Gene Regulatory Functions Research Articles

In vivo vitamin D target genes interconnect key signaling pathways of innate immunity.

The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), its nuclear receptor VDR (vitamin D receptor) and hundreds of their target genes are not only key regulators of calcium homeostasis, but also important modulators of the immune system. Innate immune cells like monocytes use VDR for efficient differentiation and are very responsive to vitamin D. So far, most information on the gene regulatory function of vitamin D and its physiological impact had been obtained from in vitro studies using supraphysiological doses of 1,25(OH)2D3. Therefore, medical experiments like the study VitDHiD (NCT03537027), where 25 healthy individuals were supplemented once with a vitamin D3 bolus (80,000 IU), provide important insight into the response to vitamin D under in vivo conditions. In this study, we inspected 452 in vivo vitamin D target genes from peripheral blood mononuclear cells (PBMCs) detected in VitDHiD and found 61 of them involved in eight major KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of innate immunity. Under in vivo conditions in healthy individuals vitamin D either silences five pathways of innate immunity, stabilizes two and increases one, so that acute inflammation is suppressed and the release of cytokines is kept under control. A ranking of the 61 target genes by inducibility, basal expression and multiple involvements in the pathways highlighted the genes NFKBIA (NFκB inhibitor alpha), NFKBIZ, FOSL2 (FOS like 2, AP1 transcription factor subunit), JDP2 (Jun dimerization protein 2), PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1), CLEC7A (C-type lectin domain containing 7A), DUSP6 (dual specificity phosphatase 6), NCF2 (neutrophil cytosolic factor 2), PLCB1 (phospholipase C beta 1), PLCG2 and TNFAIP3 (TNF alpha induced protein 3). In conclusion, vitamin D's in vivo effect on innate immunity in healthy adults is mediated by the interconnection of the pathways of neutrophil extracellular trap formation, Toll-like receptor, chemokine and phagosome signaling, NOD-like receptor, C-type lectin receptor, apoptosis and interleukin 17 through a limited set of proteins encoded by key target genes.

PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step.

RNA binding proteins (RBPs) are responsible for facilitating a wealth of post-transcriptional gene regulatory functions. The role of an RBP on regulated transcripts can be investigated through a pull-down of the RBP and high-throughput sequencing (HTS) of the associated transcripts. PhotoactivatableRibonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), is one such pull-down method that isolates, detects, and sequences the cDNA of RBP-associated transcripts. PAR-CLIP relies on a photoactivatable ribonucleoside analogue, 4-thiouridine, to facilitate covalent RNA-protein crosslinks at 365nm. These crosslinks permit stringent wash conditions and result in T to C mismatch incorporations during reverse transcription, a unique parameter for the computational analysis of high-confidence binding sites. However, until now, RBPs that bind at the 5'-termini of RNAs have been uniquely restricted from the full potential bandwidth of autoradiographic detection and HTS library preparation. The 5'-termini of RNAs are highly modified, including the most common Pol-II derived modification: the 7-methylguanosine (m7G) cap. In the conventional PAR-CLIP protocol, cap-binding proteins protect the m7G cap from the RNase treatment that generates the necessary substrate for autoradiographic detection and 5' adapter ligation-thus occluding entire populations of RNA from visualization and HTS. Here, we introduce decapping-PAR-CLIP or PAR-dCLIP. We incorporate a decapping step into the PAR-CLIP protocol to generate the necessary substrate to sequence m7G capped transcripts. While PAR-dCLIP was originally targeted towards known m7G-cap binding proteins, we argue that all RBP inquiries, and particularly those suspected to regulate translation, should incorporate this decapping step to ensure that all possible populations of bound transcripts are identified.

