Transcription Factor Target Research Articles

Transcriptional profiling from whole embryos to single neuroblast lineages in Drosophila

Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the “parts list” of cell types that comprise each organ. Here we perform single cell RNA sequencing (scRNA-seq) of the Drosophila embryo to identify the genes that characterize different cell and tissue types during development. We assay three different timepoints, revealing a coordinated change in gene expression within each tissue. Interestingly, we find that the elav and Mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, show broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. We next focus on the central nervous system (CNS), where we identify genes whose expression is enriched at each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, we ask whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, we find that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, we were able to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, neurons which share a temporal identity (e.g. Hunchback, Kruppel, Pdm, and Castor temporal transcription factors in the NB7-1 lineage) have shared transcriptional features, allowing for the identification of candidate novel temporal factors or targets of the temporal transcription factors. In conclusion, we have characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor.

Direct targeted therapy for MLL-fusion-driven high-risk acute leukaemias.

BackgroundImproving the poor prognosis of infant leukaemias remains an unmet clinical need. This disease is a prototypical fusion oncoprotein‐driven paediatric cancer, with MLL (KMT2A)‐fusions present in most cases. Direct targeting of these driving oncoproteins represents a unique therapeutic opportunity. This rationale led us to initiate a drug screening with the aim of discovering drugs that can block MLL‐fusion oncoproteins.MethodsA screen for inhibition of MLL‐fusion proteins was developed that overcomes the traditional limitations of targeting transcription factors. This luciferase reporter‐based screen, together with a secondary western blot screen, was used to prioritize compounds. We characterized the lead compound, disulfiram (DSF), based on its efficient ablation of MLL‐fusion proteins. The consequences of drug‐induced MLL‐fusion inhibition were confirmed by cell proliferation, colony formation, apoptosis assays, RT‐qPCR, in vivo assays, RNA‐seq and ChIP‐qPCR and ChIP‐seq analysis. All statistical tests were two‐sided.ResultsDrug‐induced inhibition of MLL‐fusion proteins by DSF resulted in a specific block of colony formation in MLL‐rearranged cells in vitro, induced differentiation and impeded leukaemia progression in vivo. Mechanistically, DSF abrogates MLL‐fusion protein binding to DNA, resulting in epigenetic changes and down‐regulation of leukaemic programmes setup by the MLL‐fusion protein.ConclusionDSF can directly inhibit MLL‐fusion proteins and demonstrate antitumour activity both in vitro and in vivo, providing, to our knowledge, the first evidence for a therapy that directly targets the initiating oncogenic MLL‐fusion protein.

DNA Methylation Analysis of Turner Syndrome BAV.

Turner Syndrome (TS) is a rare cytogenetic disorder caused by the complete loss or structural variation of the second sex chromosome. The most common cause of early mortality in TS results from a high incidence of left-sided congenital heart defects, including bicuspid aortic valve (BAV), which occurs in about 30% of individuals with TS. BAV is also the most common congenital heart defect in the general population with a prevalence of 0.5–2%, with males being three-times more likely to have a BAV than females. TS is associated with genome-wide hypomethylation when compared to karyotypically normal males and females. Alterations in DNA methylation in primary aortic tissue are associated with BAV in euploid individuals. Here we show significant differences in DNA methylation patterns associated with BAV in TS found in peripheral blood by comparing TS BAV (n = 12), TS TAV (n = 13), and non-syndromic BAV (n = 6). When comparing TS with BAV to TS with no heart defects we identified a differentially methylated region encompassing the BAV-associated gene MYRF, and enrichment for binding sites of two known transcription factor contributors to BAV. When comparing TS with BAV to euploid women with BAV, we found significant overlapping enrichment for ChIP-seq transcription factor targets including genes in the NOTCH1 pathway, known for involvement in the etiology of non-syndromic BAV, and other genes that are essential regulators of heart valve development. Overall, these findings suggest that altered DNA methylation affecting key aortic valve development genes contributes to the greatly increased risk for BAV in TS.

Novel regulation of the transcription factor ZHX2 by N-terminal methylation

ABSTRACT N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein–DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. The global loss of Nα-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout (Nrmt1−/− ) mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and Nrmt1−/− adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating that its function could be directly regulated by Nα-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in Nrmt1−/− livers and verified NRMT1 can methylate ZHX2 in vitro. In addition, we used a mutant of ZHX2 that cannot be methylated to directly demonstrate Nα-methylation promotes ZHX2 transcription factor activity and target promoter occupancy. Finally, we show Nrmt1−/− mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate ZHX2 misregulation as a driving force behind the liver phenotype seen in Nrmt1−/− mice.

