Fine-tune Gene Expression Research Articles

Crossing bacterial boundaries: The carbon catabolite repression system Crc-Hfq of Pseudomonas putida KT2440 as a tool to control translation in E. coli

As a global regulatory mechanism, carbon catabolite repression allows bacteria and eukaryal microbes to preferentially utilize certain substrates from a mixture of carbon sources. The mechanism varies among different species. In Pseudomonas spp., it is mainly mediated by the Crc-Hfq complex which binds to the 5′ region of the target mRNAs, thereby inhibiting their translation. This molecular mechanism enables P. putida to rapidly adjust and fine-tune gene expression in changing environments. Hfq is an RNA-binding protein that is ubiquitous and highly conserved in bacterial species. Considering the characteristics of Hfq, and the widespread use and rapid response of Crc-Hfq in P. putida, this complex has the potential to become a general toolbox for post-transcriptional multiplex regulation. In this study, we demonstrate for the first time that transplanting the pseudomonal catabolite repression protein, Crc, into E. coli causes multiplex gene repression. Under the control of Crc, the production of a diester and its precursors was significantly reduced. The effects of Crc introduction on cell growth in both minimal and rich media were evaluated. Two potential factors - off-target effects and Hfq-sequestration - could explain negative effects on cell growth. Simultaneous reduction of off-targeting and increased sequestration of Hfq by the introduction of the small RNA CrcZ, indicated that Hfq sequestration plays a more prominent role in the negative side-effects. This suggests that the negative growth effect can be mitigated by well-controlled expression of Hfq. This study reveals the feasibility of controlling gene expression using heterologous regulation systems.

1627-P: Enhancer Regulator MLL4 Programs Skeletal Muscle Metabolic Efficiency by Limiting AMPK-Mediated Fuel Catabolism

Skeletal muscle adapts to nutrient availability and physiological demands by fine-tuning gene expression patterns and metabolism to maximize metabolic efficiency. The mechanisms that enable and mediate muscle transcriptional and metabolic responses to environmental cues remain unclear, however. We have shown previously that enhancer regulator MLL4 is required for establishing and maintaining slow type I fiber program to ensure running endurance (J Clin Invest. 2020;130(9):4710-4725). Here, we report that MLL4 plays a critical role in directing muscle metabolic transcriptional adaptation that governs metabolic efficiency and systemic metabolism. MLL4 expression is regulated by nutrient availability, and skeletal muscle-specific ablation of MLL4 protects mice against diet-induced obesity and improves systemic glucose homeostasis despite reducing exercise endurance. Combining comprehensive metabolic and genomic analysis of the MLL4-depleted muscles using multidimensional ‘omics’ technologies, we gained insight into the molecular targets and biological functions of the MLL4 protein. Specifically, this seemingly paradoxical phenotype is due to markedly enhanced fuel catabolism in MLL4 deficient muscles, a favorable metabolic reprogramming caused by the activation of muscle AMPK. Isotopic 13C-glucose tracing experiments in myocyte provided further evidence that MLL4 exerts control upon muscle fuel catabolism. Remarkably, pharmacologic targeting the MLL4 pathway activates muscle AMPK, confers resistance to diet-induced obesity and ameliorates obesity-related insulin resistance and liver steatosis. These results identify an enhancer regulator limiting AMPK-mediated muscle fuel catabolism and offer a new potential therapeutic strategy for obesity-related metabolic disorders. Disclosure L.Yang: None. L.Liu: None. C.Ding: None. T.Fu: None. Z.Zhou: None. Z.Gan: None.

Splicing activates transcription from weak promoters upstream of alternative exons

Transcription and splicing are intrinsically coupled. Alternative splicing of internal exons can fine-tune gene expression through a recently described phenomenon called exon-mediated activation of transcription starts (EMATS). However, the association of this phenomenon with human diseases remains unknown. Here, we develop a strategy to activate gene expression through EMATS and demonstrate its potential for treatment of genetic diseases caused by loss of expression of essential genes. We first identified a catalog of human EMATS genes and provide a list of their pathological variants. To test if EMATS can be used to activate gene expression, we constructed stable cell lines expressing a splicing reporter based on the alternative splicing of motor neuron 2 (SMN2) gene. Using small molecules and antisense oligonucleotides (ASOs) currently used for treatment of spinal muscular atrophy, we demonstrated that increase of inclusion of alternative exons can trigger an activation of gene expression up to 45-fold by enhancing transcription in EMATS-like genes. We observed the strongest effects in genes under the regulation of weak human promoters located proximal to highly included skipped exons.

