Proximal Transcription Research Articles

Attenuated kidney oxidative metabolism in young adults with type 1 diabetes.

In type 1 diabetes (T1D), impaired insulin sensitivity may contribute to the development of diabetic kidney disease (DKD) through alterations in kidney oxidative metabolism. Young adults with T1D (n = 30) and healthy controls (HC, n = 20) underwent hyperinsulinemic-euglycemic clamp studies, MRI, 11C-acetate PET, kidney biopsies, single-cell RNA sequencing, and spatial metabolomics to assess this relationship. Participants with T1D had significantly higher glomerular basement membrane thickness compared to HC. T1D participants exhibited lower insulin sensitivity and cortical oxidative metabolism, correlating with higher insulin sensitivity. Proximal tubular transcripts of TCA cycle and oxidative phosphorylation enzymes were lower in T1D. Spatial metabolomics showed reductions in tubular TCA cycle intermediates, indicating mitochondrial dysfunction. The Slingshot algorithm identified a lineage of proximal tubular cells progressing from stable to adaptive/maladaptive subtypes, using pseudotime trajectory analysis, which computationally orders cells along a continuum of states. This analysis revealed distinct distribution patterns between T1D and HC, with attenuated oxidative metabolism in T1D attributed to a greater proportion of adaptive/maladaptive subtypes with low expression of TCA cycle and oxidative phosphorylation transcripts. Pseudotime progression associated with higher HbA1c, BMI, GBM, and lower insulin sensitivity and cortical oxidative metabolism. These early structural and metabolic changes in T1D kidneys may precede clinical DKD. gov NCT04074668.

Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (p-value < 1E-5) and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA Pol-II, including TATA-box, transcription initiator motif, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns were enriched with the Homeobox family of transcription factors and exhibited TA-rich motif sequences, suggesting potential motif-specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521 enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers, 3390 (12%) exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may dictate its function in myogenic differentiation and potentially other cellular and biological processes.

406-P: Spatial Metabolomics of Human Kidney Tissues Reveal Impaired Tricarboxylic Acid (TCA) Cycle Turnover in Type 1 Diabetes (T1D)

In abstract 2023-A-3497-Diabetes, we show impaired TCA cycle turnover using 11C acetate PET and lower proximal tubular transcripts of TCA cycle enzymes by single-cell RNA sequencing of kidney biopsies in young adults with T1D vs. healthy controls (HC). Spatial metabolomics analyses of the kidney tissue were conducted to further explore perturbations in kidney oxidative metabolism in T1D. Matrix-assisted laser desorption/ionization-mass spectrometry imaging-based spatial metabolomics was used to analyze metabolites in situ (spatial resolution: 20 μm) in kidney tissues from 8 participants with T1D and preserved kidney function and 5 HC. Univariate analysis (t-test or Wilcoxon Mann Whitney test) demonstrated that 36 of 456 METASPACE annotated metabolites were altered (P&amp;lt;0.05) in T1D vs. HC. Partial least squares-discriminant analysis (PLS-DA) and heatmaps showed clearly separated clusters of metabolites. To identify the most significant discriminators for T1D, a variable importance in projection (VIP) plot from PLS-DA model was applied. Of the top 15 metabolites, two TCA cycle intermediates, succinic acid (m/z 117.0193, -H; P = 0.016) and malic acid (m/z 133.0142, -H; P = 0.026), were reduced in kidney tissues of participants with T1D. Pathway analysis revealed that the TCA cycle, mitochondrial electron transport chain, glutamate metabolism, malate-aspartate shuttle, and purine pathway were the dominant metabolic pathways perturbed in T1D kidney tissue. Spatial metabolomics comparing T1D kidney biopsies vs. HC reveal alterations of TCA cycle intermediates indicating mitochondrial dysfunction in the subclinical stages of diabetic kidney disease. The spatial metabolomics data are consistent with the data from transcriptomics and stable isotope tracing analysis in a subset of same participants. Further analysis with pathologic features will identify potential pathways linked to disease development. Disclosure G.Zhang: None. C.Birznieks: None. I.De boer: Advisory Panel; AstraZeneca, Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Otsuka America Pharmaceutical, Inc., Bayer Inc., Consultant; George Clinical, Gilead Sciences, Inc., Medscape, Research Support; Dexcom, Inc. J.A.Schaub: None. K.J.Nadeau: None. V.Nair: None. F.Alakwaa: None. P.J.Mccown: None. A.Naik: Advisory Panel; CareDx. L.Pyle: None. D.Blondin: None. L.Liu: None. G.Richard: None. M.Kretzler: Research Support; Lilly, Boehringer Ingelheim Inc., Traveere Pharmaceuticals, Novo Nordisk, certa, Chinook Therapeutics Inc., Janssen Research &amp; Development, LLC, AstraZeneca, Moderna, Inc., Gilead Sciences, Inc., Regeneron, Ionis Pharmaceuticals, Angioin, Renalytix. P.Bjornstad: Advisory Panel; AstraZeneca, Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem, Consultant; Bayer Inc., Bristol-Myers Squibb Company. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. I.M.Tamayo: None. N.Garcia ponce de leon: None. T.B.Vigers: None. K.L.Tommerdahl: None. R.G.Nelson: None. P.E.Ladd: None. T.Alexandrov: None. Funding National Institutes of Health (UH3DK114920); JDRF (2-SRA-2019-845-S-B); Diabetes Research Center (P30DK116073)