Expression of genes encoding terpenoid biosynthesis enzymes during leaf development of Eucalyptus camaldulensis

To reveal the regulation mechanism of terpenoid biosynthesis in the leaves of Eucalyptus camaldulensis, the content of volatiles in eucalyptus leaves and the transcriptome databases of young and mature leaves were analyzed. The results showed that E. camaldulensis contains 92 and 89 kinds of volatile substances in the young and mature leaves, respectively. Among them, the content of 1,8-cineole, β-pinene, and other substances was significantly different in young and mature leaves. A total of 99 802 unigenes were obtained from the transcriptome database of young and mature leaves of E. camaldulensis and 18 441 genes displayed obviously differential expressions during both developmental stages. There were 6 982 up-regulated unigenes and 11 461 down-regulated unigenes in the young leaf stage compared to the mature leaf stage. The key genes for terpenoid biosynthesis, including limonene synthase-10, limonene synthase-11, myrcene synthase-1, α- pinene synthase-2, and 1,8-cineole synthase-2, were selected for further analysis to explore the mechanism of gene regulation and genetic transformation. The expressions of key genes were validated by RT-qPCR, and their expressions were consistent with RNA-seq data. WRKY, MYB, NAC, and bHLH transcription factors (TFs) displayed important regulatory effects on the above key genes. Thus, a regulatory network model of terpenoid biosynthesis was constructed using target genes and TFs during leaf development in E. camaldulensis. These results provide theoretical evidence for understanding the terpenoid biosynthesis in plants and reference for terpenoids utilization by genetic engineering methods in E. camaldulensis.

SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters.

Chronic hepatitis B (CHB) virus infection is one of the leading causes of cirrhosis and liver cancer. Although the major drugs against CHB including nucleos(t)ide analogs and PEG-interferon can effectively control human hepatitis B virus (HBV) infection, complete cure of HBV infection is quite rare. Targeting host factors involved in the viral life cycle contributes to developing innovative therapeutic strategies to improve HBV clearance. In this study, we found that the mRNA and protein levels of SIRT2, a class III histone deacetylase, were significantly upregulated in CHB patients, and that SIRT2 protein level was positively correlated with HBV viral load, HBsAg/HBeAg levels, HBcrAg, and ALT/AST levels. Functional analysis confirmed that ectopic SIRT2 overexpression markedly increased total HBV RNAs, 3.5-kb RNA and HBV core DNA in HBV-infected HepG2-Na+/taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, SIRT2 silencing inhibited HBV transcription and replication. In addition, we found a positive correlation between SIRT2 expression and HBV RNAs synthesis as well as HBV covalently closed circular DNA transcriptional activity. A mechanistic study suggested that SIRT2 enhances the activities of HBV enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The levels of HBV EnI/Xp and EnII/Cp-bound p53 were modulated by SIRT2. Both the mutation of p53 binding sites in EnI/Xp and EnII/Cp as well as overexpression of p53 abolished the effect of SIRT2 on HBV transcription and replication. In conclusion, our study reveals that, in terms of host factors, a SIRT2-targeted program might be a more effective therapeutic strategy for HBV infection.

Transcriptomic analysis of Simpson Golabi Behmel syndrome cells during differentiation exhibit BAT-like function

High-throughput RNA sequencing of human Simpson-Golabi-Behmel syndrome cells (SGBS) was performed during the time-course of adipogenic differentiation at day 4 (D04), 6 (D06), 8 (D08), and 10 (D10) to characterize transcriptomic changes and to identify key patterns involved in adipogenesis and browning. In the comparisons, 932 and 384 overlapping transcripts were consistently up- and down-regulated, respectively. Combining the results of protein-protein interaction network analysis MCODE and CytoHubba, 55 up-regulated hub genes from four clusters and 9 down-regulated genes were identified. The up-regulated hub genes were mainly enriched in brown adipocyte differentiation, extracellular matrix organization, and valine, leucine, and isoleucine degradation. The enrichment of downregulated hub genes was related to NRF2 signalling and glutathione metabolism, indicating that oxidative stress also plays a role. Analysis of overlapping down-regulated genes, targets of transcription factors, revealed enrichment in the IL-18 signalling pathway, which is involved in browning process and extracellular matrix organization via actomyosin mechanics and integrin-extracellular matrix interactions. Finally, the comparison transcriptomic analysis with the gene signature reported by BATLAS and PROFAT web-based tools showed an increased percentage of the brown phenotype, confirming that differentiated SGBS cells at D06, D08, and D10 gradually acquire BAT-like function.