NcOrtho: efficient and reliable identification of miRNA orthologs.

MicroRNAs (miRNAs) are post-transcriptional regulators that finetune gene expression via translational repression or degradation of their target mRNAs. Despite their functional relevance, frameworks for the scalable and accurate detection of miRNA orthologs are missing. Consequently, there is still no comprehensive picture of how miRNAs and their associated regulatory networks have evolved. Here we present ncOrtho, a synteny informed pipeline for the targeted search of miRNA orthologs in unannotated genome sequences. ncOrtho matches miRNA annotations from multi-tissue transcriptomes in precision, while scaling to the analysis of hundreds of custom-selected species. The presence-absence pattern of orthologs to 266 human miRNA families across 402 vertebrate species reveals four bursts of miRNA acquisition, of which the most recent event occurred in the last common ancestor of higher primates. miRNA families are rarely modified or lost, but notable exceptions for both events exist. miRNA co-ortholog numbers faithfully indicate lineage-specific whole genome duplications, and miRNAs are powerful markers for phylogenomic analyses. Their exceptionally low genetic diversity makes them suitable to resolve clades where the phylogenetic signal is blurred by incomplete lineage sorting of ancestral alleles. In summary, ncOrtho allows to routinely consider miRNAs in evolutionary analyses that were thus far reserved to protein-coding genes.

Mitoepigenetics and gliomas: epigenetic alterations to mitochondrial DNA and nuclear DNA alter mtDNA expression and contribute to glioma pathogenicity.

Epigenetic mechanisms allow cells to fine-tune gene expression in response to environmental stimuli. For decades, it has been known that mitochondria have genetic material. Still, only recently have studies shown that epigenetic factors regulate mitochondrial DNA (mtDNA) gene expression. Mitochondria regulate cellular proliferation, apoptosis, and energy metabolism, all critical areas of dysfunction in gliomas. Methylation of mtDNA, alterations in mtDNA packaging via mitochondrial transcription factor A (TFAM), and regulation of mtDNA transcription via the micro-RNAs (mir 23-b) and long noncoding RNAs [RNA mitochondrial RNA processing (RMRP)] have all been identified as contributing to glioma pathogenicity. Developing new interventions interfering with these pathways may improve glioma therapy.

DDT-RELATED PROTEIN4-IMITATION SWITCH alters nucleosome distribution to relieve transcriptional silencing in Arabidopsis.

DNA methylation is a conserved epigenetic modification that is typically associated with silencing of transposable elements and promoter methylated genes. However, some DNA-methylated loci are protected from silencing, allowing transcriptional flexibility in response to environmental and developmental cues. Through a genetic screen in Arabidopsis (Arabidopsis thaliana), we uncovered an antagonistic relationship between the MICRORCHIDIA (MORC) protein and the IMITATION SWITCH (ISWI) complex in regulating the DNA-methylated SUPPRESSOR OF DRM1 DRM2 CMT3 (SDC) reporter. We demonstrate that components of the plant-specific ISWI complex, including CHROMATIN REMODELING PROTEIN11 (CHR11), CHR17, DDT-RELATED PROTEIN4 (DDR4), and DDR5, function to partially de-repress silenced genes and transposable elements (TEs), through their function in regulating nucleosome distribution. This action also requires the known transcriptional activator DNAJ proteins, providing a mechanistic link between nucleosome remodeling and transcriptional activation. Genome-wide studies revealed that DDR4 causes changes in nucleosome distribution at numerous loci, a subset of which is associated with changes in DNA methylation and/or transcription. Our work reveals a mechanism for balancing transcriptional flexibility and faithful silencing of DNA-methylated loci. As both ISWI and MORC family genes are widely distributed across plant and animal species, our findings may represent a conserved eukaryotic mechanism for fine-tuning gene expression under epigenetic regulation.

Comparative Biochemical Studies of Disease-Associated Human Dicer Mutations on Processing of a Pre-microRNA and snoRNA.

Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer's catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitro biochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.

