Promoter Activity Research Articles

Effect of HNF4 on the Process of Liver Cancer and as a Potential Therapeutic Target

As a ligand-dependent transcription factor of the nuclear receptor family, HNF4 can be regulated by a variety of pathways, and can also be recruited to the DNA sequence through a variety of pathways to regulate downstream target genes by virtue of its three-dimensional structure. HNF4 is usually enriched in organs such as the liver, gallbladder, pancreas, kidneys, etc., and mutations in the HNF4A gene can cause lesions in these organs, including diseases such as liver cancer. However, the pathological role of HNF4 in different diseases is not well understood. Previous studies have shown that 12 variants of HNF4 are jointly regulated by the P1/P2 promoters. HNF4 can be recruited to specific DNA sequences by intermediates (including MLL4, KAT2B, etc.) through DNA-binding domain (DBD) after ligand-binding domain (LBD) has bound ligands. The review goes on to enumerate several pathways, pathways in which HNF4A expression is regulated, and HNF4 acts on promoters or enhancers to regulate the expression of downstream target genes. KDM1A regulates the HNF4A expression pathway, mutations in HNF1 POU domain to downregulate HNF4A promoter activity and NRF2-mediated STAT3 activation to silence HNF4A expression through miR-4, and the functional differences of different subtypes of HNF4 regulated by P1/P2, and the interaction between HNF4 and HIC1 affects the function of HNF4 in regulating FUF/FDF. This review aims to stimulate further research on HNF4, and the specific pathogenesis is clearly elucidated to find suitable targets to treat the corresponding diseases.

Correction: Yang et al. SOX2 Represses Hepatitis B Virus Replication by Binding to the Viral EnhII/Cp and Inhibiting the Promoter Activation. Viruses 2020, 12, 273

In the original publication [...]

Synthesis and biological assessment of chalcone and pyrazoline derivatives as novel inhibitor for ELF3-MED23 interaction.

HER2 overexpression significantly contributes to the aggressive nature and recurrent patterns observed in various solid tumors, notably gastric cancers. Trastuzumab, HER2-targeting monoclonal antibody drug, has shown considerable clinical success; however, readily emerging drug resistance emphasizes the pressing need for improved interventions in HER2-overexpressing cancers. To address this, we proposed targeting the protein-protein interaction (PPI) between ELF3 and MED23 as an alternative therapeutic approach to trastuzumab. In this study, we synthesized a total of 26 compounds consisting of 10 chalcones, 7 pyrazoline acetyl, and 9 pyrazoline propionyl derivatives, and evaluated their biological activity as potential ELF3-MED23 PPI inhibitors. Upon systematic analysis, candidate compound 10 was selected due to its potency in downregulating reporter gene activity of ERBB2 promoter confirmed by SEAP activity and its effect on HER2 protein and mRNA levels. Compound 10 effectively disrupted the binding interface between the ELF3 TAD domain and the 391-582 amino acid region of MED23, resulting in successful inhibition of the ELF3-MED23 PPI. This intervention led to a substantial reduction in HER2 levels and its downstream signals in the HER2-positive gastric cancer cell line. Subsequently, compound 10 induced significant apoptosis and anti-proliferative effects, demonstrating superior in vitro and in vivo anticancer activity overall. We found that the anticancer activity of compound 10 was not only restricted to trastuzumab-sensitive cases, but was also valid for trastuzumab-refractory clones. This suggests its potential as a viable therapeutic option for trastuzumab-resistant gastric cancers. In summary, compound 10 could be a novel alternative therapeutic strategy for HER2-overexpressing cancers, overcoming the limitations of trastuzumab.