NanoDam identifies Homeobrain (ARX) and Scarecrow (NKX2.1) as conserved temporal factors in the Drosophila central brain and visual system.

Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for missing factors, we developed NanoDam, which enables rapid genome-wide profiling of endogenously tagged proteins invivo with a single genetic cross. Mapping the targets of known temporal transcription factors with NanoDam revealed that Homeobrain and Scarecrow (ARX and NKX2.1 orthologs) are also temporal factors. We show that Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell-type-specific genome-wide profiling with temporal resolution and is easily adapted for use in higher organisms.

32Starch metabolism in potato Solanum tuberosum L.

Starch is a major storage carbohydrate in plants. It is an important source of calories in the human and animal diet. Also, it is widely used in various industries. Native starch consists of water-insoluble semicrystalline granules formed by natural glucose polymers amylose and amylopectin. The physicochemical properties of starch are determined by the amylose:amylopectin ratio in the granule and degrees of their polymerization and phosphorylation. Potato Solanum tuberosum L. is one of the main starch-producing crops. Growing industrial needs necessitate the breeding of plant varieties with increased starch content and specified starch properties. This task demands detailed information on starch metabolism in the producing plant. It is a complex process, requiring the orchestrated work of many enzymes, transporter and targeting proteins, transcription factors, and other regulators. Two types of starch are recognized with regard to their biological functions. Transitory starch is synthesized in chloroplasts of photosynthetic organs and degraded in the absence of light, providing carbohydrates for cell needs. Storage starch is synthesized and stored in amyloplasts of storage organs: grains and tubers. The main enzymatic reactions of starch biosynthesis and degradation, as well as carbohydrate transport and metabolism, are well known in the case of transitory starch of the model plant Arabidopsis thaliana. Less is known about features of starch metabolism in storage organs, in particular, potato tubers. Several issues remain obscure: the roles of enzyme isoforms and different regulatory factors in tissues at various plant developmental stages and under different environmental conditions; alternative enzymatic processes; targeting and transport proteins. In this review, the key enzymatic reactions of plant carbohydrate metabolism, transitory and storage starch biosynthesis, and starch degradation are discussed, and features specific for potato are outlined. Attention is also paid to the known regulatory factors affecting starch metabolism

LncRNA, miRNA and transcriptional co-regulatory network of breast and ovarian cancer reveals hub molecules

Our research focused on the development of a regulatory network of miRNA-lncRNA and miRNA-transcription factor associated with Breast Cancer (BC) and Ovarian Cancer (OC), utilising experimentally identified data from previous research works. The regulatory data from the target network helped in the identification of the most vital miRNA, lncRNA and transcription factor (TFs) hub molecules involved in the prognosis of BC and OC. Breast cancer is one of the leading types of cancer in the world whereas ovarian cancer is difficult to detect due to the lack of symptoms in the initial stages. lncRNAs are known to compete for miRNAs as per previous evidence shown in favour of the competing endogenous RNA theory. TFs are vital proteins that regulate the expression of protein coding genes. miRNAs have been known to be involved in post-transcriptional gene silencing. Thus miRNA-lncRNA interactions and miRNA-TF interactions should have a significant impact on the expression of different genes, the irregularity of which leads to diseases like cancer. Our work has revealed three lncRNAs: KCNQ1OT1, XIST and NEAT1 as well as two TFs: RELA and TP53 to have the most significant impact on both breast and ovarian cancer. We identified four miRNAs which had the maximum lncRNA targets, common to both cancers: hsa-mir-93-5p, hsa-mir-519d-3p, hsa-mir-106a-5p and hsa-mir-106b-5p. The top 15 miRNAs having maximum TF targets were found to be identical for both cancers.

Identification and validation of a novel prognostic signature based on transcription factors in breast cancer by bioinformatics analysis.

