Fine-tune Gene Expression Research Articles

Abstract 67: Functional analysis of lncRNA DUXAP10 in anaplastic thyroid cancer

Abstract Anaplastic Thyroid Cancer (ATC) is one of the most aggressive forms of thyroid cancer, with only a 4% survival rate with metastatic disease. Further, ATC is highly refractory and resistant to traditional therapeutics, resulting in poor patient outcomes. Therefore, the elucidation of molecular mechanisms that contribute to ATC’s metastatic propensity, and the identification of molecular biomarkers for the early diagnosis, prognosis, and therapeutic intervention are critical unmet needs. This is reinforced by the many challenges that accompany the use of small molecular inhibitors and targeted therapy to treat ATC. Recently, long non-coding RNAs (lncRNA) have been identified as having major regulatory roles in the fine-tuning of gene expression and cellular behavior of cancer cells, highlighting the importance of identifying non-coding profiles that potentially impact ATC behavior. Using the publicly available datasets (Gene Expression Omnibus; GSE33630, GSE85457), we have investigated gene expression profiles of ATC vs. normal thyroid tissue. Bioinformatic analyses with the GEO2R program identified the lncRNA, Double Homeobox A Pseudogene 10 (DUXAP10), to be highly upregulated in ATC patient-derived tissue samples when compared to normal thyroid tissue. DUXAP10 is structurally a pseudogene-derived lncRNA that has been identified to be upregulated in hepatocellular, bladder, esophageal, and gastric cancer, as well as papillary thyroid cancer. We found that the expression of DUXAP10 is also upregulated in the ATC cell line, T238, in comparison to Nthy-ori-3-1, an immortalized normal thyroid cell line. T238 is homozygous for the BRAF V600E mutation, which serves as a driver mutation. However, breakthrough alterations can limit the effectiveness of anti-BRAF therapeutics and highlight the utility of combination therapies. To understand the consequences of DUXAP10 overexpression in ATC, we used CRISPRi technology to determine the phenotypic and genotypic effects of DUXAP10 downregulation. CRISPRi knockdown of DUXAP10 significantly reduces the migratory capabilities of T238 by 87%. This provides evidence that elevated DUXAP10 expression may increase the probability and negative consequences of metastases. Thus, targeting DUXAP10 expression may have beneficial clinical consequences. The role of DUXAP10 on thyroid cell proliferation and invasion are currently in progress. Thus, we have identified DUXAP10 as a potential biomarker for ATC diagnosis and prognosis, as well as a potential therapeutic target. Further analysis remains to be determined if DUXAP10 can be targeted for standalone or combination therapy, as well as the elucidation of the full range of interactions that DUXAP10 has in ATC. Citation Format: Nicole DeSouza, Michelle Carnazza, Danielle Quaranto, Tara Jarboe, Kaci Kopec, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Functional analysis of lncRNA DUXAP10 in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 67.

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells.

Simple SummaryThree-dimensional (3D) cell cultures have several advantages over conventional monolayer two-dimensional (2D) cultures as they can better mimic tumor biology. This study delineates the changes in microRNAs (miRNAs) expression patterns of breast cancer cells cultured in 3D and 2D conditions. 3D organotypic cultures showed morphological changes such as cell–cell and cell–extracellular matrix interactions associated with a loss of polarity and reorganization on bulk structures in both basal Hs578T and luminal T47D breast cancer cells. Data indicate that downregulated miRNAs in Hs578T 3D cultures, relative to the 2D condition, contribute to a positive regulation of biological processes such as response to hypoxia and focal adhesion, whereas overexpressed miRNAs were related to negative regulation of the cell cycle. Remarkably, the reprogramming of miRNAs transcriptional profile was accompanied by changes in the expression of key miRNA/mRNA coregulation networks, such as miR-935/HIF-1A, which correlated with the expression found in clinical breast tumors and predicted poor patient outcomes. These data have implications in our understanding of cancer biology and impact the miRNA/mRNA regulatory axes of cells grown in 3D cultures. Our data represent a guide for novel miRNAs candidates for functional analysis, including the response to therapy and biomarkers discovery in breast cancer.The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell–cell, and cell–ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer.

