Transcriptional Function Research Articles

Correlation Analysis of Macrophage Distribution and Pathological Features of Carotid Atherosclerotic Plaque

Macrophages play a crucial role in sustaining chronic inflammation in atherosclerosis (AS) and are a hallmark of atherosclerotic plaques. They undergo differentiation into distinct subpopulations under the influence of oxidized lipids and cytokines, exerting various functions in plaque formation and progression. To examine the variations in the quantity and distribution of different macrophage subpopulations surrounding distinct pathological features of carotid plaques in human AS. Based on these findings, we seek to explore the potential correlations between specific macrophage subpopulations and AS pathological characteristics. Initially, we analyzed single-cell RNA sequencing (scRNA-Seq) data obtained from the GEO DataSets (GSE159677). The results revealed a significant infiltration of macrophages in AS, categorizing them into distinct subpopulations with diverse transcriptional features and functions. Furthermore, in different regions of AS (i.e., calcified AS core vs. proximal adjacent areas), both the total number of macrophages and the subpopulation ratios exhibited variations. Subsequently, we examined the distribution differences of macrophage subpopulations within 61 surgically resected AS plaques using marker staining techniques such as CD68, iNOS, Arg-1, CD163, and HO-1. The findings demonstrated that macrophages predominantly localized in specific pathological lesions, including necrosis, fibrous tissue degeneration, and cholesterol crystallization. Moreover, distinct subpopulations exhibited differential distribution patterns within these lesions. This study provides confirmation of extensive macrophage infiltration within atherosclerotic plaques, highlighting the presence of subtypes that exhibit variability across different pathological lesions. These results suggest that various macrophage subpopulations may contribute differently to distinct pathological lesions in AS.

Perinatal murine cytomegalovirus infection reshapes the transcriptional profile and functionality of NK cells

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow’s capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality.

A novel stop-loss mutation in NKX2-2 gene as a cause of neonatal diabetes mellitus: molecular characterization and structural analysis.

To identifythe genetic etiology of neonatal diabetes in an infant and to elucidate the molecular mechanism of the identified mutation underlying the pathogenesis. Genetic analysis was carried out by sequencing of known etiological genes associated with NDM. Molecular characterization was performed by constructing aidentified mutation inNKX2-2 geneand functional aspects wastested using transactivation, protein expression, DNA binding, nuclear localization assays. Structural analysis was performed bymodeling the NKX2-2 protein structure. A novel homozygous frameshift mutationc.772delC, p.Q258SFs*59in the NKX2-2 gene was identified in a patient with neonatal diabetes. Functional studies revealed that this mutation resulted in an elongated protein sequence, affecting DNA binding activity and transcriptional function. Structural analysis suggested alterations in the protein's tertiary structure, likely contributing to its dysfunction. This study presents the first report of a stop-loss mutation in the NKX2-2 gene associated with NDM. Our findings emphasize the importance of functional and structural characterization to understand the biological consequences of such mutations. This comprehensive analysis provides insights into the molecular mechanisms underlying NDM and its clinical phenotype, which may aid in better diagnosis and management of patients with similar variants in the future.

THU569 Steroid Receptor Coactivator 3 Is Required To Prevent Early Pregnancy Loss In The Mouse