Breast cancer (BRCA) is the leading cause of cancer mortality among women, and it is associated with many tumor suppressors and oncogenes. There is increasing evidence that transcription factors (TFs) play vital roles in human malignancies, but TFs-based biomarkers for BRCA prognosis were still rare and necessary. This study sought to develop and validate a prognostic model based on TFs for BRCA patients. Differentially expressed TFs were screened from 1,109 BRCA and 113 non-tumor samples downloaded from The Cancer Genome Atlas (TCGA). Univariate Cox regression analysis was used to identify TFs associated with overall survival (OS) of BRCA, and multivariate Cox regression analysis was performed to establish the optimal risk model. The predictive value of the TF model was established using TCGA database and validated using a Gene Expression Omnibus (GEO) data set (GSE20685). A gene set enrichment analysis was conducted to identify the enriched signaling pathways in high-risk and low-risk BRCA patients. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the TF target genes were also conducted separately. A total of 394 differentially expressed TFs were screened. A 9-TF prognostic model, comprising PAX7, POU3F2, ZIC2, WT1, ALX4, FOXJ1, SPIB, LEF1 and NFE2, was constructed and validated. Compared to those in the low-risk group, patients in the high-risk group had worse clinical outcomes (P<0.001). The areas under the curve of the prognostic model for 5-year OS were 0.722 in the training cohort and 0.651 in the testing cohort. Additionally, the risk score was an independent prediction indicator for BRCA patients both in the training cohort (HR =1.757, P<0.001) and testing cohort (HR =1.401, P=0.001). It was associated with various cancer signaling pathways. Ultimately, 9 overlapping target genes were predicted by 3 prediction nomograms. The GO and KEGG enrichment analyses of these target genes suggested that the TFs in the model may regulate the activation of some classical tumor signaling pathways to control the progression of BRCA through these target genes. Our study developed and validated a novel prognostic TF model that can effectively predict 5-year OS for BRCA patients.

Target Search Dynamics of Sox Transcription Factors

Here we investigate the impact of regions outside of the DNA‐binding domain (DBD) on target search efficiency of transcription factors (TFs). In living organisms, TFs are key for gene regulation by binding sequence DNA‐binding sites. The facilitated diffusion theory proposes a search process combining TF 1D sliding on DNA and 3D diffusion in the nucleus. Moreover, the local non‐specific TF‐DNA interactions mediated by electrostatic forces enhance the target search efficiency to reach on‐rates faster than allowed by free diffusion. While such theoretical models fit TF behaviors in prokaryotes, how TFs search the chromatinized eukaryotic genome is poorly understood. More importantly, the relevance of regions outside of the DNA‐binding domain for the search process remains to be clarified.We hypothesize that positive charges flanking the DBD accelerate TF target search by interacting non‐specifically with negatively charged DNA. We tackle this hypothesis with a comparative study between Sox2 and Sox17. While recognizing the same DNA motif, Sox2 displays higher non‐specific binding affinity, which we think is caused by a higher content in positive charges close to the DBD.To probe the kinetics of in vivo Sox‐DNA interactions in living mouse embryonic stem cells, we used highly inclined and laminated optical sheet (HiLo) microscopy. We determined that Sox2 and Sox17 have similar profiles of residence time with specific binding of ~ 12 s and non‐specific binding of ~ 0.7 s. We then quantified the search efficiency of Sox2 and Sox17 by determining their pseudo on‐rate, which we define as the time‐averaged number of specifically bound particles per time interval and per TF concentration. Since Sox2 and Sox17 bind to the same DNA motifs, they thus have the same number of targets in the mouse genome. Therefore, the pseudo on‐rate scales linearly with association rate at a given TF concentration. We found that Sox2 has a two‐fold higher pseudo on‐rate than Sox17. We also generated a Sox2 mutant in which its DBD flanking region was swapped with the one from Sox17. This led to a two‐fold decrease in pseudo on‐rate as compared to the wild‐type Sox2. These results thus directly support the hypothesis that non‐specific electrostatic interactions of positively charged residues in regions flanking the DBD enhance their DNA association rates.One limitation of in vivo experiments is that the chromatin context in which TF‐DNA interactions are observed is unknown. To dissect the differential interactions of Sox2 and Sox17 with naked versus nucleosomal DNA, we are using chemically‐synthesized in vitro chromatin combined with single‐molecule tracking via total internal reflection (TIRF) microscopy. Since Sox2 but not Sox17 is known as a pioneer factor, quantifying their interactions with chromatinized DNA will shed light on search mechanisms of pioneer TFs in compact chromatin. In that sense, we foresee that more positively‐charged DBD flanking regions may stabilize specific binding of TF to nucleosomal DNA and help TFs to invade and remodel chromatin.Taken together, our combination of in vivo and in vitro single‐molecule imaging techniques allows dissecting how the charge distribution of DBD flanking regions modulates TF target search on chromatin.