Effect of disease‐associated variants in TREM2 on splicing and gene dosage

AbstractBackgroundLoss‐of‐function variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are responsible for a spectrum of neurodegenerative disorders. In the homozygous state, they cause severe pathologies with early onset dementia, such as Nasu‐Hakola disease (NHD) and behavioral variants of frontotemporal dementia (FTD), while heterozygous variants increase risk of late onset Alzheimer’s disease (AD) and FTD. In over half of TREM2 variants found in families with recessive early onset dementia, the defect occurs at the transcript level, via premature termination codons or via aberrant splicing. The remaining ones are missense variants thought to inactivate the protein; however, the underlying pathogenic mechanism is less clear. In this work we tested whether disease‐associated TREM2 variants may contribute to the pathology via altered splicingMethodSpliceAI prediction algorithm was used to screen 56 disease‐associated/risk‐modifying variants for potential effect on splicing. Variants scored by SpliceAI were tested in the splicing reporter assay with full‐length TREM2. Constructs with the common TREM2 variant or the spliceogenic SNP allele were nucleofected into THP‐1, HMC3 and BV2 cell lines. qRT‐/RT‐PCR and western blots were used to quantify the effect of variants at the transcript and protein levels. Nanostring nCounter analysis was used to measure TREM2 RNA in brains of NHD patients who carried spliceogenic variants.ResultWe demonstrated that multiple variants causative for NHD or associated with AD/FTD affect gene splicing reduced dosage of functional transcript and protein.ConclusionOur findings point to the complex character of pathogenicity of TREM2 variants, in which effects on post‐transcriptional gene regulation and protein function are often combined. This hidden layer of complexity necessitates inclusion of computational and experimental analyses of splicing and mRNA processing for better understanding of genetic variation in disease.

ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

The constitutively active ESR1 Y537S mutation is associated with endocrine therapy (ET) resistance and progression of metastatic breast cancer through its effects on estrogen receptor (ERα) gene regulatory functions. However, the complex relationship between ERα and the progesterone receptor (PR), known as ERα/PR crosstalk, has yet to be characterized in the context of the ERα Y537S mutation. Using proximity ligation assays, we identify an increased physical interaction of ERα and PR in the context of the ERα Y537S mutation, including in the nucleus where this interaction may translate to altered gene expression. As such, more than 30 genes were differentially expressed in both patient tumor and cell line data (MCF7 and/or T47D cells) in the context of the ERα Y537S mutation compared to ERα WT. Of these, IRS1 stood out as a gene of interest, and ERα and PR occupancy at chromatin binding sites along IRS1 were uniquely altered in the context of ERα Y537S. Furthermore, siRNA knockdown of IRS1 or treatment with the IRS1 inhibitor NT-157 had a significant anti-proliferative effect in ERα Y537S cell lines, implicating IRS1 as a potential therapeutic target for restoring treatment sensitivity to patients with breast cancers harboring ERα Y537S mutations.

METTL3 promotes colorectal cancer progression through activating JAK1/STAT3 signaling pathway

The role of METTL3-mediated N6-methyladenosine (m6A) modification has been elucidated in several cancers, but the concrete mechanism underlying its function in colorectal cancer is still obscure. Here, we revealed that upregulated methyltransferase-like 3 (METTL3) in colorectal cancer exerted both methyltransferase activity-dependent and -independent functions in gene regulation. METTL3 deposited m6A on the 3’ untranslated region of the JAK1 transcript to promote JAK1 translation relying on YTHDF1 recognition. Besides, METTL3 was redistributed to the STAT3 promoter and worked in concert with NF-κB to facilitate STAT3 transcription, which was achieved independently on METTL3 methyltransferase activity. The increased JAK1 and STAT3 corporately contributed to the activation of the p-STAT3 signaling pathway and further upregulated downstream effectors expressions, including VEGFA and CCND1, which finally resulted in enhanced cancer cell proliferation and metastasis in vitro and in vivo. Collectively, our study revealed the unappreciated dual role of METTL3 as an m6A writer and a transcription regulator, which worked together in the same signaling pathway to drive colorectal cancer malignancy.

Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression

CPT-11 is one of the drugs employed in colorectal cancer treatment and has faced challenges in the form of resistance. The insulin-like growth factor 1 receptor is a tyrosine kinase receptor that mediates cancer cell survival and drug resistance. It is frequently overexpressed in colorectal cancer and has previously been identified as a microRNA target. MicroRNAs are non-coding RNA molecules that regulate gene function by suppressing messenger RNA translation. Studies have demonstrated that natural compounds can regulate microRNA function and their target genes. Therefore, combining natural compounds with existing cancer drugs can enhance the therapeutic efficacy. We investigated a natural compound, Aloin, for the potential sensitization of colorectal cancer to CPT-11. We used western blot, MTT cell viability assay, flow cytometry, and microRNA/gene knockdown and overexpression experiments, as well as an in vivo mouse model. Our investigation revealed that combining Aloin with CPT-11 exerts an enhanced anti-tumor effect in colorectal cancer. This combination reduced cell viability and induced apoptosis, both in vivo and in vitro. Furthermore, this combination upregulated miRNA-133b, while downregulating the IGF1R and its downstream MEK/ERK, and PI3K/AKT/mTOR pathways. Our findings suggests that CPT-11 and Aloin are potential combination treatment partners against colorectal cancer. MicroRNA-133b may serve as a co-therapeutic target with IGF1R against colorectal cancer, which might overcome the existing treatment limitations.

Microsatellite diversity and complexity in the viral genomes of the family Caliciviridae

BackgroundMicrosatellites or simple sequence repeats (SSR) consist of 1–6 nucleotide motifs of DNA or RNA which are ubiquitously present in tandem repeated sequences across genome in viruses: prokaryotes and eukaryotes. They may be localized to both the coding and non-coding regions. SSRs play an important role in replication, gene regulation, transcription, and protein function. The Caliciviridae (CLV) family of viruses have ss-RNA, non-enveloped, icosahedral symmetry 27–35 nm in diameter in size. The size of the genome lies between 6.4 and 8.6 kb. ResultsThe incidence, composition, diversity, complexity, and host range of different microsatellites in 62 representatives of the family of Caliciviridae were systematically analyzed. The full-length genome sequences were assessed from NCBI (https://www.ncbi.nlm.nih.gov), and microsatellites were extracted through MISA software. The average genome size is about 7538 bp ranging from 6273 (CLV61) to 8798 (CLV47) bp. The average GC content of the genomes was ~ 51%. There are a total of 1317 SSRs and 53 cSSRs in the studied genomes. CLV 41 and CLV 49 contain the highest and lowest value of SSRs with 32 and 10 respectively, while CLV16 had maximum cSSR incidence of 4. There were 29 species which do not contain any cSSR. The incidence of mono-, di-, and tri-nucleotide SSRs was 219, 884, and 206, respectively. The most prevalent mono-, di-, and tri-nucleotide repeat motifs were “C” (126 SSRs), AC/CA (240 SSRs), and TGA/ACT (23 SSRs), respectively. Most of the SSRs and cSSRs are biased toward the coding region with a minimum of ~ 90% incident SSRs in the genomes’ coding region. Viruses with similar host are found close to each other on the phylogenetic tree suggesting virus host being one of the driving forces for their evolution. ConclusionsThe Caliciviridae genomes does not conform to any pattern of SSR signature in terms of incidence, composition, and localization. This unique property of SSR plays an important role in viral evolution. Clustering of similar host in the phylogenetic tree is the evidence of the uniqueness of SSR signature.

Study on resistance of MdMYB gene family in apple

The growth and development of apples are susceptible to environmental stress. The MdMYB gene family is one of the largest gene families in apples. It is widely involved in various resistance signaling pathways in apples and can interact with a variety of genes. Through anthocyanin synthesis, lignin synthesis, epidermal wax synthesis, Na + / H + pump regulation, hormone interaction, etc., the resistance of plants to abiotic and biotic stresses is improved. The MdMYB gene is regulated by multiple factors. It is regulated by epigenetic factors such as methylation and miRNA before transcription. At the transcriptional level, it is regulated by transcription factors such as MdHY5, MdWRKY11 and MdWRKY41. After translation, it is regulated by ubiquitin ligase, which is involved in MdSIZ, MdMEIL and other proteins. This paper reviews the physiological responses and signaling pathways of MdMYB-mediated apple stress resistance and the gene function regulation mechanism of MdMYB, in order to provide new ideas for the study of apple stress resistance.