Abstract Disclosure: V. Maurya: None. L. Hai: None. M.M. Szwarc: None. W. San-Pin: None. F.J. DeMayo: None. J.P. Lydon: None. Members of the Steroid Receptor Coactivator (SRC) family enact distinct transcriptional coregulator functions within a broad spectrum of physiologies and pathologies. We previously demonstrated that endometrial SRC-3 is critical for in vitro human endometrial stromal cell decidualization, a critical cellular process in the establishment of the maternofetal interface. To address SRC-3’s in vivo role in endometrial periimplantation biology, we recently generated a SRC-3 conditional knockout mouse model (SRC-3d/d) in which SRC-3 is selectively ablated in cells that express the progesterone receptor. A six-month breeding trial with wild type proven stud males demonstrated that the SRC-3d/d female fails to produce litters (n>10 per genotype). Unlike the SRC-2d/d female, however, both embryo attachment and implantation into the stromal compartment of the SRC-3d/d endometrium are not impaired at gestation day (GD) 5 or 6. While endometrial stromal decidualization initiates with embryo invasion, pregnant SRC-3d/d mice exhibit early pregnancy loss (EPL) by GD-7.5 (before placentation). At GD-6, the SRC-3d/d decidua is significantly smaller than those of sibling controls. Histological analyses reveal extensive decidual cell death in the SRC-3d/d decidua by GD-7, which results in the sudden demise of the embryo. These histological findings are supported by recent genome-wide RNA profiling studies that show a pro-survival role for SRC-3 in this tissue. Based on these results, we conclude that endometrial SRC-3 is critical for maintaining the functionality and survival of the decidual stromal cell and embryo developmental progression to placentation. Therefore, endometrial SRC-3 (and its downstream signaling networks) are predicted to represent new molecular determinants of non-aneuploid EPL, a poorly understood pregnancy complication for which diagnostic and treatment options are limited. Presentation Date: Thursday, June 15, 2023

SAT034 Exploring The Role Of Zfp664 In Hepatic Glucose Regulation

Abstract Disclosure: A. Tsay: None. J. Wang: None. Genome wide association studies (GWAS) identify the association between variants at genomic loci and common diseases. These variants consist of single nucleotide polymorphisms (SNPs), which can be localized within coding or non-coding regions. The former could affect the functions of proteins encoded by these genes. The latter could modulate the expression of genes contained or localized nearby these SNPs. Several SNPs associated with metabolic disease such as diabetes, obesity, and dyslipidemia are found in and nearby the human ZNF664 gene, which encodes a zinc finger protein that is predicted to be a transcriptional regulator. The mouse homolog of ZNF664 is Zfp664, whose amino acid sequence is identical to ZNF664. To learn whether Zfp664 contains a transcriptional activation or repression function, the expression plasmid harboring the cDNA of full length or deletion mutants of mouse Zfp664 fused to yeast GAL4 DNA binding domain (DBD) was transfected into HEK293T cells along with a luciferase reporter plasmid containing 5 copies of the GAL4 binding sites. We saw decreased luciferase activity in cells overexpressing the GAL4 DBD and full length Zfp664 fusion protein. Thus, Zfp664 acts as a transcriptional repressor. The deletion analysis identified that amino acid 7 to 95 of Zfp664 was required for its transcriptional repressive activity. To test the metabolic function of hepatic Zfp664, 8 weeks old male C57Bl/6J mice were infected with adeno-associated virus (AAV) expressing either scramble small hairpin RNA (shRNA, as a control) or shRNA targeting Zfp664 and then fed a normal chow diet or western diet (42% kcal from fat, 0.2% total cholesterol) for 12 weeks. We found that mice infected with AAV expressing Zfp664 shRNA had a lower expression of Zfp664 in the liver but not in epididymal white adipose tissue and gastrocnemius muscle. Under chow diet feeding, not only did hepatic Zfp664 knockdown not affect glucose tolerance and insulin sensitivity, but it also did not affect fasting glucose, insulin, triglyceride, and total cholesterol levels. In contrast, in mice fed a western diet, hepatic Zfp664 knockdown worsened glucose tolerance and elevated fasting insulin levels. Moreover, the expression of hepatic gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (Pck1) and the catalytic subunit of glucose-6-phosphatase (G6pc), was increased. The fasting plasma triglyceride and total cholesterol levels were not affected by hepatic Zfp664 knockdown in western diet fed mice. Overall, these results discover the novel role of Zfp664 in the regulation of hepatic glucose homeostasis. Presentation: Saturday, June 17, 2023

Diabetes Reshapes the Circadian Transcriptome Profile in Murine Retina.