Synthesis and biological assessment of chalcone and pyrazoline derivatives as novel inhibitor for ELF3-MED23 interaction

HER2 overexpression significantly contributes to the aggressive nature and recurrent patterns observed in various solid tumors, notably gastric cancers. Trastuzumab, HER2-targeting monoclonal antibody drug, has shown considerable clinical success; however, readily emerging drug resistance emphasizes the pressing need for improved interventions in HER2-overexpressing cancers. To address this, we proposed targeting the protein-protein interaction (PPI) between ELF3 and MED23 as an alternative therapeutic approach to trastuzumab. In this study, we synthesized a total of 26 compounds consisting of 10 chalcones, 7 pyrazoline acetyl, and 9 pyrazoline propionyl derivatives, and evaluated their biological activity as potential ELF3-MED23 PPI inhibitors. Upon systematic analysis, candidate compound 10 was selected due to its potency in downregulating reporter gene activity of ERBB2 promoter confirmed by SEAP activity and its effect on HER2 protein and mRNA levels. Compound 10 effectively disrupted the binding interface between the ELF3 TAD domain and the 391–582 amino acid region of MED23, resulting in successful inhibition of the ELF3-MED23 PPI. This intervention led to a substantial reduction in HER2 levels and its downstream signals in the HER2-positive gastric cancer cell line. Subsequently, compound 10 induced significant apoptosis and anti-proliferative effects, demonstrating superior in vitro and in vivo anticancer activity overall. We found that the anticancer activity of compound 10 was not only restricted to trastuzumab-sensitive cases, but was also valid for trastuzumab-refractory clones. This suggests its potential as a viable therapeutic option for trastuzumab-resistant gastric cancers. In summary, compound 10 could be a novel alternative therapeutic strategy for HER2-overexpressing cancers, overcoming the limitations of trastuzumab.

Repressor MrERF4 and Activator MrERF34 Synergistically Regulate High Flavonol Accumulation Under UV-B Irradiation in Morella rubra Leaves.

Flavonols are important plant photoprotectants to defence UV-B irradiation, however, the underlying transcriptional regulatory mechanism of rapid flavonol accumulation in response to UV-B remains unknown. In this study, content of flavonols was significantly induced from 0.11 to 3.80 mg/g fresh weight by UV-B irradiation in leaves of Morella rubra seedlings. MrERF34 was identified as an activator that can regulate the expression of MrFLS2, and promoted flavonol biosynthesis with activator MrMYB12 under UV-B treatment. Transient overexpression of MrERF34 resulted in higher flavonol accumulation, while virus-induced gene silencing of MrERF34 reduced the content of flavonols in bayberry leaves. We further demonstrated that a repressor MrERF4 inhibited the expression of MrERF34 and MrMYB12 as well as MrFLS2 via ERF-associated-amphiphilic repression motif. Exposure to UV-B reduced the promoter activity and transcription of MrERF4, which weakened the inhibitory effect of MrERF4 on MrERF34, MrMYB12, and MrFLS2, leading to a tremendous accumulation of flavonols. Such inhibitory roles of MrERF4 in regulation of flavonol biosynthesis were further validated by transient overexpression in leaves of Nicotiana benthamiana and M. rubra. These findings enrich the synergistical regulatory mechanisms between repressor and activators in flavonol biosynthesis, and provide new insights into photoprotectants biosynthesis to mitigate UV-B stress in plants.

Berberine promotes K48-linked polyubiquitination of HNF4α, leading to the inhibition of HBV replication.

The current antiviral agents for the treatment of chronic infection with hepatitis B virus (HBV) do not completely remove covalently closed circular DNA (cccDNA) and integrated viral DNA fragments from patients. Berberine is an isoquinoline alkaloid extracted from various plants and has been reported to inhibit the replication of various types of DNA. In this study, we tested the effects of berberine and its derivatives on HBV infection. Berberine inhibited viral core promoter activity at the highest level among the compounds tested and suppressed HBV production and cccDNA synthesis in primary human hepatocytes and HBV-infected HepG2-NTCP cells at an EC50 value of 3.6 μM and a CC50 value of over 240.0 μM. Compared with other viral promoter activities, berberine treatment potently downregulated core promoter activity and reduced protein levels, but not RNA levels, of hepatic nuclear factor 4α (HNF4α), which primarily enhances enhancer II/core promoter activity. Furthermore, berberine treatment enhanced K48-linked, but not K63-linked, polyubiquitination and subsequent proteasome-dependent degradation of HNF4α. These results suggest that berberine enhances the polyubiquitination- and proteasome-dependent degradation of HNF4α and then inhibits HBV replication via the suppression of core promoter activity. The development of antiviral agents based on berberine may contribute to the amelioration of HBV-related disorders, regardless of the presence of residual cccDNA or integrated viral DNA fragments.