Selected Articles from This Issue

Abemaciclib is an oral, potent and selective CDK4 &amp; 6 inhibitor, dosed on a continuous schedule and approved as monotherapy and in combination with either fulvestrant or an aromatase inhibitor for HR+, HER2− advanced breast cancer (ABC). ESR1 mutation and PI3K pathway dysregulation are associated with resistance to endocrine therapy, and treatment with CDK4 &amp; 6 inhibitors has been hypothesized as a therapeutic strategy to overcome endocrine resistance. In this exploratory analysis, Tolaney and colleagues show in HR+, HER2− ABC, abemaciclib plus fulvestrant improved both PFS and OS, regardless of PIK3CA or ESR1 mutation status. Additionally, abemaciclib plus fulvestrant improved other endpoints, including TTC, CFS and PFS2, regardless of PIK3CA or ESR1 mutation status. These findings support further evaluation in suitably powered trials.Combined targeting of the lineage-specific master transcription factor, ETV1, and signaling factor, KIT, by the combination of KIT and MEK inhibitors, is synergistic in preclinical GIST models. Chi and colleagues report the results of a phase Ib clinical trial designed to both evaluate the safety of the combination of imatinib and binimetinib and to determine the recommended phase II dose (RP2D) and early efficacy signal of this drug combination in patients with refractory advanced KIT/PDGFRA-mutant and KIT/PDGFRA wild-type GIST including SDH-deficient GIST. The study demonstrated that the combination of imatinib/binimetinib was safe with manageable side effects and determined the RP2D. They observed durable pathological responses in a small cohort of SDH-deficient GIST patients in the phase Ib expansion at RP2D. The promising clinical activity of the combination treatment in SDH-deficient GIST observed is worthy of further clinical investigation.Cyclin-dependent kinase 4/6 inhibition may have synergistic anticancer activity when combined with taxane chemotherapy. To explore this combination in metastatic castration-resistant prostate cancer (mCRPC), de Kouchkovsky and colleagues conducted a phase Ib/IIa study of ribociclib plus docetaxel in mCRPC patients who had progressed on an androgen receptor signaling inhibitor (ARSI). With intermittent ribociclib dosing and prophylactic growth factor, the combination demonstrated an acceptable safety profile, achieved a 6-month radiographic progression-free survival (rPFS) rate of 65.8%, and a median rPFS of 8.1 months. These results compare favorably to outcomes seen with docetaxel monotherapy after ARSI therapy. Further prospective study is warranted.KRAS is the most commonly mutated driver oncogene in solid tumors, including non-small cell lung cancer (NSCLC), with mutations spanning codons 12, 13 and 61. To explore the role of different mutant KRAS isoforms in pre-clinical models and samples from patients, Ricciuti and colleagues developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability across the 6 most common KRAS mutant variants. In addition, by surveying a large institutional dataset totaling &gt;3000 NSCLC they identified clinicopathologic and genomic characteristics which correlated with unique KRAS mutant isoforms. Together, these findings revealed that KRAS mutations are highly heterogenous in terms of biological outcome and genomic profiling, and that this heterogeneity can be exploited to unveil novel potentially actionable vulnerabilities for KRAS mutant NSCLC.