Diabetic retinopathy (DR) is a common complication of diabetes and has a high prevalence. Dysregulation of circadian rhythmicity is associated with the development of DR. This research aimed to investigate rhythmical transcriptome alterations in the retina of diabetic mice. C57BL/6J mice were used to establish a diabetes model by intraperitoneal injection of streptozotocin (STZ). After 12 weeks, retinas were collected continuously at 4-hour intervals over 1day. Total RNA was extracted from normal and STZ-treated retinas and RNA sequencing was performed. Meta2d algorithm, Kyoto Encyclopedia of Genes, Phase Set Enrichment Analysis, and time-series cluster analysis were used to identify, analyze and annotate the composition, phase, and molecular functions of rhythmic transcripts in retinas. The retina exhibited powerful transcriptome rhythmicity. STZ-induced diabetes markedly modified the transcriptome characteristics of the circadian transcriptome in the retina, including composition, phase, and amplitude. Moreover, the diabetic mice led to re-organized temporal and clustering enrichment pathways in space and time and affected core clock machinery. Diabetes impairs the circadian rhythm of the transcriptomic profile of retinas. This study offers new perspectives on the negative effects of diabetes on the retina, which may provide important information for the development of new treatments for DR.

Advances in direct detection of lysine methylation and acetylation by nuclear magnetic resonance using 13C-enriched cofactors.

Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [1H, 13C]-HSQC and 13C direct-detect NMR measurements. Finally, we present 13C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.

How does NFAT3 regulate the occurrence of cardiac hypertrophy?

Cardiac hypertrophy is initially an adaptive response to physiological and pathological stimuli. Although pathological myocardial hypertrophy is the main cause of morbidity and mortality, our understanding of its mechanism is still weak. NFAT3 (nuclear factor of activated T-cell-3) is a member of the nuclear factor of the activated T cells (NFAT) family. NFAT3 plays a critical role in regulating the expression of cardiac hypertrophy genes by inducing their transcription. Recently, accumulating evidence has indicated that NFAT3 is a potent regulator of the progression of cardiac hypertrophy. This review, for the first time, summarizes the current studies on NFAT3 in cardiac hypertrophy, including the pathophysiological processes and the underlying pathological mechanism, focusing on the nuclear translocation and transcriptional function of NFAT3. This review will provide deep insight into the pathogenesis of cardiac hypertrophy and a theoretical basis for identifying new therapeutic targets in the NFAT3 network.

Genome-wide transcriptional analyses of Clarireedia jacksonii isolates associated with multi-drug resistance.

Emerging multidrug resistance (MDR) in Clarireedia spp. is a huge challenge to the management of dollar spot (DS) disease on turfgrass. Insight into the molecular basis of resistance mechanisms may help identify key molecular targets for developing novel effective chemicals. Previously, a MDR isolate (LT586) of C. jacksonii with significantly reduced sensitivities to propiconazole, boscalid, and iprodione, and a fungicide-sensitive isolate (LT15) of the same species were isolated from creeping bentgrass (Agrostis stolonifera L.). The present study aimed to further explore the molecular mechanisms of resistance by using genome-wide transcriptional analyses of the two isolates. A total of 619 and 475 differentially expressed genes (DEGs) were significantly down and upregulated in the MDR isolate LT586, compared with the sensitive isolate LT15 without fungicide treatment. Three hundreds and six and 153 DEGs showed significantly lower and higher expression in the MDR isolate LT586 than those in the sensitive isolate LT15, which were commonly induced by the three fungicides. Most of the 153 upregulated DEGs were xenobiotic detoxification-related genes and genes with transcriptional functions. Fifty and 17 upregulated DEGs were also commonly observed in HRI11 (a MDR isolate of the C. jacksonii) compared with the HRS10 (a fungicide-sensitive isolate of same species) from a previous study without and with the treatment of propiconazole, respectively. The reliability of RNA-seq data was further verified by qRT-PCR method using a few select potentially MDR-related genes. Results of this study indicated that there were multiple uncharacterized genes, possibly responsible for MDR phenotypes in Clarireedia spp., which may have important implications in understanding the molecular mechanisms underlying MDR resistance.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response.

Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg-expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses.