BRG1 improves reprogramming efficiency by enhancing glycolytic metabolism

BRG1 has been found to promote the generation of induced pluripotent stem cells (iPSCs) by regulating epigenetic modifications or binding to transcription factors, however, the role of BRG1 on the cellular metabolism during reprogramming has not been reported. In this study, we found that BRG1 improved the efficiency of porcine iPSC generation, and upregulated the expression of pluripotency-related factors. Further analysis revealed that BRG1 promoted cellular glycolysis, and increased levels of glycolysis-related metabolites. It enhanced the transcriptional activity of glycolysis-related gene HK2, PKM2, and PFK-1 promoters, and decreased the enrichment of H3K9me3 in glycolysis- and pluripotency-related gene promoters. BRG1 also increased the phosphorylation level at the Ser473 site of AKT protein. The specific PI3K/AKT signaling pathway inhibitor, LY294002, impaired the generation of porcine iPSCs, downregulated the expression of pluripotency-related factors, and inhibited cellular glycolysis, overexpressing BRG1 rescued those changes caused by LY294002 treatment. In addition, the glycolysis inhibitor 2-DG and BRG1 inhibitor PFI-3 had similar effects to LY294002. The above results suggest that overexpression of BRG1 promotes the generation of porcine iPSCs by facilitating glycolytic reprogramming through the PI3K/AKT signaling pathway.

Roles of S100A1 and S100A10 from hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) in the immune response to Vibrio infection

The S100 proteins are highly conserved EF-hand calcium-binding proteins found only in vertebrates. In the current study, two S100 genes (S100A1 and S100A10) were successfully identified and characterized from hybrid grouper Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀. The deduced S100A10 protein contained two EF-hand domains, and S100A1 only possessed the N-terminal EF-hand. Phylogenetic analysis revealed that S100A1 and S100A10 from hybrid grouper were evolutionarily closely related to their counterparts in other selected vertebrates. Quantitative real-time PCR results revealed that the transcripts of S100A1 and S100A10 mRNA were ubiquitously distributed in all the examined tissues. After Vibrio alginolyticus infection, the expression of S100A1 and S100A10 in the spleen increased significantly. Moreover, overexpression of S100A1 and S100A10 could not only regulate the expression of interleukin 8 (IL-8), IL-10, and IL-16 in the head kidney, liver, and spleen, change the activities of acid phosphatase, catalase, lysozyme, and superoxide dismutase in serum, but also reduce the promoter activities of interferon 3 and nuclear factor kappa-B in vitro. Taken together, this study indicated that S100A1 and S100A10 participate in the immune response of hybrid grouper against bacterial infection.

The R2R3 MYB700 activates the expression of biosynthetic genes involved in the accumulation of (+)-catechin but not (-)-epicatechin in Chinese plum fruits (Prunus salicina Lindl.)