Cell origin–dependent cooperativity of mutant Dnmt3a and Npm1 in clonal hematopoiesis and myeloid malignancy

AbstractIn adult acute myeloid leukemia (AML), the acquisition of driver somatic mutations may be preceded by a benign state termed clonal hematopoiesis (CH). To develop therapeutic strategies to prevent leukemia development from CH, it is important to understand the mechanisms by which CH-driving and AML-driving mutations cooperate. Here, we use mice with inducible mutant alleles common in human CH (DNMT3AR882; mouse Dnmt3aR878H) and AML (NPM1c; mouse Npm1cA). We find that Dnmt3aR878H/+ hematopoietic stem cells (HSCs), but not multipotent progenitor cell (MPP) subsets, have reduced cytokine expression and proinflammatory transcriptional signatures and a functional competitive advantage over their wild-type counterparts. Dnmt3aR878H/+ HSCs are the most potent cell type transformed by Npm1cA, generating myeloid malignancies in which few additional cooperating somatic mutation events were detected. At a molecular level, Npm1cA, in cooperation with Dnmt3aR878H, acutely increased the accessibility of a distinct set of promoters in HSCs compared with MPP cells. These promoters were enriched for cell cycling, PI3K/AKT/mTOR signaling, stem cell signatures, and targets of transcription factors, including NFAT and the chromatin binding factor HMGB1, which have been implicated in human AML. These results demonstrate cooperativity between preexisting Dnmt3aR878H and Npm1cA at the chromatin level, where specific loci altered in accessibility by Npm1cA are dependent on cell context as well as Dnmt3a mutation status. These findings have implications for biological understanding and therapeutic intervention in the transformation from CH to AML.

Adaptive Response of Saccharomyces Hosts to Totiviridae L-A dsRNA Viruses Is Achieved through Intrinsically Balanced Action of Targeted Transcription Factors.

Totiviridae L-A virus is a widespread yeast dsRNA virus. The persistence of the L-A virus alone appears to be symptomless, but the concomitant presence of a satellite M virus provides a killer trait for the host cell. The presence of L-A dsRNA is common in laboratory, industrial, and wild yeasts, but little is known about the impact of the L-A virus on the host’s gene expression. In this work, based on high-throughput RNA sequencing data analysis, the impact of the L-A virus on whole-genome expression in three different Saccharomyces paradoxus and S. cerevisiae host strains was analyzed. In the presence of the L-A virus, moderate alterations in gene expression were detected, with the least impact on respiration-deficient cells. Remarkably, the transcriptional adaptation of essential genes was limited to genes involved in ribosome biogenesis. Transcriptional responses to L-A maintenance were, nevertheless, similar to those induced upon stress or nutrient availability. Based on these data, we further dissected yeast transcriptional regulators that, in turn, modulate the cellular L-A dsRNA levels. Our findings point to totivirus-driven fine-tuning of the transcriptional landscape in yeasts and uncover signaling pathways employed by dsRNA viruses to establish the stable, yet allegedly profitless, viral infection of fungi.

Chromatin interaction-aware gene regulatory modeling with graph attention networks.

Linking distal enhancers to genes and modeling their impact on target gene expression are longstanding unresolved problems in regulatory genomics and critical for interpreting noncoding genetic variation. Here, we present a new deep learning approach called GraphReg that exploits 3D interactions from chromosome conformation capture assays to predict gene expression from 1D epigenomic data or genomic DNA sequence. By using graph attention networks to exploit the connectivity of distal elements up to 2 Mb away in the genome, GraphReg more faithfully models gene regulation and more accurately predicts gene expression levels than the state-of-the-art deep learning methods for this task. Feature attribution used with GraphReg accurately identifies functional enhancers of genes, as validated by CRISPRi-FlowFISH and TAP-seq assays, outperforming both convolutional neural networks (CNNs) and the recently proposed activity-by-contact model. Sequence-based GraphReg also accurately predicts direct transcription factor (TF) targets as validated by CRISPRi TF knockout experiments via in silico ablation of TF binding motifs. GraphReg therefore represents an important advance in modeling the regulatory impact of epigenomic and sequence elements.

FindIT2: an R/Bioconductor package to identify influential transcription factor and targets based on multi-omics data

BackgroundTranscription factors (TFs) play central roles in regulating gene expression. With the rapid growth in the use of high-throughput sequencing methods, there is a need to develop a comprehensive data processing and analyzing framework for inferring influential TFs based on ChIP-seq/ATAC-seq datasets.ResultsHere, we introduce FindIT2 (Find Influential TFs and Targets), an R/Bioconductor package for annotating and processing high-throughput multi-omics data. FindIT2 supports a complete framework for annotating ChIP-seq/ATAC-seq peaks, identifying TF targets by the combination of ChIP-seq and RNA-seq datasets, and inferring influential TFs based on different types of data input. Moreover, benefited from the annotation framework based on Bioconductor, FindIT2 can be applied to any species with genomic annotations, which is particularly useful for the non-model species that are less well-studied.ConclusionFindIT2 provides a user-friendly and flexible framework to generate results at different levels according to the richness of the annotation information of user’s species. FindIT2 is compatible with all the operating systems and is released under Artistic-2.0 License. The source code and documents are freely available through Bioconductor (https://bioconductor.org/packages/devel/bioc/html/FindIT2.html).