Transcriptomic Analysis to Uncover the Mechanism of Radiosensitization of AR-Positive Triple Negative Breast Cancers with AR Inhibition

The androgen receptor (AR) has been shown to drive tumor growth in triple negative breast cancers (TNBC), and previous work demonstrated AR inhibition as a strategy for radiosensitization in AR-positive (AR+) TNBC. Despite its role in radioresistance, the mechanistic role of AR in response to radiation therapy (RT) remains unknown, as does the benefit of 2nd generation anti-androgens in this context. We hypothesized that all 2nd generation anti-AR therapy would radiosensitize similarly and that canonical AR transcriptional function was responsible for radioresistance in these models. Radiosensitization was assessed using 2nd generation AR antagonists (apalutamide, enzalutamide, and darolutamide) using clonogenic survival assays in MDA-MB-453, SUM185, MFM-223, and MDA-MB-231 cells at 2-6Gy. Cellular fractionation experiments were performed and quantitated to determine the location of the AR protein in cells treated with AR agonists +/- RT. RNA Seq was performed and transcriptomic approaches were used (Advaita iPathway analysis) to investigate AR-mediated effects in response to RT. Inhibition with the 2nd generation anti-androgens enzalutamide and apalutamide is sufficient to radiosensitize AR+ TNBC models (rER: 1.34-1.41); while darolutamide had no effect on radiosensitivity (rER: 0.96-1.11). Additionally, TNBC cells with low AR expression were not radiosensitized by AR inhibition with any drug (rER: 0.96-1.03). While stimulation with the synthetic androgen methyltrienolone R1881 is sufficient to induce nuclear translocation of AR in AR+ TNBC cells, AR inhibition with enzalutamide, apalutamide, or darolutamide blocked AR nuclear translocation under growth conditions with charcoal stripped serum or fetal bovine serum. When cells are treated with R1881+RT, nuclear translocation of AR was induced at similar or greater levels compared to R1881 alone in AR+ TNBC cells. Combination treatment of RT with enzalutamide in the presence of hormones reduced nuclear localization of AR (32-39% reduction) compared to RT alone. RNA-sequencing after RT identified transcriptional changes potentially regulated by AR+RT, including changes in the NHEJ pathway genes. Additionally, pathway analyses in these models demonstrated changes in the MAPK/ERK signaling pathway, among others, that may regulate RT resistance in AR+ TNBC models. Most 2nd generation anti-androgens confer radiosensitization in AR+ TNBC models with cellular localization changes of AR noted after RT. The known structural differences amongst 2nd generation anti-androgens may account for differences in radiosensitization noted. Furthermore, AR-mediated radioresistance may be due, at least in part, to downstream MAPK/ERK signaling. This work builds on the mechanistic understanding of AR-mediated radioresistance in AR+ TNBC and may expose vulnerabilities to overcome resistance to combination treatment with AR inhibition and RT.

Dual Functions of T Lymphocytes in Breast Carcinoma: From Immune Protection to Orchestrating Tumor Progression and Metastasis.

Breast cancer (BC) is the most common cancer type in women and the second leading cause of death. Despite recent advances, the mortality rate of BC is still high, highlighting a need to develop new treatment strategies including the modulation of the immune system and immunotherapies. In this regard, understanding the complex function of the involved immune cells and their crosstalk with tumor cells is of great importance. T-cells are recognized as the most important cells in the tumor microenvironment and are divided into several subtypes including helper, cytotoxic, and regulatory T-cells according to their transcription factors, markers, and functions. This article attempts to provide a comprehensive review of the role of T-cell subsets in the prognosis and treatment of patients with BC, and crosstalk between tumor cells and T-cells. The literature overwhelmingly contains controversial findings mainly due to the plasticity of T-cell subsets within the inflammatory conditions and the use of different panels for their phenotyping. However, investigating the role of T-cells in BC immunity depends on a variety of factors including tumor types or subtypes, the stage of the disease, the localization of the cells in the tumor tissue and the presence of different cells or cytokines.

Two galactinol synthases contribute to the drought response of Camellia sinensis.