Chinese plum fruits are rich in phenolic compounds from the flavan-3-ol family, including (+)-catechin and (-)-epicatechin, widely associated with sensory attributes such as bitterness and astringency, beneficial for human health, and involved in enzymatic browning. Leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) enzymes catalyze (+)-catechin and (-)-epicatechin biosynthesis, respectively. It has been described that genes encoding for LAR and ANR are transcriptionally regulated by transcription factors belonging to MYB, bHLH, and bZIP families. In this study, we examined two Chinese plum cultivars (‘98–99’ and ‘Angeleno’) with different flavan-3-ol profiles to characterize MYB transcription factors involved in malus the accumulation of (+)-catechin and (-)-epicatechin in fruits. We observed that fruits of ‘98–99’ accumulate a higher content of (+)-catechin than (-)-epicatechin, while ‘Angeleno’ displays an opposite profile. We identified PsaMYBPA1, PsaMYB7, and a MYB transcription factor not previously described in the literature, which we named PsaMYB700. The expression of PsaMYB700 was up to 10-fold higher in ‘98–99’ than ‘Angeleno’ at the earliest fruit developmental stage. Promoter activation assays indicated that PsaMYBPA1 activates the flavan-3-ol-related genes LAR, anthocyanidin synthase (ANS), and ANR, while PsaMYB7 and PsaMYB700 activate LAR and ANS. In contrast to PsaMYBPA1 and PsaMYB7, PsaMYB700 displays high activation levels without the presence of a bHLH co-regulator. Further, the expression levels of PsaMY700 positively correlate with the higher (+)-catechin accumulation in fruits of ‘98–99,’ compared with ‘Angeleno.’ Finally, we detected a PsabZIP5 with similar expression levels between cultivars, which activates the promoters of PsaMYBPA1, PsaMYB7, and PsaMYB700. This study elucidates the roles of PsaMYBPA1, PsaMYB7, PsaMYB700 and PsabZIP5 in flavan-3-ol biosynthesis, highlighting a hierarchy of transcriptional regulation of these compounds.

Development of a Myogenin minimal promoter-based system for visualizing the degree of myogenic differentiation

Myogenic differentiation plays a fundamental role in myogenesis during development and in muscle regeneration. Sequential expression of myogenic regulatory factors (MRFs) including myogenin in the progenitor cells triggers the expression of effector proteins such as myosin heavy chain (MHC), leading to the terminal muscle differentiation. Although we have a snapshot-like understanding of molecules at each stage of the differentiation, how these molecules are interrelated in the continuum of myogenic differentiation remains poorly understood. In this study, we analyzed the dynamics of the minimal Myogenin promoter activity in live myoblasts. With the development of a new co-expression analysis method, we were able to reveal in detail the relationship between this Myogenin promoter activity and the expression of endogenous myogenin or MHC, as differentiation markers. Consequently, we found that our visualization system of myogenic differentiation is suitable for monitoring the transition from myoblasts to myotubes, in which the Myogenin promoter activity quantitatively represents the degree of myogenic differentiation. Thus, this system allows simultaneous observation of the degree of myoblast differentiation in relation to other molecules, which would contribute to deepening our understanding of myogenic differentiation as a continuous process.

Genome-wide survey and expression analysis of JAZ genes in watermelon (Citrullus lanatus).

BACKGROUND JAZ: (Jasmonate ZIM-domain) genes play important roles in plant growth and JA signaling pathway which is correlated with fruit ripening process. However, there have been few reports on the genome-wide identification of JAZ genes in watermelon and its relationship with fruit ripening. METHODS AND RESULTS: In this study, bioinformatics approaches were employed to identify ClaJAZ genes of watermelon at the genome-wide levels. Further exploration delved into the phylogenetic relationships, chromosomal mappings, promoter dynamics, expression, and architectural features of the JAZ genes. The results showed that a total of 9 ClaJAZ genes unevenly distributed across six chromosomes were identified in the watermelon genome, and they all have conserved Jas and TIFY domains. These JAZ genes were divided into four distinct groups with five genes involved in inter-chromosomal tandem duplication events, and members of the same subgroup exhibited a high degree of similarity in their gene structure and protein motif patterns. Analysis of the promoter regions of the ClaJAZ genes indicated the presence of cis-acting elements associated with hormonal responses, stress, and developmental processes. Gene expression analysis through real-time quantitative PCR (qRT-PCR) showed that there were spatiotemporal differences in the expression of ClaJAZ genes at various stages of fruit development. Among them, ClaJAZ7 has the highest level of transcriptional expression and showed strong promoter activity. CONCLUSIONS: This study conducted a comprehensive analysis of the ClaJAZ genes and provided insights into the role of ClaJAZ in the development and ripening of watermelon fruit.