Nuclear Tkt promotes ischemic heart failure via the cleaved Parp1/Aif axis.

Transketolase (Tkt), an enzyme in pentose phosphate pathway, has been reported to regulate genome instability and cell survival in cancers. Yet, the role of Tkt after myocardial ischemic injury remains to be elucidated. Label-free proteomics revealed dramatic elevation of Tkt in murine hearts after myocardial infarction (MI). Lentivirus-mediated Tkt knockdown ameliorated cardiomyocyte apoptosis and preserved the systolic function after myocardial ischemic injury. In contrast, Tkt overexpression led to the opposite effects. Inducible conditional cardiomyocyte Tkt-knockout mice were generated, and cardiomyocyte-expressed Tkt was found to play an intrinsic role in the ischemic heart failure of these model mice. Furthermore, through luciferase assay and chromatin immunoprecipitation, Tkt was shown to be a direct target of transcription factor Krüppel-like factor 5 (Klf5). In cardiomyocytes under ischemic stress, Tkt redistributed into the nucleus. By binding with the full-length poly(ADP-ribose) polymerase 1 (Parp1), facilitating its cleavage, and activating apoptosis inducible factor (Aif) subsequently, nuclear Tkt demonstrated its non-metabolic functions. Overall, our study confirmed that elevated nuclear Tkt plays a noncanonical role in promoting cardiomyocyte apoptosis via the cleaved Parp1/Aif pathway, leading to the deterioration of cardiac dysfunction.

Active unfolding of the glucocorticoid receptor by the Hsp70/Hsp40 chaperone system in single-molecule mechanical experiments

The glucocorticoid receptor (GR) is an important transcription factor and drug target linked to a variety of biological functions and diseases. It is one of the most stringent physiological clients of the Hsp90/Hsp70/Hsp40 chaperone system. In this study, we used single-molecule force spectroscopy by optical tweezers to observe the interaction of the GR’s ligand-binding domain (GR-LBD) with the Hsp70/Hsp40 chaperone system (Hsp70/40). We show in real time that Hsp70/40 can unfold the complete GR-LBD in a stepwise manner. Each unfolding step involves binding of an Hsp70 to the GR-LBD and subsequent adenosine triphosphate (ATP) hydrolysis, stimulated by Hsp40. The kinetics of chaperone-mediated unfolding depend on chaperone concentrations as well as the presence of the nucleotide exchange factor BAG1. We find that Hsp70/40 can stabilize new unfolding intermediates, showing that Hsp70/40 can directly interact with the folded core of the protein when working as an unfoldase. Our results support an unfolding mechanism where Hsp70 can directly bind to folded protein structures and unfold them upon ATP hydrolysis. These results provide important insights into the regulation of GR by Hsp70/40.

The MIEL1-ABI5/MYB30 regulatory module fine tunes abscisic acid signaling during seed germination.

The transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) plays a crucial role in abscisic acid (ABA) signaling during seed germination. However, how ABI5 is regulated during this process is poorly understood. Here, we report that the ubiquitin E3 ligase MIEL1 and its target transcription factor MYB30 modulate ABA responses in Arabidopsis thaliana during seed germination and seedling establishment via the precise regulation of ABI5. MIEL1 interacts with and ubiquitinates ABI5 to facilitate its degradation during germination. The transcription factor MYB30, whose turnover is mediated by MIEL1 during seed germination, also interacts with ABI5 to interfere with its transcriptional activity. MYB30 functions downstream of MIEL1 in the ABA response, and both are epistatic to ABI5 in ABA-mediated inhibition of seed germination and postgerminative growth. ABA treatment induces the degradation of MIEL1 and represses the interaction between MIEL1 and ABI5/MYB30, thus releasing both ABI5 and MYB30. Our results demonstrate that MIEL1 directly mediates the proteasomal degradation of ABI5 and inhibits its activity via the release of its target protein MYB30, thus ensuring precise ABA signaling during seed germination and seedling establishment.