9-OR: Insulin Resistance (IR) and Kidney Oxidative Metabolism in People with Type 1 Diabetes (T1D)

IR has been linked to kidney injury in T1D. Animal models show that IR associates with impaired TCA cycle turnover and oxidative phosphorylation, collectively termed oxidative metabolism, but little is known about this relationship in humans with T1D. Thirty young adults with T1D (age: 23±3 years, diabetes duration: 13±5 years, 53% female, HbA1c: 7.9±1.1%, BMI: 25±3 kg/m2, UACR: 5 [3, 8] mg/g) and 20 healthy controls (HC) (age: 25±3, 50% female, HbA1c: 5.2±0.3%, BMI: 23±2 kg/m2, UACR: 5 [3, 9] mg/g) underwent hyperinsulinemic-euglycemic clamps to assess whole-body insulin sensitivity (IS), and MRI to assess kidney perfusion. A subset underwent voxel-wise and region-of-interest (ROI) pharmacokinetic (PK) 11C-acetate PET analyses (n=16 T1D; n=10 HC) to quantify kidney cortical oxidative metabolism (k 2), and research kidney biopsies with single-cell RNA sequencing (n=28 T1D; n=13 HC). Compared to HC, participants with T1D exhibited lower IS (7.8±2.6 vs. 14.3±4.0 mg/kg/min, p&amp;lt;0.0001), cortical perfusion (196±68 vs. 243±46 ml/min/100g, p=0.01) and lower cortical k 2 (0.16±0.02 vs. HC 0.18±0.02 min-1, p=0.04) in voxel-wise models, although significance was not reached in the ROI PK analyses. IS associated with cortical k 2 (r:0.43, p=0.03) and the associations remained significant after adjusting for age, sex, and HbA1c (p=0.04). No significant interaction observed between T1D and HC for IS and cortical k2 (p=0.78). Proximal tubular transcripts of the enzymes catalyzing the proximal steps of the TCA cycle (e.g., ACO1, IDH1, SUCLG1) were lower in T1D vs. HC (all FDR-adjusted p&amp;lt;0.0001). Kidney oxidative metabolism is impaired in young people with T1D and is linked to lower whole-body IS. Statistical differences in k 2 from ROI and voxel-wise analyses suggest regional variations in kidney oxidative metabolism that may not be apparent in global analysis. Spatial metabolomic analyses of kidney tissue in a subset of these participants are shown in abstract #2023-A-3407-Diabetes. Disclosure G.Richard: None. S.Gross: None. V.N.Shah: Advisory Panel; LifeScan Diabetes Institute, Medscape, Consultant; DKSH, Research Support; Novo Nordisk, Tandem Diabetes Care, Inc., Dexcom, Inc., Insulet Corporation, JDRF, National Institutes of Health, Speaker's Bureau; Dexcom, Inc., Insulet Corporation. L.Pyle: None. T.B.Vigers: None. J.K.Snell-bergeon: None. I.De boer: Advisory Panel; AstraZeneca, Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Otsuka America Pharmaceutical, Inc., Bayer Inc., Consultant; George Clinical, Gilead Sciences, Inc., Medscape, Research Support; Dexcom, Inc. D.Van raalte: Consultant; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck &amp; Co., Inc., Research Support; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck &amp; Co., Inc. L.Li: None. P.V.Prasad: None. P.E.Ladd: None. C.Birznieks: None. B.B.Chin: None. D.Cherney: Other Relationship; Boehringer Ingelheim-Lilly, Merck, AstraZeneca, Sanofi, Mitsubishi-Tanabe, Abbvie, Janssen, Bayer, Prometic, BMS, Maze, Gilead, CSL-Behring, Otsuka, Novartis, Youngene, Lexicon and Novo-Nordisk, Research Support; Boehringer Ingelheim-Lilly, Merck, Janssen, Sanofi, AstraZeneca, CSL-Behring and Novo-Nordisk. P.J.Mccown: None. F.Alakwaa: None. M.Kretzler: Research Support; Lilly, Boehringer Ingelheim Inc., Traveere Pharmaceuticals, Novo Nordisk, certa, Chinook Therapeutics Inc., Janssen Research &amp; Development, LLC, AstraZeneca, Moderna, Inc., Gilead Sciences, Inc., Regeneron, Ionis Pharmaceuticals, Angioin, Renalytix. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. F.C.Brosius: Advisory Panel; Gilead Sciences, Inc. R.G.Nelson: None. K.J.Nadeau: None. P.Bjornstad: Advisory Panel; AstraZeneca, Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem, Consultant; Bayer Inc., Bristol-Myers Squibb Company. G.Zhang: None. L.Driscoll: None. K.L.Tommerdahl: None. J.A.Schaub: None. A.Naik: Advisory Panel; CareDx. V.Nair: None. A.A.Macdonald: None. Funding JDRF; National Institute of Diabetes and Digestive and Kidney Diseases

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF’s RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

Computational modeling of chromatin accessibility identified important epigenomic regulators

Chromatin accessibility is essential for transcriptional activation of genomic regions. It is well established that transcription factors (TFs) and histone modifications (HMs) play critical roles in chromatin accessibility regulation. However, there is a lack of studies that quantify these relationships. Here we constructed a two-layer model to predict chromatin accessibility by integrating DNA sequence, TF binding, and HM signals. By applying the model to two human cell lines (GM12878 and HepG2), we found that DNA sequences had limited power for accessibility prediction, while both TF binding and HM signals predicted chromatin accessibility with high accuracy. According to the HM model, HM features determined chromatin accessibility in a cell line shared manner, with the prediction power attributing to five core HM types. Results from the TF model indicated that chromatin accessibility was determined by a subset of informative TFs including both cell line-specific and generic TFs. The combined model of both TF and HM signals did not further improve the prediction accuracy, indicating that they provide redundant information in terms of chromatin accessibility prediction. The TFs and HM models can also distinguish the chromatin accessibility of proximal versus distal transcription start sites with high accuracy.

Retroposition of the Long Transcript from Multiexon IFN-β Homologs in Ancestry Vertebrate Gave Rise to the Proximal Transcription Elements of Intronless IFN-β Promoter in Humans.

