Eukaryotic Gene Transcription Research Articles

Methyl-dependent auto-regulation of the DNA N6-adenine methyltransferase AMT1 in the unicellular eukaryote Tetrahymena thermophila.

DNA N6-methyladenine (6mA) is a potential epigenetic mark involved in gene transcription in eukaryotes, yet the regulatory mechanism governing its methyltransferase (MTase) activity remains obscure. Here, we exploited the 6mA MTase AMT1 to elucidate its auto-regulation in the unicellular eukaryote Tetrahymena thermophila. The detailed endogenous localization of AMT1 in vegetative and sexual stages revealed a correlation between the 6mA reestablishment in the new MAC and the occurrence of zygotically expressed AMT1. Catalytically inactive AMT1 reduced 6mA level on the AMT1 gene and its expression, suggesting that AMT1 modulated its own transcription via 6mA. Furthermore, AMT1-dependent 6mA regulated the transcription of its target genes, thereby affecting cell fitness. Our findings unveil a positive feedback loop of transcriptional activation on the AMT1 gene and highlight the crucial role of AMT1-dependent 6mA in gene transcription.

The carboxy-terminal domain of RNA polymerase II large subunit: simple repeats are not simple.

Transcription, as a crucial step in the transmission of genetic information, is completed by DNA-dependent RNA polymerase. In eukaryotes, the transcription of protein-coding genes is completed by RNA polymerase II (Pol II). A distinctive feature of Pol II is the carboxy-terminal domain (CTD) of its largest subunit, RPB1, which is composed of a series of heptapeptide repeats that play a vital role in transcription. Here, we provide a comprehensive review and summary of the sequence characteristics and evolutionary trajectory of the eukaryotic RPB1 CTD, as well as its regulatory function within the transcription cycle. We particularly focus on the mechanisms by which the CTD participates in the regulation of transcription and co-transcriptional processing through post-translational modifications. This deepens our understanding of the intricate regulatory mechanisms governing gene transcription in eukaryotes and lays the groundwork for further investigation into the role of the RPB1 CTD.

Structural Features of DNA in tRNA Genes and Their Upstream Sequences.

RNA polymerase III (Pol III) transcribes tRNA genes using type II promoters. The internal control regions contain a Box A and a Box B, which are recognized by TFIIIC. The 5'-flanking regions of tRNA genes clearly play a role in the regulation of transcription, but consensus sequences in it have been found only in some plants and S. pombe; although, the TATA binding protein (TBP) is a component of the TFIIIB complex in all eukaryotes. Archaea utilize an ortholog of the TBP. The goal of this work is the detection of the positions of intragenic and extragenic promoters of Pol III, which regulate the transcription of tRNA genes in eukaryotes and archaea. For this purpose, we analyzed textual and some structural, mechanical, and physicochemical properties of the DNA in the 5'-flanking regions of tRNA genes, as well as in 30 bp at the beginning of genes and 60 bp at the end of genes in organisms possessing the TBP or its analog (eukaryotes, archaea) and organisms not possessing the TBP (bacteria). Representative tRNA gene sets of 11 organisms were taken from the GtRNAdb database. We found that the consensuses of A- and B-boxes in organisms from all three domains are identical; although, they differ in the conservativism of some positions. Their location relative to the ends of tRNA genes is also identical. In contrast, the structural and mechanical properties of DNA in the 5'-flanking regions of tRNA genes differ not only between organisms from different domains, but also between organisms from the same domain. Well-expressed TBP binding positions are found only in S. pombe and A. thaliana. We discuss possible reasons for the variability of the 5'-flanking regions of tRNA genes.

Functional Characterization of Accessible Chromatin in Common Wheat.

Eukaryotic gene transcription is fine-tuned by precise spatiotemporal interactions between cis-regulatory elements (CREs) and trans-acting factors. However, how CREs individually or coordinated with epigenetic marks function in regulating homoeolog bias expression is still largely unknown in wheat. In this study, through comprehensively characterizing open chromatin coupled with DNA methylation in the seedling and spikelet of common wheat, we observed that differential chromatin openness occurred between the seedling and spikelet, which plays important roles in tissue development through regulating the expression of related genes or through the transcription factor (TF)-centered regulatory network. Moreover, we found that CHH methylation may act as a key determinant affecting the differential binding of TFs, thereby resulting in differential expression of target genes. In addition, we found that sequence variations in MNase hypersensitive sites (MHSs) result in the differential expression of key genes responsible for important agronomic traits. Thus, our study provides new insights into the roles of CREs in regulating tissue or homoeolog bias expression, and controlling important agronomic traits in common wheat. It also provides potential CREs for genetic and epigenetic manipulation toward improving desirable traits for wheat molecule breeding.

Light regulates widespread plant alternative polyadenylation through the chloroplast

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

The jet-like chromatin structure defines active secondary metabolism in fungi.