A new pipeline denoted SPICE identifies novel JUN-IKZF1 composite elements

Abstract DNA composite elements allow combinatorial transcription factor binding, which is important for fine tuning gene expression in response to physiological stimuli and in pathological conditions. Moreover, composite elements may contribute to epigenetic regulation. Accordingly, developing screening algorithms to identify novel composite elements and their compositions is crucial. Here we report a pipeline termed Spacing Preference Identification of Composite Elements (SPICE), that systematically predicts novel protein binding partners along with their DNA motifs and spacing preferences in a genome wide fashion. Besides predicting known composite elements like AP1-IRF composite elements (AICE) and STAT5 tetramers, SPICE de novo predicted a range of novel binding partners, including JUN-IKZF1 composite elements. We validated JUN-IKZF1 association by co-immunoprecipitation and showed cooperative binding of these factors to an upstream conserved noncoding region, CNS9, in the human IL10 gene based on electrophoretic mobility shift assays. Furthermore, the activity of an IL10 luciferase reporter depended on both AP1 and IKZF1 binding sites within this composite element in mouse B and T cells, confirming its transcriptional relevance. These data establish SPICE as a valuable pipeline for predicting novel transcription factor complexes that regulate gene transcription and provide insights into the biological cooperation of IKZF1 and AP1. Division of Intramural Research, NHLBI.

Abstract IA15: Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer

Abstract The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacological inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor, Trametinib (MEKi), we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition. Citation Format: Rushika M. Perera. Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA15.

The Archer and the Prey: The Duality of PAF1C in Antiviral Immunity.

In the ongoing arms race between virus and host, fine-tuned gene expression plays a critical role in antiviral signaling. However, viruses have evolved to disrupt this process and promote their own replication by targeting host restriction factors. Polymerase-associated factor 1 complex (PAF1C) is a key player in this relationship, recruiting other host factors to regulate transcription and modulate innate immune gene expression. Consequently, PAF1C is consistently targeted by a diverse range of viruses, either to suppress its antiviral functions or co-opt them for their own benefit. In this review, we delve into the current mechanisms through which PAF1C restricts viruses by activating interferon and inflammatory responses at the transcriptional level. We also highlight how the ubiquity of these mechanisms makes PAF1C especially vulnerable to viral hijacking and antagonism. Indeed, as often as PAF1C is revealed to be a restriction factor, viruses are found to have targeted the complex in reply.

YBX2 modulates mRNA stability via interaction with YTHDF2 in endometrial cancer cells

RNA-binding proteins (RBPs) fine-tune gene expression by modulating RNA stability, translation, and degradation. RBPs are involved in the development of endometrial cancer. In particular, Y-box-binding protein 2 (YBX2), a germ cell-specific member of the YBX family, has been reported to maintain cancer stem cell-like phenotypes in endometrial cancer. However, the mechanism by which YBX2 modulates mRNA stability in endometrial cancer cells remains unknown. In this study, we examined the effects of the ectopic expression of YBX2 in endometrial adenocarcinoma-derived Ishikawa cells. We found that elevated levels of YBX2 delayed cell proliferation, without increasing cell apoptosis. Transcriptomic analysis revealed disturbances in gene expression caused by YBX2. Interestingly, heat shock protein family A (Hsp70) member 6 (HSPA6) levels were downregulated due to the reduced mRNA stability after YBX2 binding. YBX2 facilitated the formation of relatively stable cytoplasmic granules in tumor cells via its mRNA binding domain. Moreover, N6-methyladenosine (m6A) reader proteins are recruited by YBX2 granules via the cold-shock domain. Notably, knockdown of YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2), an m6A reader, ameliorated the reduction in HSPA6 mRNA levels induced by YBX2, indicating the synergistic effects of YBX2 and YTHDF2 on mRNA stability. Therefore, YBX2 regulates RNA stability by interacting with the m6A reader proteins.

Single-cytosine methylation at W-boxes repels binding of WRKY transcription factors through steric hindrance.

DNA methylation is an epigenetic mark that fine-tunes gene expression, notably by negatively or positively regulating transcription factor (TF)-DNA binding. In plants, DNA methylation has primarily been shown to inhibit TF-DNA binding. However, little is known about the underlying mechanisms. Here, we show that DNA methylation decreases the binding of several Arabidopsis (Arabidopsis thaliana) WRKY TFs to their genomic regions and their binding sites in vitro. We also provide evidence that DNA methylation at a single cytosine located in a functional core W-box motif repels DNA binding of AtWRKY40 in vitro. Using structural modelling, we further demonstrate that this cytosine interacts through van der Waals contacts with the conserved tyrosine of WRKY-DNA binding domains. Importantly, our model predicts steric hindrance when a 5-methyl group is present on this specific cytosine, thereby likely preventing tight binding of WRKY-DNA binding domains. Finally, because the WRKY motif and the residues involved in DNA contacts are conserved across Arabidopsis and rice (Oryza sativa) WRKY TFs, we propose that this methylation-dependent WRKY-DNA binding inhibitory mechanism could be widespread across plant species.

