Gene Expression In Eukaryotic Cells Research Articles

Transcription bodies regulate gene expression by sequestering CDK9

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9—the kinase that releases paused polymerase II—is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.

A circular RNA-gawky-chromatin regulatory axis modulates stress-induced transcription.

In response to heavy metal stress, the RNA-binding protein (RBP) gawky translocates into the nucleus and acts as a chromatin-interacting factor to activate the transcription of many stress-responsive genes. However, the upstream regulators of gawky-mediated transcription and their mechanistic details remain unknown. Here, we identified a class of metal-responsive element-containing circRNAs (MRE circRNAs) which specifically interact with gawky during copper stress. Using classic stress-responsive genes as a readout (Drosophila MT), we found that overexpression of MRE circRNAs led to a significant repression in stress-induced transcription. Mechanistically, MRE circRNAs promote the dissociation of gawky from chromatin and increase its aberrant cytoplasmic accumulation, which ultimately impedes the loading of RNA polymerase II to the active gene loci. The MRE motif serves as an important RNA regulon for maintaining the circRNA-gawky interaction, loss of which impaired the inhibitory effects of MRE circRNAs on gawky. Through RNA-seq analyses, we then identified over 500 additional stress-responsive genes whose induced transcription was attenuated upon MRE circRNA overexpression. Finally, we uncovered the physiological relevance of MRE circRNA-mediated regulation in cellular defense against copper overloading. Taken together, this study proposes that the circRNA-RBP-chromatin axis may represent a fundamental regulatory network for gene expression in eukaryotic cells.

Combined 3D DNA FISH, Single-Molecule RNA FISH, and Immunofluorescence.

Nuclear architecture is a potential regulator of gene expression in eukaryotic cells. Studies connecting nuclear architecture to gene expression are often population-averaged and do not report on the cell-level heterogeneity in genome organization and associated gene expression. In this report we present a simple way to combine fluorescence in situ hybridization (FISH)-based detection of DNA, with single-molecule RNA FISH (smFISH) and immunofluorescence (IF), while also preserving the three-dimensional (3D) nuclear architecture of a cell. Recently developed smFISH techniques enable the detection of individual RNA molecules; while using 3D DNA FISH, copy numbers and positions of genes inside the nucleus can be interrogated without interfering with 3D nuclear architecture. Our method to combine 3D DNA FISH with smFISH and IF enables a unique quantitative handle on the central dogma of molecular biology.

An intramolecular disulphide bond in human 4E-T affects its binding to eIF4E1a protein.

The cap at the 5'terminus of mRNA is a key determinant of gene expression in eukaryotic cells, which among others is required for cap dependent translation and protects mRNA from degradation. These properties of cap are mediated by several proteins. One of them is 4E-Transporter (4E-T), which plays an important role in translational repression, mRNA decay and P-bodies formation. 4E-T is also one of several proteins that interact with eukaryotic initiation factor 4E (eIF4E), a cap binding protein which is a key component of the translation initiation machinery. The molecular mechanisms underlying the interactions of these two proteins are crucial for mRNA processing. Studying the interactions between human eIF4E1a and the N-terminal fragment of 4E-T that possesses unstructured 4E-binding motifs under non-reducing conditions, we observed that 4E-T preferentially forms an intramolecular disulphide bond. This "disulphide loop" reduces affinity of 4E-T for eIF4E1a by about 300-fold. Considering that only human 4E-T possesses two cysteines located between the 4E binding motifs, we proposed that the disulphide bond may act as a switch to regulate interactions between the two proteins.

The presence of an intron relieves gene repression caused by promoter-proximal four-bp specific sequences in yeast

Introns can enhance gene expression in eukaryotic cells in a process called intron-mediated enhancement (IME). The levels of enhancement are affected not only by the intron sequence but also by coding sequences (CDSs). However, the parts of CDSs responsible for mediating IME have not yet been identified. In this study, we identified an IME-mediating sequence by analyzing three pairs of IME-sensitive and -insensitive CDSs in Saccharomyces cerevisiae. Expression of the CDSs yCLuc, yRoGLU1, and KmBGA1 was enhanced by the presence of an intron (i.e., they were IME sensitive), but the expression of each corresponding codon-changed CDS, which encoded the identical amino acid sequence, was not enhanced (i.e., they were IME insensitive). Interestingly, the IME-insensitive CDSs showed higher expression levels that were like intron-enhanced expression of IME-sensitive CDSs, suggesting that expression of IME-sensitive CDSs was repressed. A four-nucleotide sequence (TCTT) located in the promoter-proximal position of either the untranslated or coding region was found to be responsible for repression in IME-sensitive CDSs, and repression caused by the TCTT sequence was relieved by the presence of an intron. Further, it was found that the expression of intron-containing yeast-native genes, UBC4 and MPT5, was repressed by TCTT in the CDS but relieved by the introns. These results indicate that TCTT sequences in promoter-proximal positions repress gene expression and that introns play a role in relieving gene repression caused by sequences such as TCTT.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Vorinostat-induced acetylation of RUNX3 reshapes transcriptional profile through long-range enhancer-promoter interactions in natural killer cells