ZmEREB25 transcription factor mediates transactivation of core starch synthetic genes in maize endosperm via interaction with ZmARF27

Starch, as the primary storage material in maize endosperm, is essential in determining yield and quality. Although the starch biosynthetic pathway in maize has been well-documented, the transcriptional network underlying endosperm starch synthesis remains elusive. Through a comprehensive co-expression analysis, we screened an endosperm-preferential AP2/ERF transcription factor ZmEREB25, which exhibited a strong correlation with the expression pattern of starch biosynthetic genes in maize endosperm. ZmEREB25 enhanced the promoter activities of the core starch biosynthetic genes, namely Sh2, SSIIIa and SSI, through specific binding to the GCCGAC-containing elements present in their promoters. Given that ZmEREB25 lacked the transactivation capacity, we further identified an ARF transcription factor, ZmARF27, that interacted with ZmEREB25 to coordinately transactivate the promoters of Sh2, SSIIIa and SSI genes via direct binding to these promoters. Our present study demonstrated that the ZmEREB25-ZmARF27 complex is crucial for transactivating core starch synthetic genes in maize endosperm and uncovered a novel regulatory pathway for starch synthesis in maize endosperm.

Subunits Med12 and Med13 of Mediator Cooperate with Subunits SAYP and Bap170 of SWI/SNF in Active Transcription in Drosophila

SAYP and Bap170, subunits of the SWI/SNF remodeling complex, have the ability to support enhancer-dependent transcription when artificially recruited to the promoter on a transgene. We found that the phenomenon critically depends on two subunits of the Mediator kinase module, Med12 and Med13 but does not require the two other subunits of the module (Cdk8 and CycC) or other subunits of the core part of the complex. A cooperation of the above proteins in active transcription was also observed at endogenous loci, but the contribution of the subunits to the activity of a particular gene differed in different loci. The factors SAYP/Bap170 and Med12/Med13 did not form sufficiently stable interactions in the extract, and their cooperation was apparently local at regulatory elements, the presence of SAYP and Bap170 in a locus being necessary for stable recruitment of Med12 and Med13 to the locus. In addition to the above factors, the Nelf-A protein was found to participate in the process. The cooperation of the factors, independent of enzymatic activities of the complexes they are part of, appears to be a novel mechanism that maintains promoter activity and may be used in many loci of the genome. Extended intrinsically disordered regions of the factors were assumed to sustain the mechanism.

HNF-1β alleviates podocyte injury in lupus nephritis by maintaining endoplasmic reticulum homeostasis

ObjectiveThe current study aims to elucidate the critical function of hepatocyte nuclear factor 1-beta (HNF1-β) in lupus nephritis (LN) by investigating its modulation of the Derlin-1/valosin-containing protein (VCP)/VCP-interacting membrane selenoprotein...

LDHB Mediates Histone Lactylation to Activate PD-L1 and Promote Ovarian Cancer Immune Escape