Rapid-Kinetics Degron Benchmarking Reveals Off-Target Activities and Mixed Agonism-Antagonism of MYB Inhibitors

The transcription factor (TF) MYB is a critical transcriptional dependency of acute myeloid leukemia (AML) where it has been a long-term focus of drug development efforts. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We reasoned that the specificity and on-target activity of MYB inhibitors would be best evaluated in a rapid kinetics system where their immediate transcriptional effects are benchmarked against targeted MYB degradation. We began by engineering a chemical degron model and established the direct gene-regulatory functions of MYB in a nascent transcriptomics assay. We fused the C-terminus of MYB with an FKBP12 F36V (dTAG) domain and a fluorescent tag by a homozygous knock-in of the FKBP12 F36V-coding DNA sequence into the endogenous MYB locus in MV411 cells. The resulting fusion protein was nearly completely degraded after a 1-hour treatment with dTAG V-1, a highly specific VHL-engaging PROTAC. As expected, degradation of MYB resulted in a profound loss of cell viability, consistent with the effects of a genetic MYB knockout in AML cells. To establish MYB's direct gene-regulatory functions, we measured genome-wide rates of nascent mRNA synthesis by thiol (SH)-linked alkylation metabolic sequencing of RNA (SLAM-seq) after a 1-hour MYB degradation. Defining direct targets as those genes which displayed significant changes in transcription rates (FDR<0.0), we detected 450 genes directly regulated by MYB. Of these, 319 genes were downregulated and 131 genes were upregulated, indicating that MYB acts as a transcriptional activator and repressor of these genes, respectively. In parallel, we performed SLAM-seq in AML cells treated with six agents reported as MYB inhibitors: MYBMIM, celastrol, naphthol AS-E phosphate, mebendazole, plumbagin and all-trans retinoic acid (ATRA). For comparison, we treated MV411 cells with the BET bromodomain inhibitor JQ1, which has been reported to indirectly inhibit MYB by interfering with its expression and function. The MYB inhibitors displayed a dramatic variability in the number of dysregulated genes, varying from 19 (naphthol) to 1123 (MYBMIM). In contrast, JQ1 caused widespread and bimodal effects, altering the transcription rates of >2000 genes in both directions. On pairwise overlap, the MYB inhibitors captured a relatively minor portion of the direct MYB program (between 5-155, or 1-34%, of the 450 direct MYB targets), compared with 43% of the MYB program captured by JQ1. Nonetheless, the MYB inhibitors displayed a stronger specificity for MYB target genes compared to JQ1, because they elicited much narrower responses. Thus, while MYB inhibitors displayed strong enrichments for primary MYB targets, they did not attenuate the entire MYB transcriptional program, and significant portions of their activities appeared to be off-target. In addition, the inhibitors displayed bimodal effects on the transcription of MYB-regulated genes, further activating subsets of genes that were repressed by MYB degradation, and vice versa. These observations uncovered unexpected activities of MYB inhibitors as context-dependent mixed agonists-antagonists. We conclude that MYB has a narrow direct transcriptional program in AML cells, which is only partially captured by the existing MYB inhibitors, which display partial specificity for MYB targets and act as mixed agonists-antagonists. Our work will serve as a proof-of-principle demonstration of the use of rapid kinetic resolution of TF degron models for a more precise characterization of the target specificity and efficacy of TF-directed inhibitors. Benchmarking of TF modulators against degron models in nascent transcriptomics assays should be considered as an important criterion in their functional characterization.

Cross-tissue patterns of DNA hypomethylation reveal genetically distinct histories of cell development