Natural killer (NK) cells are an essential part of the innate immune system that helps control infections and tumors. Recent studies have shown that Vorinostat, a histone deacetylase (HDAC) inhibitor, can cause significant changes in gene expression and signaling pathways in NK cells. Since gene expression in eukaryotic cells is closely linked to the complex three-dimensional (3D) chromatin architecture, an integrative analysis of the transcriptome, histone profiling, chromatin accessibility, and 3D genome organization is needed to gain a more comprehensive understanding of how Vorinostat impacts transcription regulation of NK cells from a chromatin-based perspective. The results demonstrate that Vorinostat treatment reprograms the enhancer landscapes of the human NK-92 NK cell line while overall 3D genome organization remains largely stable. Moreover, we identified that the Vorinostat-induced RUNX3 acetylation is linked to the increased enhancer activity, leading to elevated expression of immune response-related genes via long-range enhancerpromoter chromatin interactions. In summary, these findings have important implications in the development of new therapies for cancer and immune-related diseases by shedding light on the mechanisms underlying Vorinostat's impact on transcriptional regulation in NK cells within the context of 3D enhancer network.

UPF3A is dispensable for nonsense-mediated mRNA decay in mouse pluripotent and somatic cells.

Nonsense-mediated mRNA decay (NMD) is a highly conserved regulatory mechanism of post-transcriptional gene expression in eukaryotic cells. NMD plays essential roles in mRNA quality and quantity control and thus safeguards multiple biological processes including embryonic stem cell differentiation and organogenesis. UPF3A and UPF3B in vertebrate species, originated from a single UPF3 gene in yeast, are key factors in the NMD machinery. Although UPF3B is a well-recognized weak NMD-promoting factor, whether UPF3A functions in promoting or suppressing NMD is under debate. In this study, we generated a Upf3a conditional knockout mouse strain and established multiple lines of embryonic stem cells and somatic cells without UPF3A. Through extensive analysis on the expressions of 33 NMD targets, we found UPF3A neither represses NMD in mouse embryonic stem cells, somatic cells, nor in major organs including the liver, spleen, and thymus. Our study reinforces that UPF3A is dispensable for NMD when UPF3B is present. Furthermore, UPF3A may weakly and selectively promote NMD in certain murine organs.

RNA deadenylation complexes in development and diseases.

RNA deadenylation, the process of shortening of the 3' poly(A) tail of an RNA molecule, is one of the key steps of post-transcriptional regulation of gene expression in eukaryotic cells. PAN2/3 and CCR4-NOT (CNOT) are the two dominant RNA deadenylation complexes, which play central roles in mediating mRNA decay and translation. While degradation is the final fate of virtually all RNAs in their life cycles, selection of RNA targets as well as control of the rate and timing of RNA decay, in coordination with other molecular pathways, including translation, can be modulated in certain contexts. Such regulation influences cell growth, proliferation, and differentiation at the cellular level; and contributes to establish polarity and regulate signaling at the tissue level. Dysregulation of deadenylation processes have also been implicated in human diseases ranging from cardiac diseases and neurodevelopmental disorders to cancers. In this review, we will discuss mechanisms of gene expression control mediated by the RNA deadenylation complexes and highlight relevant evidence supporting the emerging roles of RNA deadenylation and its regulatory proteins during development and in diseases. A systemic understanding of these mechanisms will be a critical foundation for development of effective strategies to therapeutically target them.

Genome Editing and Obesity.

Defined as a condition of body fat excess leading to significant morbidity, obesity is a chronic metabolic illness associated with a significant number of diseases. The incidence of obesity does not solely depend on dietary habits, with energy balance being regulated by the complex interactions between genetic, behavioural and environmental factors. Genome-wide association studies (GWASs) have demonstrated that several genes are linked to obesity, and these findings shed light on a growing number of novel potential therapeutics for weight management, including genome editing. In this regard, the cutting-edge technology known as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), thanks to its ability to edit DNA or modulate gene expression in eukaryotic cells, undoubtedly enables to understand the genetic mechanisms implicated in obesity and could be a promising tool for its treatment. This chapter summarizes the genetics underlying obesity and currently available obesity treatments, further discussing the research progress of genome editing in the knowledge and treatment of body fat excess.