Mining and application of constitutive promoters from Rhodosporidium toruloides

Rhodosporidium toruloides is an oleaginous yeast under development with promising industrial applications. Since promoters of different strengths have been demonstrated as an efficient strategy to fine-tune gene expression in synthetic biology, a set of constitutive promoters with strengths varying over 2 orders of magnitude were identified in R. toruloides through transcriptome analysis under different growth conditions. Promoter candidates were first cloned and characterized using an enhanced green fluorescent protein (EGFP) as a reporter under eight conditions, and 31 promoters were identified with strength varied from 0.1 to 19.0 folds of the commonly used strong promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGPD1). The resultant promoters were then used to optimize the linoleic acid biosynthetic pathway in R. toruloides in different media, including the use of lignocellulosic hydrolysate as the fermentation substrate, and improved the production of linoleic acid by up to 214.2% in minimal medium, with the highest production of 350.3 mg/L in Yeast Peptone Dextrose medium. This work has enriched the promoter library of R. toruloides, and helped develop R. toruloides as a platform organism for applications in biomanufacturing and synthetic biology.

Coupling epigenetics and RNA polyadenylation: missing links.

Precise regulation of gene expression is crucial for plant survival. As a cotranscriptional regulatory mechanism, pre-mRNA polyadenylation is essential for fine-tuning gene expression. Polyadenylation can be alternatively projected at various sites of a transcript, which contributes to transcriptome diversity. Epigenetic modification is another mechanism of transcriptional control. Recent studies have uncovered crosstalk between cotranscriptional polyadenylation processes and both epigenomic and epitranscriptomic markers. Genetic analyses have demonstrated that DNA methylation, histone modifications, and epitranscriptomic modification are involved in regulating polyadenylation in plants. Here we summarize current understanding of the links between epigenetics and polyadenylation and their novel biological efficacy for plant development and environmental responses. Unresolved issues and future directions are discussed to shed light on the field.

Messenger-RNA modification standards and machine learning models facilitate absolute site-specific quantification of uridine modifications.

Enzyme-mediated chemical modifications to mRNA are important for fine-tuning gene expression, but they are challenging to quantify due to low copy number and limited tools for accurate detection. The pseudouridine (ψ) modification is highly abundant in mRNAs but is difficult to detect and quantify because there is no available antibody, it is mass silent, and maintains canonical basepairing with adenine. The presence of another uridine modification, dihydrouridine (DHU), was recently demonstrated in mRNA through chemical labeling. We and others have recently shown that nanopores may be used to qualitatively identify uridine modifications in direct RNA sequencing by alignment to an unmodified transcriptome. In this work, we apply supervised machine learning models that are trained on sequence-specific modified and unmodified synthetic controls to endogenous transcriptome data, to achieve site-specific uridine modification quantification. Our models reveal that for every site studied, different signal parameters are required to maximize the accuracy of ψ and DHU classification, suggesting that application transcriptome-wide would require more standards. We show that applying our model is critical for quantification, particularly for low-abundance mRNAs. Our engine, used here to quantitatively profile ψ and DHU occupancy in specific human mRNA sites, can be implemented across cell types and cell states to determine the occupancy of uridine modifications, thus providing critical insights into physiological relevance of ψ and DHU to mRNAs. With a further library of appropriate molecular standards, our method has the potential to quantify all putative uridine modifications in the human transcriptome.

Real-time tracking of transcription-translation coupling.