BackgroundEstablishment of DNA methylation (DNAme) patterns is essential for balanced multi-lineage cellular differentiation, but exactly how these patterns drive cellular phenotypes is unclear. While > 80% of CpG sites are stably methylated, tens of thousands of discrete CpG loci form hypomethylated regions (HMRs). Because they lack DNAme, HMRs are considered transcriptionally permissive, but not all HMRs actively regulate genes. Unlike promoter HMRs, a subset of non-coding HMRs is cell type-specific and enriched for tissue-specific gene regulatory functions. Our data further argues not only that HMR establishment is an important step in enforcing cell identity, but also that cross-cell type and spatial HMR patterns are functionally informative of gene regulation.ResultsTo understand the significance of non-coding HMRs, we systematically dissected HMR patterns across diverse human cell types and developmental timepoints, including embryonic, fetal, and adult tissues. Unsupervised clustering of 126,104 distinct HMRs revealed that levels of HMR specificity reflects a developmental hierarchy supported by enrichment of stage-specific transcription factors and gene ontologies. Using a pseudo-time course of development from embryonic stem cells to adult stem and mature hematopoietic cells, we find that most HMRs observed in differentiated cells (~ 60%) are established at early developmental stages and accumulate as development progresses. HMRs that arise during differentiation frequently (~ 35%) establish near existing HMRs (≤ 6 kb away), leading to the formation of HMR clusters associated with stronger enhancer activity. Using SNP-based partitioned heritability from GWAS summary statistics across diverse traits and clinical lab values, we discovered that genetic contribution to trait heritability is enriched within HMRs. Moreover, the contribution of heritability to cell-relevant traits increases with both increasing HMR specificity and HMR clustering, supporting the role of distinct HMR subsets in regulating normal cell function.ConclusionsOur results demonstrate that the entire HMR repertoire within a cell-type, rather than just the cell type-specific HMRs, stores information that is key to understanding and predicting cellular phenotypes. Ultimately, these data provide novel insights into how DNA hypo-methylation provides genetically distinct historical records of a cell’s journey through development, highlighting HMRs as functionally distinct from other epigenomic annotations.

Characterization of human transcription factor function and patterns of gene regulation in HepG2 cells.

Transcription factors (TFs) are trans-acting proteins that bind cis-regulatory elements (CREs) in DNA to control gene expression. Here, we analyzed the genomic localization profiles of 529 sequence-specific TFs and 151 cofactors and chromatin regulators in the human cancer cell line HepG2, for a total of 680 broadly termed DNA-associated proteins (DAPs). We used this deep collection to model each TF's impact on gene expression, and identified a cohort of 26 candidate transcriptional repressors. We examine high occupancy target (HOT) sites in the context of three-dimensional genome organization and show biased motif placement in distal-promoter connections involving HOT sites. We also found a substantial number of closed chromatin regions with multiple DAPs bound, and explored their properties, finding that a MAFF/MAFK TF pair correlates with transcriptional repression. Altogether, these analyses provide novel insights into the regulatory logic of the human cell line HepG2 genome and show the usefulness of large genomic analyses for elucidation of individual TF functions.

Structural Evolution of Gene Promoters Driven by Primate-Specific KRAB Zinc Finger Proteins.

Krüppel-associated box (KRAB) zinc finger proteins (KZNFs) recognize and repress transposable elements (TEs); TEs are DNA elements that are capable of replicating themselves throughout our genomes with potentially harmful consequences. However, genes from this family of transcription factors have a much wider potential for genomic regulation. KZNFs have become integrated into gene-regulatory networks through the control of TEs that function as enhancers and gene promoters; some KZNFs also bind directly to gene promoters, suggesting an additional, more direct layer of KZNF co-option into gene-regulatory networks. Binding site analysis of ZNF519, ZNF441, and ZNF468 suggests the structural evolution of KZNFs to recognize TEs can result in coincidental binding to gene promoters independent of TE sequences. We show a higher rate of sequence turnover in gene promoter KZNF binding sites than neighboring regions, implying a selective pressure is being applied by the binding of a KZNF. Through CRISPR/Cas9 mediated genetic deletion of ZNF519, ZNF441, and ZNF468, we provide further evidence for genome-wide co-option of the KZNF-mediated gene-regulatory functions; KZNF knockout leads to changes in expression of KZNF-bound genes in neuronal lineages. Finally, we show that the opposite can be established upon KZNF overexpression, further strengthening the support for the role of KZNFs as bona-fide gene regulators. With no eminent role for ZNF519 in controlling its TE target, our study may provide a snapshot into the early stages of the completed co-option of a KZNF, showing the lasting, multilayered impact that retrovirus invasions and host response mechanisms can have upon the evolution of our genomes.