Eukaryotic genomes are spatially organized within the nucleus in a nonrandom manner. However, fungal genome arrangement and its function in development and adaptation remain largely unexplored. Here, we show that the high-order chromosome structure of Fusarium graminearum is sculpted by both H3K27me3 modification and ancient genome rearrangements. Active secondary metabolic gene clusters form a structure resembling chromatin jets. We demonstrate that these jet-like domains, which can propagate symmetrically for 54 kb, are prevalent in the genome and correlate with active gene transcription and histone acetylation. Deletion of GCN5, which encodes a core and functionally conserved histone acetyltransferase, blocks the formation of the domains. Insertion of an exogenous gene within the jet-like domain significantly augments its transcription. These findings uncover an interesting link between alterations in chromatin structure and the activation of fungal secondary metabolism, which could be a general mechanism for fungi to rapidly respond to environmental cues, and highlight the utility of leveraging three-dimensional genome organization in improving gene transcription in eukaryotes.

Chromatin Remodeling Complex PBAF Activates and Represses Inflammatory Genes.

The PBAF chromatin remodeling complex regulates chromatin state and gene transcription in higher eukaryotes. In this work, we studied the role of PBAF in the regulation of NF-κB-and JAK/STAT-dependent activation of inflammatory genes. We performed knockdown of specific module subunit BAF200, which resulted in destruction of the entire PBAF specific module and changed the level of the genes transcription of both pathways. PBAF can be both an activator and a repressor of inflammatory genes. Thus, PBAF is an important regulator of inflammatory gene expression.

CHROMATIN REMODELING COMPLEX PBAF ACTIVATES AND REPRESSES INFLAMMATORY GENES

The PBAF chromatin remodeling complex regulates chromatin state and gene transcription in higher eukaryotes. In this work, we studied the role of PBAF in the regulation of NF–κB-and JAK/STAT-dependent activation of inflammatory genes. We performed knockdown of specific module subunit BAF200 resulted in destruction of the entire PBAF specific module and changed the level of the genes transcription of both pathways. PBAF can be both an activator and a repressor of inflammatory genes. Thus, PBAF is one of the important regulators of inflammatory gene expression.

Histone acetyltransferase Gcn5-mediated histone H3 acetylation facilitates cryptococcal morphogenesis and sexual reproduction.

Eukaryotic gene transcription is typically regulated by a series of histone modifications, which play a crucial role in adapting to complex environmental stresses. In the ubiquitous human fungal pathogen Cryptococcus neoformans, sexual life cycle is a continuous intracellular differentiation process that strictly occurs in response to mating stimulation. Despite the comprehensive identification of the regulatory factors and genetic pathways involved in its sexual cycle, understanding of the epigenetic modifications involved in this process remains quite limited. In this research, we found that histone acetyltransferase Gcn5-mediated histone H3 acetylation plays a crucial role in completing the cryptococcal sexual cycle, including yeast-hyphae morphogenesis and the subsequent sexual reproduction. Furthermore, we demonstrated that Gcn5 participates in this process primarily through regulating the key morphogenesis regulator Znf2 and its targets. This study thus provided a comprehensive understanding of how histone acetylation modification impacts sexual life cycle in a high-risk human pathogenic fungus.

Dual roles of the Arabidopsis PEAT complex in histone H2A deubiquitination and H4K5 acetylation

Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes. However, the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified. In this study, we found that the previously identified PWWP–EPCR–ARID–TRB (PEAT) complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2 (HAM1/2) to form a larger version of PEAT complex in Arabidopsis thaliana. UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo. HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase complex, and are responsible for histone H4K5 acetylation. Within the PEAT complex, the PWWP components (PWWP1, PWWP2, and PWWP3) directly interact with UBP5 and are necessary for UBP5-mediated H2A deubiquitination, while the EPCR components (EPCR1 and EPCR2) directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5 acetylation. Collectively, our study not only identifies dual roles of the PEAT complex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants.

Driving forces behind phase separation of the carboxy-terminal domain of RNA polymerase II

Eukaryotic gene regulation and pre-mRNA transcription depend on the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II. Due to its highly repetitive, intrinsically disordered sequence, the CTD enables clustering and phase separation of Pol II. The molecular interactions that drive CTD phase separation and Pol II clustering are unclear. Here, we show that multivalent interactions involving tyrosine impart temperature- and concentration-dependent self-coacervation of the CTD. NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations demonstrate the presence of diverse tyrosine-engaging interactions, including tyrosine-proline contacts, in condensed states of human CTD and other low-complexity proteins. We further show that the network of multivalent interactions involving tyrosine is responsible for the co-recruitment of the human Mediator complex and CTD during phase separation. Our work advances the understanding of the driving forces of CTD phase separation and thus provides the basis to better understand CTD-mediated Pol II clustering in eukaryotic gene transcription.