IFN-β is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-β and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-β in chicken and humans.

Organochlorine pesticide dieldrin upregulate proximal promoter (PII) driven CYP19A1 gene expression and increases estrogen production in granulosa cells

Organochlorine pesticides are highly persistent environmental pollutants, generally shown to act through estrogen receptor alpha and alter estrogen biosynthesis. However, the molecular mechanism of regulation of estrogen biosynthesis by these pesticides is not clear. Estrogen is main female fertility hormone regulated by rate-limiting enzyme aromatase. It is encoded by the CYP19A1 gene, which is expressed using specific promoters. In the present study, the attempt has been made to elucidate the effect of dieldrin on the promoter-specific CYP19A1 gene expression and estrogen hormone production in buffalo granulosa cells. The buffalo granulosa cells were cultured and treated with dieldrin in a dose (100,150 and 200 ng/mL) and time (6, 12, and 24 h) dependent manner, followed by analysis of CYP19A1, promoter-specific CYP19A1 transcript expression, and estrogen production. Results showed that dieldrin significantly increased the expression of the CYP19A1 gene after 6 and 12 h while its expression was decreased after 24 h. To understand the upregulation of CYP19A1 gene, promoters’ specific CYP19A1 transcript analysis was done. The finding showed that dieldrin significantly increased the proximal promoter specific CYP19A1 transcript while there was no effect on distal promoter specific CYP19A1 transcripts. This specific-promoter activity was quantified by chromatin immunoprecipitation assay (ChIP). Results confirmed the involvement of the proximal promoter in the overexpression of CYP19A1 gene. Furthermore, a significant increase in estradiol-17β level was also observed. Overall, the present study demonstrated the stimulatory effect of dieldrin on the CYP19A1 gene and will help to understand the toxicological role of dieldrin on the reproductive system.

Loss of proximal tubular transcription factor Krüppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation

Loss of fatty acid β-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.

PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

SUMMARYPBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.

Network Pharmacological Mechanism Research of Herba Drynariae Rhizoma-Epimedii Folium in Treating Osteoarthritis

Objective: To investigate the potential mechanism of Drynariae Rhizoma-Epimedii Folium in the treatment of osteoarthritis (OA) based on network pharmacology. Methods: The potential active constituents and targets of Drynariae Rhizoma-Epimedii Folium were screened through the traditional Chinese medicine (TCM) systems pharmacology database and analysis platform (TCMSP). Genecards database is used to find relevant targets of OA. The targets of “Drynariae Rhizoma-Epimedii Folium” were mapped to the targets of OA, and used Cytoscape software to build a “drug-ingredient-target-di- sease” regulatory network and protein protein interaction (PPI) network. R software was used to analyze the Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment of traditional Chinese medicine-disease targets. Results: Thirty-four effective ingredients and 130 traditional Chinese medicine-disease targets were screened out for the treatment of OA. The GO functions of traditional Chinese medicine-disease targets mainly included cytokine activity, cytokine receptor binding, nuclear receptor activity, transcription factor activity, proximal promoter DNA-binding transcription activator activity, DNA-binding transcription activator activity, phosphatase binding and so on. KEGG pathways involved in traditional Chinese medicine-disease targets mainly included TLR4 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, PI3K/AKT signaling pathway, apoptotic signaling pathway and so on. Conclusion: Network pharmacology may predict the multiple targets and multiple signaling pathways in Drynariae Rhizoma-Epimedii Folium treatment for OA, providing new ideas for future research.

Genome-wide prioritization reveals novel gene signatures associated with cardiotoxic effects of tyrosine kinase inhibitors.