Roles and regulation of histone acetylation in hepatocellular carcinoma.

Hepatocellular Carcinoma (HCC) is the most frequent malignant tumor of the liver, but its prognosis is poor. Histone acetylation is an important epigenetic regulatory mode that modulates chromatin structure and transcriptional status to control gene expression in eukaryotic cells. Generally, histone acetylation and deacetylation processes are controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Dysregulation of histone modification is reported to drive aberrant transcriptional programmes that facilitate liver cancer onset and progression. Emerging studies have demonstrated that several HDAC inhibitors exert tumor-suppressive properties via activation of various cell death molecular pathways in HCC. However, the complexity involved in the epigenetic transcription modifications and non-epigenetic cellular signaling processes limit their potential clinical applications. This review brings an in-depth view of the oncogenic mechanisms reported to be related to aberrant HCC-associated histone acetylation, which might provide new insights into the effective therapeutic strategies to prevent and treat HCC.

Dysregulated RNA polyadenylation contributes to metabolic impairment in non-alcoholic fatty liver disease.

Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyadenylation, two major steps in pre-mRNA processing, are significantly altered in non-alcoholic fatty liver disease (NAFLD). Moreover, we find that Serine and Arginine Rich Splicing Factor 10 (SRSF10) binding is enriched adjacent to consensus polyadenylation motifs and its expression is significantly decreased in NAFLD, suggesting a role mediating pre-mRNA dysregulation in this condition. Consistently, inactivation of SRSF10 in mouse and human hepatocytes in vitro, and in mouse liver in vivo, was found to dysregulate polyadenylation of key metabolic genes such as peroxisome proliferator-activated receptor alpha (PPARA) and exacerbate diet-induced metabolic dysfunction. Collectively our work implicates dysregulated pre-mRNA polyadenylation in obesity-induced liver disease and uncovers a novel role for SRSF10 in this process.

Analysis of RNA by Northern Blotting.

Northern hybridization is used to measure the amount and size of RNAs transcribed from eukaryotic genes and to estimate their abundance. No other method is capable of obtaining these pieces of information simultaneously from a large number of RNA preparations; northern analysis is therefore fundamental to studies of gene expression in eukaryotic cells. To prepare a northern blot for hybridization, RNA must be separated according to size through a denaturing agarose or polyacrylamide gel and transferred to a solid support in a way that preserves its topological distribution within the gel. These important steps in northern analysis are discussed here.

Sequence, structural and functional conservation among the human and fission yeast ELL and EAF transcription elongation factors.

Transcription elongation is a dynamic and tightly regulated step of gene expression in eukaryotic cells. Eleven nineteen Lysine rich Leukemia (ELL) and ELL Associated Factors (EAF) family of conserved proteins are required for efficient RNA polymerase II-mediated transcription elongation. Orthologs of these proteins have been identified in different organisms, including fission yeast and humans. In the present study, we have examined the sequence, structural and functional conservation between the fission yeast and human ELL and EAF orthologs. Our computational analysis revealed that these proteins share some sequence characteristics, and were predominantly disordered in both organisms. Our functional complementation assays revealed that both human ELL and EAF proteins could complement the lack of ell1+ or eaf1+ in Schizosaccharomyces pombe respectively. Furthermore, our domain mapping experiments demonstrated that both the amino and carboxyl terminal domains of human EAF proteins could functionally complement the S. pombe eaf1 deletion phenotypes. However, only the carboxyl-terminus domain of human ELL was able to partially rescue the phenotypes associated with lack of ell1+ in S. pombe. Collectively, our work adds ELL-EAF to the increasing list of human-yeast complementation gene pairs, wherein the simpler fission yeast can be used to further enhance our understanding of the role of these proteins in transcription elongation and human disease.

Phosphorylation within Intrinsic Disordered Region Discriminates Histone Variant macroH2A1 Splicing Isoforms-macroH2A1.1 and macroH2A1.2.