Design and Synthesis of Bi‐aryl Methylated Lactam Derivatives to Inhibit the BRD7 Bromodomain Function in Prostate Cancer

Targeting bromodomain‐containing proteins has emerged as a promising therapeutic strategy and multiple clinical studies indicate that bromodomain (BD) inhibitors can act as anticancer and anti‐inflammatory agents. Bromodomain‐containing protein 7 (BRD7) is a subunit of the SWI/SNF chromatin remodeling complex, which is involved in the fine‐tuning of gene expression through changes in the organization of DNA. It regulates the progression of different types of cancers such as nasopharyngeal carcinoma and hormone‐dependent breast and ovarian cancers. BRD7 is also implicated in progression of prostate cancer, another hormone‐dependent malignancy; however, the role of BRD7 in this type of cancer is undefined. (Figure 1) We have characterized BRD7 as an oncogenic driver across a variety of prostate cancer cell lines, implicating BRD7 inhibitors as a potential therapeutic strategy to combat therapy resistant cancers.To develop inhibitors of BRD7 we are developing small molecules that bind selectively to the bromodomain of BRD7. The main challenge when developing BRD7 BD inhibitors is the high similarity to the BRD9 BD, its closest homolog. Several dual inhibitors of BRD7 and BRD9 have been developed, all of them being more selective for the BRD9 BD over the BRD7 BD. Using the X‐ray crystal structure of both BDs bound to a BRD9‐selective inhibitor, we identified an accessible hydrophobic region in the binding pocket of BRD7 that is not present in BRD9, which we hypothesized could be exploited to develop BRD7‐selective inhibitors. Based on this structural difference in the BRD7 binding pocket, we designed a library of compounds that share a bi‐aryl methylated lactam core. (Figure 2)From in silicomolecular docking simulations and in vitro affinity screenings, we have identified several ligands selective for BRD7. These compounds also display potent anti‐proliferative effects in prostate cancer cell lines, providing further evidence for proposing BRD7 as a therapeutic target in prostate cancer. Our on‐going work focuses on optimizing the potency and solubility properties of BRD7 inhibitors, and characterizing their selectivity using cell‐based models.

Dataset for GB_SynP: a modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants

Dataset for GB_SynP: a modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants

Characterization of aquaporin-1ab (Aqp1ab) mRNA in mud loach (Misgurnus mizolepis) exposed to heavy metal and immunostimulant stimuli

Aquaporins (AQPs) are key proteins that regulate fluid homeostasis in cells via modulating osmotic water transport. In the present study, we identified three variants of Aqp1ab transcript (mmAQP1ab x1, mmAQP1ab x2, and mmAQP1ab x3) in mud loaches (Misgurnus mizolepis), and their expression patterns were examined in response to heavy metal and immunostimulant exposure. Mud loach Aqp1ab gene has a somewhat different organizational structure (i.e. five exons interrupted by four introns) compared to most other teleostean Aqp1ab orthologues, which have four exons. The 5′-flanking regulatory region of Aqp gene showed diverse transcription factor binding motifs, particularly those associated with stress/immune responses. Developmental expression patterns indicated that Aqp1ab mRNA was maternally inherited, presumably important for fine-tuning gene expression during embryonic and early larval developments. Expression of mud loach Aqp1ab mRNA was significantly and differentially modulated in several tissues (intestine, kidneys, spleen, and liver) in response to various heavy metal treatments. In addition, Aqp1ab gene expression was highly induced in response to immune challenge (LPS and polyI:C injections). Collectively, our results suggested that AQPs are multifunctional effectors playing diverse roles in cellular pathways relevant to immune and/or stress adaptation responses, in addition to their involvement in osmoregulation.

The story of rRNA expansion segments: Finding functionality amidst diversity.

Expansion segments (ESs) are multinucleotide insertions present across phyla at specific conserved positions in eukaryotic rRNAs. ESs are generally absent in bacterial rRNAs with some exceptions, while the archaeal rRNAs have microexpansions at regions that coincide with those of eukaryotic ESs. Although there is an increasing prominence of ribosomes, especially the ribosomal proteins, in fine-tuning gene expression through translation regulation, the role of rRNA ESs is relatively underexplored. While rRNAs have been established as the major catalytic hub in ribosome function, the presence of ESs widens their scope as a species-specific regulatory hub of protein synthesis. In this comprehensive review, we have elaborately discussed the current understanding of the functional aspects of rRNA ESs of cytoplasmic eukaryotic ribosomes and discuss their past, present, and future. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems Translation > Ribosome Structure/Function Translation > Regulation.