PHF5A is a potential diagnostic, prognostic, and immunological biomarker in pan-cancer

Studying the molecular mechanisms and regulatory functions of genes is crucial for exploring new approaches and tactics in cancer therapy. Studies have shown that the aberrant expression of PHF5A in tumors is linked to the origin and advancement of multiple cancers. However, its role in diagnosis, prognosis, and immunological prediction has not been comprehensively investigated in a pan-cancer analysis. Using several bioinformatic tools, we conducted a systematic examination of the potential carcinogenesis of PHF5A in various tumors from multiple aspects. Our analysis indicated that PHF5A expression varied between normal and tumor tissues and was linked to clinical diagnosis and prognosis in various cancers. The results confirmed a notable variation in the levels of PHF5A promoter methylation among several types of primary tumor and normal tissues and methylation of the PHF5A promoter played a guiding role in prognosis in some cancers. According to our findings, PHF5A played a critical role in tumor immunity and it might be an excellent target for anticancer immunotherapy. To sum up, PHF5A can be used in pan-cancer diagnostics, prognostics, and immunotherapy.

PML modulates epigenetic composition of chromatin to regulate expression of pro-metastatic genes in triple-negative breast cancer.

The promyelocytic leukemia (PML) protein organizes nuclear aggregates known as PML nuclear bodies (PML-NBs), where many transcription factors localize to be regulated. In addition, associations of PML and PML-NBs with chromatin are described in various cell types, further implicating PML in transcriptional regulation. However, a complete understanding of the functional consequences of PML association to DNA in cellular contexts where it promotes relevant phenotypes is still lacking. We examined PML chromatin association in triple-negative breast cancer (TNBC) cell lines, where it exerts important oncogenic functions. We find that PML associates discontinuously with large heterochromatic PML-associated domains (PADs) that contain discrete gene-rich euchromatic sub-domains locally depleted of PML. PML promotes heterochromatic organization in PADs and expression of pro-metastatic genes embedded in these sub-domains. Importantly, this occurs outside PML-NBs, suggesting that nucleoplasmic PML exerts a relevant gene regulatory function. We also find that PML plays indirect regulatory roles in TNBC cells by promoting the expression of pro-metastatic genes outside PADs. Our findings suggest that PML is an important transcriptional regulator of pro-oncogenic metagenes in TNBC cells, via transcriptional regulation and epigenetic organization of heterochromatin domains that embed regions of local transcriptional activity.

Dysregulated expression of miR‑367 in disease development and its prospects as a therapeutic target and diagnostic biomarker (Review).

MicroRNA (miR)-367 has a wide range of functions in gene regulation and as such plays a critical role in cell proliferation, differentiation and development, making it an essential molecule in various physiological processes. miR-367 belongs to the miR-302/367 cluster and is located in the intronic region of human chromosome 4 on the 4q25 locus. Dysregulation of miR-367 is associated with various disease conditions, including cancer, inflammation and cardiac conditions. Moreover, miR-367 has shown promise both as a tumor suppressor and a potential diagnostic biomarker for breast, gastric and prostate cancer. The elucidation of the essential role of miR-367 in inflammation, development and cardiac diseases emphasizes its versatility in regulating various physiological processes beyond cancer biology. However, further research is necessary to fully understand the complex regulatory mechanisms involving miR-367 in different physiological and pathological contexts. In conclusion, the versatility and significance of miR-367 makes it a promising candidate for further study and in the development of new diagnostic and therapeutic strategies.

Isoforskolin modulates AQP4-SPP1-PIK3C3 related pathway for chronic obstructive pulmonary disease via cAMP signaling

BackgroundCyclic adenosine monophosphate (cAMP) levels are directly activated by adenylate cyclase (AC) and play an anti-inflammatory role in chronic obstructive pulmonary disease (COPD). Previously, we have shown that isoforskolin (ISOF) can effectively activate AC1 and AC2 in vitro, improve pulmonary ventilation and reduce the inflammatory response in COPD model rats, supporting that ISOF may be a potential drug for the prevention and treatment of COPD, but the mechanism has not been explored in detail.MethodsThe potential pharmacological mechanisms of ISOF against COPD were analyzed by network pharmacology and multi-omics based on pharmacodynamic study. To use specific agonists, inhibitors and/or SiRNA for gene regulation function studies, combined qPCR, WB were applied to detect changes in mRNA and protein expression of important targets PIK3C3, AKT, mTOR, SPP1 and AQP4 which related to ISOF effect on COPD. And the key inflammatory factors detected by ELISA.ResultsBioinformatics suggested that the anti-COPD pharmacological mechanism of ISOF was related to PI3K-AKT signaling pathway, and suggested target protein like PIK3C3, AQP4, SPP1, AKT, mTOR. Using the AQP4 inhibitor,or inhibiting SPP1 expression by siRNA-SPP1 could block the PIK3C3-AKT-mTOR pathway and ameliorate chronic inflammation. ISOF showed cAMP-promoting effect then suppressed AQP4 expression, together with decreased level of IL-1β, IL-6, and IL-8.ConclusionsThese findings demonstrate ISOF controlled the cAMP-regulated PIK3C3-AKT-mTOR pathway, thereby alleviating inflammatory development in COPD. The cAMP/AQP4/PIK3C3 axis also modulate Th17/Treg differentiation, revealed potential therapeutic targets for this disease.