CsGolS2-1 and CsGolS2-2 are involved in the transcriptional mechanism and play an important role in the drought response of tea plants. GolS is critical for the biosynthesis of galactinol and has been suggested to contribute to drought tolerance in various plants. However, whether GolS plays a role in drought response and the underlying transcriptional mechanism of GolS genes in response to drought stress in tea plants is still unclear. In this study, we found that drought stress promotes the accumulation of galactinol in tea leaves and that the expression of CsGolS2-1 and CsGolS2-2, which encode proteins capable of catalyzing galactinol biosynthesis, is continuously and dramatically induced by drought stress. Moreover, transgenic Arabidopsis plants expressing CsGolS2-1 and CsGolS2-2 were more drought-tolerant than WT plants, as evidenced by increased cell membrane stability. In addition, the drought-responsive transcription factor CsWRKY2 has been shown to positively regulate the expression of CsGolS2-1 and CsGolS2-2 by directly binding to their promoters. Furthermore, CsVQ9 was found to interact with CsWRKY2 and promote its transcriptional function to activate CsGolS2-1 and CsGolS2-2 expression. Taken together, our findings provide insights not only into the positive role played by CsGolS2-1 and CsGolS2-2 in the drought response of tea plants but also into the transcriptional mechanisms involved.

Cyclin-dependent kinases: Masters of the eukaryotic universe.

A family of structurally related cyclin-dependent protein kinases (CDKs) drives many aspects of eukaryotic cell function. Much of the literature in this area has considered individual members of this family to act primarily either as regulators of the cell cycle, the context in which CDKs were first discovered, or as regulators of transcription. Until recently, CDK7 was the only clear example of a CDK that functions in both processes. However, new data points to several "cell-cycle" CDKs having important roles in transcription and some "transcriptional" CDKs having cell cycle-related targets. For example, novel functions in transcription have been demonstrated for the archetypal cell cycle regulator CDK1. The increasing evidence of the overlap between these two CDK types suggests that they might play a critical role in coordinating the two processes. Here we review the canonical functions of cell-cycle and transcriptional CDKs, and provide an update on how these kinases collaborate to perform important cellular functions. We also provide a brief overview of how dysregulation of CDKs contributes to carcinogenesis, and possible treatment avenues. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Processing > 3' End Processing RNA Processing > Splicing Regulation/Alternative Splicing.

The sequencing of the key genes and end products in the TLR4 signaling pathway from the kidney of Rana dybowskii exposed to Aeromonas hydrophila.

Infectious diseases caused by Aeromonas hydrophila (AH) have reduced the populations of Rana dybowskii). However, little is known about the immune response of R. dybowskii against AH infections. The toll-like receptor (TLR) signaling pathway has been identified as a critical component in innate immunity, responsible for identifying pathogen-associated molecular patterns in pathogens. Our study used the next-generation sequencing technique and single-molecule long-read sequencing to determine the structures of transcript isoforms and functions of genes in the kidneys of R. dybowskii, as well as identify and validate the related genes in the TLR4 signaling pathway. In total, 628,774 reads of inserts were identified, including 300,053 full-length non-chimeric reads and 233,592 non-full-length reads. Among the transcriptome sequences, 124 genes were identified as homologs of known genes in the TLR4 pathway especially inflammatory cytokines and receptors. Our findings shed light on the structures and functions of R. dybowskii genes exposed to AH and confirm the presence of both MyD88-dependent and independent pathways in R. dybowskii. Our work reveals how various functional proteins in amphibians at the initial stage of immune response are activated and complete their corresponding functions in a short time.

Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases.

Reversible post-translational modifications (PTMs) are key to establishing protein-protein and protein-nucleic acid interactions that govern a majority of the signaling pathways in cells. Sequence-specific PTMs are catalyzed by transferases, and their removal is carried out by a class of reverse-acting enzymes termed "detransferases". Currently available chemoproteomic approaches have been valuable in characterizing substrates of transferases. However, proteome-wide cataloging of the substrates of detransferases is challenging, mostly due to the loss of the epitope, rendering immunoprecipitation and activity-based methods ineffective. Herein, we develop a general chemoproteomic strategy called crosslinking-assisted substrate identification (CASI) for systematic characterization of cellular targets of detransferases and successfully apply it to lysine demethylases (KDMs) which catalyze the removal of methyl groups from lysine sidechain in histones to modulate gene transcription. By setting up a targeted azido-methylamino photo-reaction deep inside the active site of KDM4, engineered to carry p-azido phenylalanine, we reveal a novel "demethylome" that has escaped the traditional methods. The proteomic survey led to the identification of a battery of nonhistone substrates of KDM4, extending the biological footprint of KDM4 beyond its canonical functions in gene transcription. A notable finding of KDM4A-mediated demethylation of an evolutionarily conserved lysine residue in eukaryotic translational initiation factor argues for a much broader role of KDM4A in ribosomal processes. CASI, representing a substantive departure from earlier approaches by shifting focus from simple peptide-based probes to employing full-length photo-activatable demethylases, is poised to be applied to >400 human detransferases, many of which have remained poorly understood due to the lack of knowledge about their cellular targets.