Somatic Mutations in Core Spliceosome Components Promote Tumorigenesis and Generate an Exploitable Vulnerability in Human Cancer

Simple SummaryHigh throughput exome sequencing approaches have disclosed recurrent cancer-associated mutations in spliceosomal components, which drive aberrant pre-mRNA processing events and support the tumor phenotype. At the same time, mutations in spliceosome genes and aberrant splicing regulation establish a selective vulnerability of cancer cells to splicing-targeting approaches, which could be exploited therapeutically. It is conceivable that a better understanding of the mechanisms and roles of abnormal splicing in tumor metabolism will facilitate the development of a novel generation of tumor-targeting drugs. In this review, we describe recent advances in the elucidation of the biological impact and biochemical effects of somatic mutations in core spliceosome components on splicing choices and their associated targetable vulnerabilities.Alternative pre-mRNA processing enables the production of distinct mRNA and protein isoforms from a single gene, thus greatly expanding the coding potential of eukaryotic genomes and fine-tuning gene expression programs. Splicing is carried out by the spliceosome, a complex molecular machinery which assembles step-wise on mRNA precursors in the nucleus of eukaryotic cells. In the last decade, exome sequencing technologies have allowed the identification of point mutations in genes encoding splicing factors as a recurrent hallmark of human cancers, with higher incidence in hematological malignancies. These mutations lead to production of splicing factors that reduce the fidelity of the splicing process and yield splicing variants that are often advantageous for cancer cells. However, at the same time, these mutations increase the sensitivity of transformed cells to splicing inhibitors, thus offering a therapeutic opportunity for novel targeted strategies. Herein, we review the recent literature documenting cancer-associated mutations in components of the early spliceosome complex and discuss novel therapeutic strategies based on small-molecule spliceosome inhibitors that exhibit strong anti-tumor effects, particularly against cancer cells harboring mutations in spliceosomal components.

Internal Transcription Terminators Control Stoichiometry of ABC Transporters in Cellulolytic Clostridia.

ABSTRACTThe extracellular substrate-binding proteins (SBPs) of ATP-binding cassette (ABC) importers tend to be expressed in excess relative to their cognate translocators, but how the stoichiometry of ABC transporters is controlled remains unclear. Here, we elucidated a mechanism contributing to differential gene expression in operons encoding ABC importers by employing cellulolytic Clostridia species, specifically Ruminiclostridium cellulolyticum. We found that there were usually stem-loop structures downstream of SBP genes, which could prematurely terminate the transcription of ABC importers and were putative internal intrinsic terminators, resulting in high transcript levels of upstream SBP genes and low transcript levels of downstream cognate translocator genes. This was determined by their termination efficiencies. Internal terminators had a lower U content in their 3′ U-rich tracts and longer GC-rich stems, which distinguishes them from canonical terminators and potentially endows them with special termination efficiencies. The pairing of U-rich tracts and the formation of unpaired regions in these internal terminators contributed to their folding energies, affecting the stability of their upstream SBP transcripts. Our findings revealed a strategy of internal transcriptional terminators controlling in vivo stoichiometry of their flanking transcripts.IMPORTANCE Operons encoding protein complexes or metabolic pathways usually require fine-tuned gene expression ratios to create and maintain the appropriate stoichiometry for biological functions. In this study, a strategy for controlling differential expression of genes in an operon was proposed by utilizing ABC importers from Ruminiclostridium cellulolyticum. We found that a stem-loop structure is introduced into the intergenic regions of operons encoding ABC importers as the putative internal terminator, which results in the premature termination of transcription. Consequently, the stoichiometric ratio of genes flanking terminators is precisely determined by their termination efficiencies and folding energies at the transcriptional level. Thus, it can be utilized as a promising synthetic biology tool to control the differential expression of genes in an operon.

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.

Development of a novel expression system in lactic acid bacteria controlled by a broad-host-range promoter PsrfA

BackgroundLatic acid bacteria (LAB) are exploited for development of gene expression system owing to its health promoting properties and a high degree of safety status. Most of the expression systems were constructed in Lactobacillus lactis with inducible promoters. It is necessary to exploit novel promoters to develop LAB host platforms which are indispensable in dairy and health application to satisfy the production demand of increased number of target-genes. Previously, promoter PsrfA had been displayed broad host range and used to construct auto-inducible expression system in B. subtilis and E. coli. In this work, the feasibility of PsrfA in LAB was estimated.ResultsPlasmid with the green fluorescent protein (GFP) inserting downstream of PsrfA was transformed into L. casei 5257, L. plantarum 97, L. fermentum 087 and Weissella confusa 10, respectively. The recombinant strains grew well and displayed different fluorescence which could be detected by spectrophotometer and laser scanning confocal microscope. Moreover, the promoter activity was strain- specifically influenced by particular carbon and nitrogen sources. Heterologous laccase CotA could be expressed by PsrfA in L. casei 5257-05 and L. plantarum 97-06. By adjusting the pH value from 4.5 to 6.5 during incubation, the CotA activity detected from L. plantarum 97-05 and L. casei 5257-05 was increased by 137.7% and 61.5%, respectively. Finally, the fermentation pH was variably up-regulated along with the production of NADH oxidase which was controlled by the PsrfA and its derivative mutated with core regions.ConclusionsThese data suggested that PsrfA was valid for gene expression in different species of LAB. Moreover, PsrfA could be used as an attractive candidate for fine-tuning gene expression in a broad range of prokaryotic expression plants.