Simple SummaryMacroH2A1, a histone H2A variant, is present as two alternative splicing isoforms, macroH2A1.1 and macroH2A1.2, which are finely regulated through several mechanisms, including post-translational modifications (PTM). In this article, the authors provide the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting through mass spec analysis. They report a different phosphorylation level in their intrinsically disordered linker region, which can be responsible for their different biological role, as computational analysis shows.Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

Functional Role of circRNAs in the Regulation of Fetal Development, Muscle Development, and Lactation in Livestock.

circRNAs are a class of endogenous noncoding RNA molecules with closed loop structures. They are mainly responsible for regulating gene expression in eukaryotic cells. With the emergence of high-throughput RNA sequencing (RNA-Seq) and new types of bioinformatics tools, thousands of circRNAs have been discovered, making circRNA one of the research hotspots. Recent studies have shown that circRNAs play an important regulatory role in the growth, reproduction, and formation of livestock products. They can not only regulate mammalian fetal growth and development but also have important regulatory effects on livestock muscle development and lactation. In this review, we briefly introduce the putative biogenic pathways and regulatory functions of circRNA and highlight our understanding of circRNA and its latest advances in fetal development, muscle development, and lactation biogenesis as well as expression in livestock. This review will provide a theoretical basis for the research and development of related industries.

RECOGNICER: A coarse-graining approach for identifying broad domains from ChIP-seq data.

Histone modifications are major factors that define chromatin states and have functions in regulating gene expression in eukaryotic cells. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) technique has been widely used for profiling the genome-wide distribution of chromatin-associating protein factors. Some histone modifications, such as H3K27me3 and H3K9me3, usually mark broad domains in the genome ranging from kilobases (kb) to megabases (Mb) long, resulting in diffuse patterns in the ChIP-seq data that are challenging for signal separation. While most existing ChIP-seq peak-calling algorithms are based on local statistical models without account of multi-scale features, a principled method to identify scale-free board domains has been lacking. Here we present RECOGNICER (Recursive coarse-graining identification for ChIP-seq enriched regions), a computational method for identifying ChIP-seq enriched domains on a large range of scales. The algorithm is based on a coarse-graining approach, which uses recursive block transformations to determine spatial clustering of local enriched elements across multiple length scales. We apply RECOGNICER to call H3K27me3 domains from ChIP-seq data, and validate the results based on H3K27me3's association with repressive gene expression. We show that RECOGNICER outperforms existing ChIP-seq broad domain calling tools in identifying more whole domains than separated pieces. RECOGNICER can be a useful bioinformatics tool for next-generation sequencing data analysis in epigenomics research.

Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR).

The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.

Inferring the transcriptional regulatory mechanism of signal-dependent gene expression via an integrative computational approach.

Eukaryotic transcription factors (TFs) coordinate different upstream signals to regulate the expression of their target genes. To unveil this regulatory network in B-cell receptor signaling, we developed a computational pipeline to systematically analyze the extracellular signal-regulated kinase (ERK)- and IκB kinase (IKK)-dependent transcriptome responses. We combined a bilinear regression method and kinetic modeling to identify the signal-to-TF and TF-to-gene dynamics, respectively. We input a set of time-course experimental data for B cells and concentrated on transcriptional activators. The results show that the combination of TFs differentially controlled by ERK and IKK could contribute divergent expression dynamics in orchestrating the B-cell response. Our findings provide insights into the regulatory mechanisms underlying signal-dependent gene expression in eukaryotic cells.

Analytical distributions for detailed models of stochastic gene expression in eukaryotic cells

The stochasticity of gene expression presents significant challenges to the modeling of genetic networks. A two-state model describing promoter switching, transcription, and messenger RNA (mRNA) decay is the standard model of stochastic mRNA dynamics in eukaryotic cells. Here, we extend this model to include mRNA maturation, cell division, gene replication, dosage compensation, and growth-dependent transcription. We derive expressions for the time-dependent distributions of nascent mRNA and mature mRNA numbers, provided two assumptions hold: 1) nascent mRNA dynamics are much faster than those of mature mRNA; and 2) gene-inactivation events occur far more frequently than gene-activation events. We confirm that thousands of eukaryotic genes satisfy these assumptions by using data from yeast, mouse, and human cells. We use the expressions to perform a sensitivity analysis of the coefficient of variation of mRNA fluctuations averaged over the cell cycle, for a large number of genes in mouse embryonic stem cells, identifying degradation and gene-activation rates as the most sensitive parameters. Furthermore, it is shown that, despite the model's complexity, the time-dependent distributions predicted by our model are generally well approximated by the negative binomial distribution. Finally, we extend our model to include translation, protein decay, and auto-regulatory feedback, and derive expressions for the approximate time-dependent protein-number distributions, assuming slow protein decay. Our expressions enable us to study how complex biological processes contribute to the fluctuations of gene products in eukaryotic cells, as well as allowing a detailed quantitative comparison with experimental data via maximum-likelihood methods.