Genome wide identification and expression profiling of PYL genes in barley

PYLs (pyrabactin resistance1/PYR1-like) sense ABA, an essential phytohormone that regulates plant growth and stress responses. PYLs act as the main controllers of ABA stress signaling in plants. In this study, a total of 10 HvPYLs were discovered in the barley genome using an in silico genome search technique. These HvPYLs were then grouped into 3 subfamilies based on a phylogenetic analysis that compared them to the genomes of Arabidopsis, Brachypodium, rice, and maize. These HvPYLs demonstrated conserved motif compositions and identical protein structures across clades. Additionally, this study includes detailed investigations of gene structure variations, chromosomal distributions, cis-regulatory elements, protein-protein interactions, expression profiles in different tissues, and stress responses. We identified various cis-elements in the HvPYL promoter regions related to plant development and stresses indicating their potential roles in development and stress management. Our analysis of the interaction network has identified that HvPYLs can interact with key components of the ABA signaling pathway, demonstrating the critical regulatory functions of HvPYL genes in managing stress and growth in barley. These findings provide a basis for future research aimed at exploring the functions of PYL genes, with the ultimate goal of enhancing stress tolerance in barley and other related species.

Effect of Fermentation Scale on Microbiota Dynamics and Metabolic Functions for Indigo Reduction.

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

Mitotic genome-bookmarking by nuclear hormone receptors: A novel dimension in epigenetic reprogramming and disease assessment

Arrival of multi-colored fluorescent proteins and advances in live cell imaging has immensely contributed to our understanding of intracellular trafficking of nuclear receptors and their roles in gene regulatory functions. These regulatory events need to be faithfully propagated from progenitor to progeny cells. This is corroborated by multiple converging mechanisms that include histone modifications and lately, the phenomenon of ‘mitotic genome-bookmarking’ by specific transcription factors. This phenomenon refers to the retention and feed-forward transmission of progenitor's architectural blueprint of active transcription status which is silenced and preserved during mitosis. Upon mitotic exit, this phenomenon ensures accurate reactivation of transcriptome, proteome, cellular traits and phenotypes in the progeny cells. In addition to diverse modes of genome-bookmarking by nuclear receptors, a correlation between disease-associated receptor polymorphism and disruption of this phenomenon is apparent. However, breakthrough technologies shall reveal finer details of this phenomenon to help achieve normalcy in receptor-specific diseases.

You are who your friends are-nuclear pore proteins as components of chromatin-binding complexes.

Nuclear pore complexes are large multicomponent protein complexes that are embedded in the nuclear envelope, where they mediate nucleocytoplasmic transport. In addition to supporting transport, nuclear pore components, termed nucleoporins (Nups), can interact with chromatin and influence genome function. A subset of Nups can also localize to the nuclear interior and bind chromatin intranuclearly, providing an opportunity to investigate chromatin-associated functions of Nups outside of the transport context. This review focuses on the gene regulatory functions of such intranuclear Nups, with a particular emphasis on their identity as components of several chromatin regulatory complexes. Recent proteomic screens have identified Nups as interacting partners of active and repressive epigenetic machinery, architectural proteins, and DNA replication complexes, providing insight into molecular mechanisms via which Nups regulate gene expression programs. This review summarizes these interactions and discusses their potential functions in the broader framework of nuclear genome organization.