVE: 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.

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.

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.

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.

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.

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.

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.