Tyrosine kinase inhibitors (TKIs) are characterized as multi-targeted anticancer agents that lack specificity, leading to cardiovascular adverse effects. To date, there are no reliable means to predict the cardiotoxicity of TKIs under development. The present study assessed the usual variants of genes to determine the molecular targets of TKIs associated with heart failure (HF). Gene or gene products affected by TKIs were assessed using the Drug Gene Interaction Database. These genes were investigated in genome-wide association studies (GWAS) datasets associated with HF at a genome-wide significant level (P<1×10−5). Subsequently, single-nucleotide polymorphisms (SNPs) that reached the established GWAS threshold (P<5×10−8) were investigated for genome-wide significance. Based on a threshold score of 3, nine gene loci yielded associations according to their biological function using RegulomeDB. Finally, comprehensive functional analysis of SNPs was performed using bioinformatics databases to identify potential drug targets. Using rSNPBase, rs7115242, rs143160639 and rs870064 were found to interfere with proximal transcription regulation, while rs7115242, rs143160639 and rs117153772 were involved in distal regulation, and most SNPs participated in post-transcriptional RNA binding protein-mediated regulation. rs191188930 on platelet-derived growth factor receptor (PDGFR) α was associated with numerous TKI drugs, including sunitinib, pazopanib, sorafenib, dasatinib and nilotinib. Using RegulomeDB and HaploReg v4.1, rs191188930 was predicted to be located in enhancer histone markers. PhenoScanner GWAS analysis revealed that rs191188930 was associated with other diseases or phenotypes, in addition to HF. Genotype-Tissue Expression analysis indicated that the PDGFRα gene had the highest median expression in ‘Cells-Transformed fibroblasts’, and the Search Tool for the Retrieval of Interacting Genes/Proteins revealed the protein-protein interaction network of PDGFRα. The present findings demonstrated the overlap of TKI-induced genes and those mediating HF risk, suggesting molecular mechanisms potentially responsible for TKI-induced HF risk. Additionally, the present genetic study may be helpful to further investigate off-target drug effects.

Integrated Multichip Analysis Identifies Potential Key Genes in the Pathogenesis of Nonalcoholic Steatohepatitis.

BackgroundNonalcoholic steatohepatitis (NASH) is rapidly becoming a major chronic liver disease worldwide. However, little is known concerning the pathogenesis and progression mechanism of NASH. Our aim here is to identify key genes and elucidate their biological function in the progression from hepatic steatosis to NASH.MethodsGene expression datasets containing NASH patients, hepatic steatosis patients, and healthy subjects were downloaded from the Gene Expression Omnibus database, using the R packages biobase and GEOquery. Differentially expressed genes (DEGs) were identified using the R limma package. Functional annotation and enrichment analysis of DEGs were undertaken using the R package ClusterProfile. Protein-protein interaction (PPI) networks were constructed using the STRING database.ResultsThree microarray datasets GSE48452, GSE63067 and GSE89632 were selected. They included 45 NASH patients, 31 hepatic steatosis patients, and 43 healthy subjects. Two up-regulated and 24 down-regulated DEGs were found in both NASH patients vs. healthy controls and in steatosis subjects vs. healthy controls. The most significantly differentially expressed genes were FOSB (P = 3.43×10-15), followed by CYP7A1 (P = 2.87×10-11), and FOS (P = 6.26×10-11). Proximal promoter DNA-binding transcription activator activity, RNA polymerase II-specific (P = 1.30×10-5) was the most significantly enriched functional term in the gene ontology analysis. KEGG pathway enrichment analysis indicated that the MAPK signaling pathway (P = 3.11×10-4) was significantly enriched.ConclusionThis study characterized hub genes of the liver transcriptome, which may contribute functionally to NASH progression from hepatic steatosis.

The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.

Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

Transcription of the WUSCHEL-RELATED HOMEOBOX 4 gene in Arabidopsis thaliana

Phylogenetic shadowing and chromatin accessibility data suggested that essential regulatory elements are absent in the 2.9 kb immediate upstream region of the published WOX4pro::YFP cambium marker. Inclusion of an additional 6.3 kb of upstream promoter sequence and confocal imaging with different fluorophores in transgenic Arabidopsis lines revealed a much wider cell-type-specific expression pattern in parenchymous cells of the aerial plant body. The previously demonstrated activity of the WOX4pro::YFP marker in the cambium of vascular strands in the young Arabidopsis inflorescence stem depicts only sectors of a circular subcortical layer of parenchymous AtWOX4-positive cells. Transcription starts in subepidermal cells within the inflorescence apex in a phyllotactic pattern and extends into successively branching lateral organs, which are connected via small tube-like domains of AtWOX4-expressing cells with the circular subcortical parenchymal layer that extends basipetally down the stem. AtWOX4 expression is most dynamic in leaves, where promoter activity is observed transiently at the adaxial side of the lamina and remains detectable later in the palisade parenchyma, although at a weaker level than in the vasculature. In the root the extended AtWOX4 promoter is active through the proximal root meristem, i.e. in the quiescent centre (QC) and its surrounding initials, a pattern that is broader than transcription of its stem cell promoting relative AtWOX5 in the QC. Outside the proximal meristem AtWOX4 transcription is observed in upper cell layers of the columella root cap beneath or above within the stele in proto- and metaxylem cells, in a ribbon-type pattern which divides the central cylinder in two equal halves. This xylem-specific expression it the root stele relates to established AtWOX4 activity in xylem parenchyma specificity within vascular bundles of the stem.

DNA methylation and its effects on gene expression during primary to secondary growth in poplar stems

BackgroundAs an important epigenetic mark, 5-methylcytosine (5mC) methylation is involved in many DNA-dependent biological processes and plays a role during development and differentiation of multicellular organisms. However, there is still a lack of knowledge about the dynamic aspects and the roles of global 5mC methylation in wood formation in tree trunks. In this study, we not only scrutinized single-base resolution methylomes of primary stems (PS), transitional stems (TS), and secondary stems (SS) of Populus trichocarpa using a high-throughput bisulfite sequencing technique, but also analyzed the effects of 5mC methylation on the expression of genes involved in wood formation.ResultsThe overall average percentages of CG, CHG, and CHH methylation in poplar stems were ~ 53.6%, ~ 37.7%, and ~ 8.5%, respectively, and the differences of 5mC in genome-wide CG/CHG/CHH contexts among PS, TS, and SS were statistically significant (p < 0.05). The evident differences in CG, CHG, and CHH methylation contexts among 2 kb proximal promoters, gene bodies, and 2 kb downstream regions were observed among PS, TS, and SS. Further analysis revealed a perceptible global correlation between 5mC methylation levels of gene bodies and transcript levels but failed to reveal a correlation between 5mC methylation levels of proximal promoter regions and transcript levels. We identified 653 and 858 DMGs and 4978 and 4780 DEGs in PS vs TS and TS vs SS comparisons, respectively. Only 113 genes of 653 DMGs and 4978 DEGs, and 114 genes of 858 DMGs and 4780 DEG were common. Counterparts of some of these common genes in other species, including Arabidopsis thaliana, are known to be involved in secondary cell wall biosynthesis and hormone signaling. This indicates that methylation may directly modulate wood formation genes and indirectly attune hormone signaling genes, which in turn impact wood formation.ConclusionsDNA methylation only marginally affects pathway genes or regulators involved in wood formation, suggesting that further studies of wood formation should lean towards the indirect effects of methylation. The information and data we provide here will be instrumental for understanding the roles of methylation in wood formation in tree species.