Model-driven design of synthetic N-terminal coding sequences for regulating gene expression in yeast and bacteria.

N-terminal coding sequences (NCSs) are key regulatory elements for fine-tuning gene expression during translation initiation-the rate-limiting step of translation. However, owing to the complex combinatory effects of NCS biophysical factors and endogenous regulation, designing NCSs remains challenging. In this study, a multi-view learning strategy for model-driven generation of synthetic NCSs for Saccharomyces cerevisiae and Bacillus subtilis are implemented, which are widely used in laboratories and industries. NCS libraries for S. cerevisiae and B. subtilis with nearly 150,000 cells were sorted. Next, model training was performed with NCS deep features extracted from DNA, codon, and amino acid sequences, as well as calculated features from the minimum free energy (MFE) and tRNA adaption index. Two models were separately developed for generating synthetic NCSs for both up- and down-regulating gene expression with accuracies higher than 65% for S. cerevisiae and B. subtilis. Synthetic NCSs were then applied to enhance bioproduction, yielding 1.48- and 1.71-fold production improvements of D-limonene by S. cerevisiae and ovalbumin by B. subtilis, respectively. This work provides model-driven design of synthetic NCSs as a toolbox for regulating gene expression in S. cerevisiae and B. subtilis. The machine learning-based modeling approach can be used for NCS design in other microorganisms.

Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production

Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system.

Kingdom-Wide Analysis of FungalProtein-Coding and tRNA Genes Reveals Conserved Patterns of Adaptive Evolution.

Protein-coding genes evolved codon usage bias due to the combined but uneven effects of adaptive and nonadaptive influences. Studies in model fungi agree on codon usage bias as an adaptation for fine-tuning gene expression levels; however, such knowledge is lacking for most other fungi. Our comparative genomics analysis of over 450 species supports codon usage and transfer RNAs (tRNAs) as coadapted for translation speed and this is most likely a realization of convergent evolution. Rather than drift, phylogenetic reconstruction inferred adaptive radiation as the best explanation for the variation of interspecific codon usage bias. Although the phylogenetic signals for individual codon and tRNAs frequencies are lower than expected by genetic drift, we found remarkable conservation of highly expressed genes being codon optimized for translation by the most abundant tRNAs, especially by inosine-modified tRNAs. As an application, we present a sequence-to-expression neural network that uses codons to reliably predict highly expressed transcripts. The kingdom Fungi, with over a million species, includes many key players in various ecosystems and good targets for biotechnology. Collectively, our results have implications for better understanding the evolutionary success of fungi, as well as informing the biosynthetic manipulation of fungal genes.

Construction of Strong Promoters by Assembling Sigma Factor Binding Motifs.

Development of strong promoters is of growing interest in the field of biotechnology and synthetic biology. Here we present a protocol for the construction of strong prokaryotic promoters that can be recognized by designated multiple sigma factors by interlocking their cognate binding motifs on DNA strands. Strong and stress responsive promoters for Escherichia coli and Bacillus subtilis have been created following the presented protocol. Customized promoters could be easily developed for fine-tuning gene expression or overproducing enzymes with prokaryotic cell factories.

Fine-Tuning Plant Gene Expression with Synthetic Trans-Acting Small Interfering RNAs.

RNAi-based tools are widely used in gene function studies and for crop improvement. However, no effective methods for precisely controlling the degree of induced silencing have been reported until recently. Here we report a detailed protocol for designing and generating synthetic trans-acting small interfering RNA (syn-tasiRNA) constructs for fine-tuning gene expression in plants. Recently developed high-throughput AtTAS1c-D2-B/c-based vectors are used to clone and express syn-tasiRNAs that possess different efficacies depending on their precursor location and on their degree of base-pairing with the 5' end of target RNAs.

Pulsed Electric Fields Alter Expression of NF-κB Promoter-Controlled Gene.