Regulation of 5-Aminolevunilic Acid and Its Application in Agroforestry

The review briefly introduces the natural occurrence, physicochemical properties, and biosynthesis of 5-aminolevuinic acid (ALA) and highlights a variety of applications in the planting industry and its possible mechanisms. It has been known that ALA can be used as biological pesticides, fungicides, and herbicides when the concentrations are higher than 838 mg L−1 (about 5 mmol L−1). When ALA concentrations are 100–300 mg L−1, it can be used to thin surplus flowers in the spring of orchards and promote fruit coloration before maturation. When the concentrations are lower than 100 mg L−1, especially not higher than 10 mg L−1, ALA can be used as a new plant growth regulator to promote seed germination, plant (including root and shoot) growth, enhance stress tolerance, increase crop yield, and improve product quality. In photosynthesis, ALA is involved in the regulation of the whole process. In stress tolerance, ALA induces plant preventive and protective systems through the NO/H2O2 signaling network. In secondary metabolism, ALA regulates many gene expressions encoding transcription factors or function proteins to promote anthocyanin and flavonol biosynthesis and accumulation. In general, ALA promotes plant health and robustness, reduces the use of chemical fertilizers and pesticides—which is conducive to improving the ecological environment, human production, and living conditions—and has a broad application prospect in agroforestry production. As a new plant growth regulator with multiple and powerful functions, the underlying regulatory mechanisms need more study.

Murine leukemia virus (MLV) P50 protein induces cell transformation via transcriptional regulatory function

BackgroundThe murine leukemia virus (MLV) has been a powerful model of pathogenesis for the discovery of genes involved in cancer. Its splice donor (SD’)-associated retroelement (SDARE) is important for infectivity and tumorigenesis, but the mechanism remains poorly characterized. Here, we show for the first time that P50 protein, which is produced from SDARE, acts as an accessory protein that transregulates transcription and induces cell transformation.ResultsBy infecting cells with MLV particles containing SDARE transcript alone (lacking genomic RNA), we show that SDARE can spread to neighbouring cells as shown by the presence of P50 in infected cells. Furthermore, a role for P50 in cell transformation was demonstrated by CCK8, TUNEL and anchorage-independent growth assays. We identified the integrase domain of P50 as being responsible for transregulation of the MLV promoter using luciferase assay and RTqPCR with P50 deleted mutants. Transcriptomic analysis furthermore revealed that the expression of hundreds of cellular RNAs involved in cancerogenesis were deregulated in the presence of P50, suggesting that P50 induces carcinogenic processes via its transcriptional regulatory function.ConclusionWe propose a novel SDARE-mediated mode of propagation of the P50 accessory protein in surrounding cells. Moreover, due to its transforming properties, P50 expression could lead to a cellular and tissue microenvironment that is conducive to cancer development.

Establishment of paternal methylation imprint at the H19/Igf2 imprinting control region.

The insulator model explains the workings of the H19 and Igf2 imprinted domain in the soma, where insulation of the Igf2 promoter from its enhancers occurs by CTCF in the maternally inherited unmethylated chromosome but not the paternally inherited methylated allele. The molecular mechanism that targets paternal methylation imprint establishment to the imprinting control region (ICR) in the male germline is unknown. We tested the function of prospermatogonia-specific broad low-level transcription in this process using mouse genetics. Paternal imprint establishment was abnormal when transcription was stopped at the entry point to the ICR. The germline epimutation persisted into the paternal allele of the soma, resulting in reduced Igf2 in fetal organs and reduced fetal growth, consistent with the insulator model and insulin-like growth factor 2 (IGF2)'s role as fetal growth factor. These results collectively support the role of broad low-level transcription through the H19/Igf2 ICR in the establishment of its paternal methylation imprint in the male germ line, with implications for Silver-Russell syndrome.