In prokaryotic cells, transcription and translation occur in the same compartment, enabling a functional interaction between both gene expression machineries. Several studies, including recent high-resolution structures, demonstrate a physical link that establishes coupling between both machineries either mediated by direct interactions between the RNAP and the ribosomeor by factor-mediated interactions via the transcription factors NusA and NusG. In contrast, recent work challenges the notion that transcription and translation are generally coupled. Here, to understand what promotes coupling and how it is regulated, we reconstituted in vitro the complete active transcription-translation coupling system and developed multicolor single-molecule fluorescence microscopy assays to track in real-time how single RNA molecules are actively transcribed (Duss et al., Cell, 2019) and simultaneously translated by the ribosome. Furthermore, by site-specifically labeling the ribosome and the RNAP, we can detect the direct physical interaction between both machineries. Our data shows that physical coupling between RNAP and the ribosome can be transient and depends on the mRNA length between both machineries. We also detect different coupled states while the polymerase moves away from the ribosome, likely representing the different states found by cryoEM (Wang & Ebright, Science, 2020; Webster & Weixlbaumer, Science, 2020; O’Reilly & Rappsilber, Science, 2020). RNAP and the ribosome can also remain physically coupled even if the transcription machinery moves more than 100 nucleotides away from the ribosome suggesting that physical and functional coupling can be unrelated. Overall, by tracking the conformation and interaction dynamics of different macromolecular machines over time, our methods allow us to investigate how different macromolecular machines cooperate to fine-tune gene expression.

Differential chromatin binding preference is the result of the neo-functionalization of the TB1 clade of TCP transcription factors in grasses.

The understanding of neo-functionalization of plant transcription factors (TFs) after gene duplication has been extensively focused on changes in protein-protein interactions, the expression pattern of TFs, or the variation of cis-elements bound by TFs. Yet, the main molecular role of a TF, that is, its specific chromatin binding for the direct regulation of target gene expression, continues to be mostly overlooked. Here, we studied the TB1 clade of the TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTORS (TCP) TF family within the grasses (Poaceae). We identified an Asp/Gly amino acid replacement within the TCP domain, originated within a paralog TIG1 clade exclusive for grasses. The heterologous expression of Zea mays TB1 and its two paralogs BAD1 and TIG1 in Arabidopsis mutant plants lacking the TB1 ortholog BRC1 revealed distinct functions in plant development. Notably, the Gly acquired in the TIG1 clade does not impair TF homodimerization and heterodimerization, while it modulates chromatin binding preferences. We found that invivo TF recognition of target promoters depends on this Asp/Gly mutation and directly impacts downstream gene expression and subsequent plant development. These results provided new insights into how natural selection fine-tunes gene expression regulation after duplication of TFs to define plant architecture.

Engineered PROTAC-CID Systems for Mammalian Inducible Gene Regulation.

Gene regulation via chemically induced dimerization (CID) is useful for biomedical research. However, the number, type, versatility, and in vivo applications of CID tools remain limited. Here, we demonstrate the development of proteolysis-targeting chimera-based scalable CID (PROTAC-CID) platforms by systematically engineering the available PROTAC systems for inducible gene regulation and gene editing. Further, we show orthogonal PROTAC-CIDs that can fine-tune gene expression at gradient levels or multiplex biological signals with different logic gating operations. Coupling the PROTAC-CID platform with genetic circuits, we achieve digitally inducible expression of DNA recombinases, base- and prime-editors for transient genome manipulation. Finally, we package a compact PROTAC-CID system into adeno-associated viral vectors for inducible and reversible gene activation in vivo. This work provides a versatile molecular toolbox that expands the scope of chemically inducible gene regulation in human cells and mice.

Comparison of neuronal GAL4 drivers along with the AGES (auxin-inducible gene expression system) and TARGET (temporal and regional gene expression targeting) systems for fine tuning of neuronal gene expression in Drosophila.

Spatial and temporal control of gene expression in Drosophila is essential in elucidating gene function. Spatial control is facilitated by the UAS/GAL4 system, and this can be coupled with additional adaptations for precise temporal control and fine tuning of gene expression levels. Here we directly compare the level of pan-neuronal transgene expression governed by nSyb-GAL4 and elav-GAL4, as well as mushroom body-specific expression alongside OK107-GAL4. We also compare the temporal modulation of gene expression in neurons with the auxin-inducible gene expression system (AGES) and temporal and regional gene expression targeting (TARGET) systems.

Plant Promoters and Terminators for High-Precision Bioengineering.

High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels. Gene transcription is tightly regulated by promoters and terminators. Promoters determine the timing, tissues and cells, and levels of the expression of genes. Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally, e.g., the 3'-end processing, stability, translation efficiency, and nuclear to cytoplasmic export of mRNAs. The promoter and terminator combination affects gene expression. In the present article, we review the function and features of plant core promoters, proximal and distal promoters, and terminators, and their effects on and benchmarking strategies for regulating gene expression.