Background To investigate the effects of LDHB on lactylation of programmed cell death 1 ligand (PD-L1) and immune evasion of ovarian cancer. Methods Ovarian cancer cells were transfected with LDHB siRNA and cultured with primed T cells. Cell proliferation and viability were measured by cell counting kit 8 (CCK-8) and colony formation assay. The production of immune factors was detected by enzyme-linked immunosorbent assay (ELISA). The histone lactylation and activity of PD-L1 promoter were measured by chromatin immunoprecipitation (ChIP)-qPCR assay and luciferase reporter gene assay, respectively. Results Knockdown of LDHB notably inhibited the growth, glucose uptake, lactate production, and ATP production of ovarian cancer cells. Knockdown of LDHB enhanced the killing effects of T cells, led to increased production of immune activation factors IL-2, TNF-α, and IFN-γ, as well as elevated the levels of granzyme B and perforin. Mechanical study identified that LDHB regulated the H3K18 lactylation (H3K18la) modification on PD-L1 promoter region to promote its expression. Overexpression of PD-L1 abolished the immune activation effects that induced by siLDHB. Conclusion The LDHB modulated lactate production and the histone lactylation on PD-L1 promoter, which ultimately regulated its expression and participated in the immune evasion of ovarian cancer cells.

Evaluating the predictive power of combined gene expression dynamics from single cells on antibiotic survival.

Heteroresistance can allow otherwise drug-susceptible bacteria to survive and resume growth after antibiotic exposure. This temporary form of antibiotic tolerance can be caused by the upregulation of stress response genes or a decrease in cell growth rate. However, it is not clear how expression of multiple genes contributes to the tolerance phenotype. By using fluorescent reporters for stress related genes, we conducted real time measurements of expression prior to, during, and after antibiotic exposure. We first identified relationships between growth rate and reporter levels based on auto and cross correlation analysis, revealing consistent patterns where changes in growth rate were anticorrelated with fluorescence following a delay. We then used pairs of stress gene reporters and time lapse fluorescence microcopy to measure the growth rate and reporter levels in cells that survived or died following antibiotic exposure. Using these data, we asked whether combined information about reporter expression and growth rate could improve our ability to predict whether a cell would survive or die following antibiotic exposure. We developed a Bayesian inference model to predict how the combination of dual reporter expression levels and growth rate impact ciprofloxacin survival in Escherichia coli . We found clear evidence of the impact of growth rate and the gadX promoter activity on survival. Unexpectedly, our results also revealed examples where additional information from multiple genes decreased prediction accuracy, highlighting an important and underappreciated effect that can occur when integrating data from multiple simultaneous measurements.

HDAC4 Inhibits NMDA Receptor-mediated Stimulation of Neurogranin Expression.

The coordination of neuronal wiring and activity within the central nervous system (CNS) is crucial for cognitive function, particularly in the context of aging and neurological disorders. Neurogranin (Ng), an abundant forebrain protein, modulates calmodulin (CaM) activity and deeply influences synaptic plasticity and neuronal processing. This study investigates the regulatory mechanisms of Ng expression, a critical but underexplored area for combating cognitive impairment. Utilizing both in vitro and in vivo hippocampal models, we show that Ng expression arises during late developmental stages, coinciding with the processes of synaptic maturation and neuronal circuit consolidation. We observed that Ng expression increases in neuronal networks with heightened synaptic activity and identified GluN2B-containing N-methyl-D-aspartate (NMDA) receptors as key drivers of this expression. Additionally, we discovered that nuclear-localized HDAC4 inhibits Ng expression, establishing a regulatory axis that is counteracted by NMDA receptor stimulation. Analysis of the Ng gene promoter activity revealed regulatory elements between the - 2.4 and - 0.85 Kbp region, including a binding site for RE1-Silencing Transcription factor (REST), which may mediate HDAC4's repressive effect on Ng expression. Further analysis of the promoter sequence revealed conserved binding sites for the myocyte enhancer factor-2 (MEF2) transcription factor, a target of HDAC4-mediated transcription regulation. Our findings elucidate the interplay between synaptic activity, NMDAR function, and transcriptional regulation in controlling Ng expression, offering insights into synaptic plasticity mechanisms and potential therapeutic strategies to prevent cognitive dysfunction.

Advancing protein display on bacterial spores through an extensive survey of coat components.