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Nucleus pulposus primary cilia alter their length in response to changes in extracellular osmolarity but do not control TonEBP-mediated osmoregulation

The nucleus pulposus (NP) cells adapt to their physiologically hyperosmotic microenvironment through Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cell5 [NFAT5])-mediated osmoregulation. Primary cilia in different organs serve diverse roles including osmosensing, but its contribution to NP cell osmoadaptive response is unknown. A high percentage of cultured primary NP cells possessed primary cilia that changed length in response to osmotic stimuli. Stable silencing of Intraflagellar Transport 88 (Ift88) or Kinesin Family Member 3 A (Kif3a) to inhibit the formation of primary cilia did not affect hyperosmotic upregulation of TonEBP. While ShKif3a blocked hyperosmotic increase of TonEBP-Transactivation Domain (TAD) activity, overall the knockdown of either gene did not alter the hyperosmotic status of proximal promoter activities and transcription of key TonEBP targets. On the other hand, a small decrease in TonEBP level under hypoosmotic condition was attenuated by Ift88 or Kif3a knockdown. Noteworthy, none of the TonEBP target genes were responsive to hypoosmotic stimulus in control and Ift88 or Kif3a knockdown cells, suggesting the primary role of TonEBP in the hyperosmotic adaptation of NP cells. Similarly, in Kif3a null mouse embryonic fibroblasts (MEFs), the overall TonEBP-dependent hyperosmotic responses were preserved. Unlike NP cells, TonEBP targets were responsive to hypoosmolarity in wild-type MEFs, and these responses remained intact in Kif3a null MEFs. Together, these results suggest that primary cilia are dispensable for TonEBP-dependent osmoadaptive response.

CDK4/6 Inhibition Suppresses Myeloma Cell Growth and Viability via Perturbation of E2F Proliferative Program

Cancer cells are characterized by perturbed transcriptional profiles. These dysregulations depend on transcriptional regulators unpredicted by genetic changes. Multiple myeloma cells also tend to develop striking dependencies on super-enhancer regulatory elements. Also, promoter proximal transcription factors like E2F1 and SP1 play prominent roles in myeloma cell biology by controlling proliferative and survival genes. Importantly, our genome-wide profiling of the MM epigenome revealed that promoter and enhancer-associated factors govern distinct biological functions providing a non-overlapping control of the myeloma transcriptome.

Development of a new promoter to avoid the silencing of genes in the production of recombinant antibodies in chinese hamster ovary cells

BackgroundThe production of recombinant proteins in mammalian cell lines is one of the most important areas in biopharmaceutical industry. Viral transcriptional promoters are widely used to express recombinant proteins in mammalian cell lines. However, these promoters are susceptible to silencing, thus limiting protein productivity. Some CpG islands can avoid the silencing of housekeeping genes; for that reason, they have been used to increase the production of recombinant genes in cells of animal origin. In this study, we evaluated the CpG island of the promoter region of the β-actin gene of Cricetulus griseous (Chinese hamster), associated to the Cytomegalovirus (CMV) promoter, to increase recombinant antibodies production in Chinese Hamster Ovary (CHO) cells.ResultsWe focused on the non-coding region of CpG island, which we called RegCG. RegCG behaved as a promoter, whose transcriptional activity was mainly commanded by the CAAT and CArG boxes of the proximal promoter. However, the transcription started mainly at the intronic region before the proximal transcription start site. While the CMV promoter was initially more powerful than RegCG, the latter promoter was more resistant to silencing than the CMV promoter in stable cell lines, and its activity was improved when combined with the CMV promoter. Thereby, the chimeric promoter was able to maintain the expression of recombinant antibodies in stable clones for 40 days at an average level 4 times higher than the CMV promoter. Finally, the chimeric promoter showed compatibility with a genetic amplification system by induction with methotrexate in cells deficient in the dihydrofolate reductase gene.ConclusionsWe have generated an efficient synthetic hybrid transcription promoter through the combination of RegCG with CMV, which, in stable cell lines, shows greater activity than when both promoters are used separately. Our chimeric promoter is compatible with a genetic amplification system in CHO DG44 cells and makes possible the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be a good alternative for the generation of clones expressing high amount of recombinant proteins, essential for industrial applications.