The possibility to artificially adjust and fine-tune gene expression is one of the key milestones in bioengineering, synthetic biology, and advanced medicine. Since the effects of proteins or other transgene products depend on the dosage, controlled gene expression is required for any applications, where even slight fluctuations of the transgene product impact its function or other critical cell parameters. In this context, physical techniques demonstrate optimistic perspectives, and pulsed electric field technology is a potential candidate for a noninvasive, biophysical gene regulator, exploiting an easily adjustable pulse generating device. We exposed mammalian cells, transfected with a NF-κB pathway-controlled transcription system, to a range of microsecond-duration pulsed electric field parameters. To prevent toxicity, we used protocols that would generate relatively mild physical stimulation. The present study, for the first time, proves the principle that microsecond-duration pulsed electric fields can alter single-gene expression in plasmid context in mammalian cells without significant damage to cell integrity or viability. Gene expression might be upregulated or downregulated depending on the cell line and parameters applied. This noninvasive, ligand-, cofactor-, nanoparticle-free approach enables easily controlled direct electrostimulation of the construct carrying the gene of interest; the discovery may contribute towards the path of simplification of the complexity of physical systems in gene regulation and create further synergies between electronics, synthetic biology, and medicine.

RNA m6A Modification in Immunocytes and DNA Repair: The Biological Functions and Prospects in Clinical Application.

As the most prevalent internal modification in mRNA, N 6-methyladenosine (m6A) plays broad biological functions via fine-tuning gene expression at the post-transcription level. Such modifications are deposited by methyltransferases (i.e., m6A Writers), removed by demethylases (i.e., m6A Erasers), and recognized by m6A binding proteins (i.e., m6A Readers). The m6A decorations regulate the stability, splicing, translocation, and translation efficiency of mRNAs, and exert crucial effects on proliferation, differentiation, and immunologic functions of immunocytes, such as T lymphocyte, B lymphocyte, dendritic cell (DC), and macrophage. Recent studies have revealed the association of dysregulated m6A modification machinery with various types of diseases, including AIDS, cancer, autoimmune disease, and atherosclerosis. Given the crucial roles of m6A modification in activating immunocytes and promoting DNA repair in cells under physiological or pathological states, targeting dysregulated m6A machinery holds therapeutic potential in clinical application. Here, we summarize the biological functions of m6A machinery in immunocytes and the potential clinical applications via targeting m6A machinery.

MED12 and BRD4 cooperate to sustain cancer growth upon loss of mediator kinase

Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.

MicroRNA Cues from Nature: A Roadmap to Decipher and Combat Challenges in Human Health and Disease?

MicroRNAs are small non-coding RNA (18–24 nt long) that fine-tune gene expression at the post-transcriptional level. With the advent of “multi-omics” analysis and sequencing approaches, they have now been implicated in every facet of basic molecular networks, including metabolism, homeostasis, and cell survival to aid cellular machinery in adapting to changing environmental cues. Many animals must endure harsh environmental conditions in nature, including cold/freezing temperatures, oxygen limitation (anoxia/hypoxia), and food or water scarcity, often requiring them to revamp their metabolic organization, frequently on a seasonal or life stage basis. MicroRNAs are important regulatory molecules in such processes, just as they are now well-known to be involved in many human responses to stress or disease. The present review outlines the role of miRNAs in natural animal models of environmental stress and adaptation including torpor/hibernation, anoxia/hypoxia tolerance, and freeze tolerance. We also discuss putative medical applications of advances in miRNA biology including organ preservation for transplant, inflammation, ageing, metabolic disorders (e.g., obesity), mitochondrial dysfunction (mitoMirs) as well as specialized miRNA subgroups respective to low temperature (CryomiRs) and low oxygen (OxymiRs). The review also covers differential regulation of conserved and novel miRNAs involved at cell, tissue, and stress specific levels across multiple species and their roles in survival. Ultimately, the species-specific comparison and conserved miRNA responses seen in evolutionarily disparate animal species can help us to understand the complex miRNA network involved in regulating and reorganizing metabolism to achieve diverse outcomes, not just in nature, but in human health and disease.

Metabolic Fuel for Epigenetic: Nuclear Production Meets Local Consumption.

Epigenetic modifications are responsible for finetuning gene expression profiles to the needs of cells, tissues, and organisms. To rapidly respond to environmental changes, the activity of chromatin modifiers critically depends on the concentration of a handful of metabolites that act as substrates and co-factors. In this way, these enzymes act as metabolic sensors that directly link gene expression to metabolic states. Although metabolites can easily diffuse through the nuclear pore, molecular mechanisms must be in place to regulate epigenetic marker deposition in specific nuclear subdomains or even on single loci. In this review, I explore the possible subcellular sites of metabolite production that influence the epigenome. From the relationship between cytoplasmic metabolism and nuclear metabolite deposition, I converse to the description of a compartmentalized nuclear metabolism. Last, I elaborate on the possibility of metabolic enzymes to operate in phase-separated nuclear microdomains formed by multienzyme and chromatin-bound protein complexes.