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SP investigated, 26 formed functional fusions with β-glucuronidase (GUS) - a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed the 1) SP promoter activation dynamics during growth, 2) supported GUS activity on spores, 3) surface availability, 4) protection from thermal and proteolytic degradation, and 5) compatibility of co-expression of many of these fusions. Multi-copy expression and pairwise co-expression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and difficulty in predicting compatibility between different SPs fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA ( s mall s pore c oat assembly protein A ) as an effective spore display carrier. SscA supported enzyme activity at least fourfold higher than any other SP, including the well-established and popular anchor, CotY. We attribute this to the biochemical and genetic features of SscA; its promoter demonstrated early and sustained activation relative to other SPs and its small size (∼3 KDa) likely minimally interferes with enzyme folding, oligomerization, and activity. Although the specific localization of SscA within the spore coat has not been established, its hydrophobic nature and low surface availability suggests that it assembles internally within the spore coat, which makes it highly stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledgebase to advance the biotechnological utility of B. subtilis spores.

PERM1 regulates mitochondrial energetics through O-GlcNAcylation in the heart

PERM1 was initially identified as a new downstream target of PGC-1α and ERRs that regulates mitochondrial bioenergetics in skeletal muscle. Subsequently, we and other groups demonstrated that PERM1 is also a positive regulator of mitochondrial bioenergetics in the heart. However, the exact mechanisms of regulatory functions of PERM1 remain poorly understood. O-GlcNAcylation is a post-translational modification of proteins that are regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. O-GlcNAcylation is a powerful signaling mechanism mediating cellular responses to stressors and nutrient availability, which, among other targets, may influence cardiac metabolism. We hypothesized that PERM1 regulates mitochondrial energetics in cardiomyocytes through modulation of O-GlcNAcylation. We found that overexpression of PERM1 decreased the total levels of O-GlcNAcylated proteins, concomitant with decreased OGT and increased OGA expression levels. Luciferase gene reporter assay showed that PERM1 significantly decreases the promoter activity of Ogt without changing the promoter activity of Oga. The downregulation of OGT by PERM1 overexpression was mediated through its interaction with E2F1, a known transcription repressor of Ogt. A deliberate increase of O-GlcNAcylation through Oga silencing in cardiomyocytes decreased the basal and maximal mitochondrial respiration and ATP production rates, all of which were completely restored by PERM1 overexpression. Furthermore, excessive O-GlcNAcylation caused by the loss of PERM1 led to the increase of O-GlcNAcylated PGC-1α, a master regulator of mitochondrial bioenergetics, concurrent with the dissociation of PGC-1α from PPARα, a well-known transcription factor that regulates fatty acid β-oxidation. We conclude that PERM1 positively regulates mitochondrial energetics, in part, via suppressing O-GlcNAcylation in cardiac myocytes.

Expression and regulation of SETBP1 in the song system of male zebra finches (Taeniopygia guttata) during singing

Rare de novo heterozygous loss-of-function SETBP1 variants lead to a neurodevelopmental disorder characterized by speech deficits, indicating a potential involvement of SETBP1 in human speech. However, the expression pattern of SETBP1 in brain regions associated with vocal learning remains poorly understood, along with the underlying molecular mechanisms linking it to vocal production. In this study, we examined SETBP1 expression in the brain of male zebra finches, a well-established model for studying vocal production learning. We demonstrated that zebra finch SETBP1 exhibits a greater number of exons and isoforms compared to its human counterpart. We characterized a SETBP1 antibody and showed that SETBP1 colocalized with FoxP1, FoxP2, and Parvalbumin in key song nuclei. Moreover, SETBP1 expression in neurons in Area X is significantly higher in zebra finches singing alone, than those singing courtship song to a female, or non-singers. Importantly, we found a distinctive neuronal protein expression of SETBP1 and FoxP2 in Area X only in zebra finches singing alone, but not in the other conditions. We demonstrated SETBP1´s regulatory role on FoxP2 promoter activity in vitro. Taken together, these findings provide compelling evidence for SETBP1 expression in brain regions to be crucial for vocal learning and its modulation by singing behavior.