Functional Verification of the Four Splice Variants from Ajania purpurea NST1 in Transgenic Tobacco

Ajania purpurea is a small semi-shrub in the Asteraceae family. Its corolla is purplish red from the middle to the top, and its leaves and flowers are all fragrant. It can be introduced and cultivated as ornamental plants. In order to survive adversity, plants actively regulate the expression of stress response genes and transcripts. Alternative splicing is a common phenomenon and an important regulation mode of eukaryotic gene transcription, which plays an important role in various biological processes. In this study, four splice variants of the NST1 gene were identified from A. purpurea, and the molecular mechanism of NST1 alternative splice variants involved in abiotic stress was explored through bioinformatics, transgenics and paraffin sectionalization. The analysis of amino acid sequences showed that ApNST1.1 had alternative 5′splicing, ApNST1.2 had alternative 3′splicing and ApNST1 had the two splicing types. The main conclusions from studying transgenic tobacco seedlings and adult seedlings under abiotic stress were as follows: ApNST1, ApNST1.1 and ApNST1.3 showed salt tolerance at seedling stage, especially ApNST1.3. At the mature seedling stage, the stem height of ApNST1.1 increased significantly, and ApNST1.1 showed obvious salt tolerance, while ApNST1.2 showed obvious cold resistance. Compared to Super35S::GFP, the xylem of ApNST1 thickened by 94 μm, and the cell wall thickened by 0.215 μm. These results are of great significance to the breeding and application of ApNST1 to select splice variants with more resistance to abiotic stress, and to future study in this area. At the same time, they provide a new direction for A. purpurea breeding, and increase the possibility of garden applications.

Genomic variations combined with epigenetic modifications rewire open chromatin in rice.

Cis-regulatory elements (CREs) fine-tune gene transcription in eukaryotes. CREs with sequence variations play vital roles in driving plant or crop domestication. However, how global sequence and structural variations are responsible for multi-level changes between indica and japonica rice (Oryza sativa) is still not fully elucidated. To address this, we conducted multi-omics studies using MH-seq (MNase hypersensitivity sequencing) in combination with RNA-seq, ChIP-seq and BS-seq between the japonica rice variety Nipponbare (NIP) and indica rice variety 93-11. We found that differential MHSs exhibited some distinct intrinsic genomic sequence features between NIP and 93-11. Notably, through MHS-GWAS integration, we found that key sequence variations may be associated with differences of agronomic traits between NIP and 93-11, which is partly achieved by MHSs harboring CREs. In addition, SV-derived differential MHSs caused by TE insertion, especially by non-common TEs among rice varieties, were associated with genes with distinct functions, indicating that TE driven gene neo- or subfunctionalization is mediated by changes of chromatin openness. This study thus provides insights into how sequence and genomic structural variations control agronomic traits of NIP and 93-11; it also provides genome editing targets for molecular breeding aiming at improving favorable agronomic properties.

Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification.

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires nascent RNA synthesis and translation. Analysis of the coordinated regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprogramming of nascent RNA synthesis and translation. Our nascent P-TRAP (nP-TRAP) method may serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes.

One of the first biological machineries to be created seems to have been the ribosome. Since then, organisms have dedicated great efforts to optimize this apparatus. The ribosomal RNA (rRNA) contained within ribosomes is crucial for protein synthesis and maintenance of cellular function in all known organisms. In eukaryotic cells, rRNA is produced from ribosomal DNA clusters of tandem rRNA genes, whose organization in the nucleolus, maintenance and transcription are strictly regulated to satisfy the substantial demand for rRNA required for ribosome biogenesis. Recent studies have elucidated mechanisms underlying the integrity of ribosomal DNA and regulation of its transcription, including epigenetic mechanisms and a unique recombination and copy-number control system to stably maintain high rRNA gene copy number. In this Review, we disucss how the crucial maintenance of rRNA gene copy number through control of gene amplification and of rRNA production by RNA polymeraseI are orchestrated. We also discuss how liquid-liquid phase separation controls the architecture and function of the nucleolus and the relationship between rRNA production, cell senescence and disease.

Comparative Study of Quantitative TSS Maps among Yeast Strains Demonstrates Key Genetic Sites Required for TSS Selection

Gene transcription is finely regulated to ensure proper cellular functions. Transcription initiation is a critical step of gene regulation as it integrates most regulatory signals, determines the location of transcription start sites (TSS), and establishes the transcript abundance. Recent studies have shown that transcription of most genes in eukaryotes can be initiated from multiple spatially located TSSs. Furthermore, different TSSs were used in various cell types as well as in response to environmental stress. How TSSs are selected during transcription, however, remains largely unknown. We aimed to infer important genetic variations associated with divergence of TSSs to better understand the role of genomic context in TSS selection. Specifically, we used no‐amplification non‐tagging cap analysis of gene expression (nAnT‐iCAGE) sequencing to accurately determine TSSs on a genome‐wide scale for three different populations of budding yeast (Saccharomyces cerevisiae). Comparative studies of these quantitative TSS maps and their genomic sequences showed that transversion substitutions at several positions, such as ‐8, ‐1, and +1 position relative to the TSS, coincided with divergence of the dominant TSS in many promoters. Additionally, a range of transversion substitutions upstream of the TSS at around the ‐150 position showed divergence of the dominant TSS in many promoters as well. However, many divergent TSSs did not have genetic variations nearby, suggesting that transcription factors likely played a major role in TSS selection. This study lays a solid foundation for further functional characterization of these genetic variations, contributing to a better understanding of the genetic mechanisms underlying the regulation of transcription initiation.

A capped Tudor domain within a core subunit of the Sin3L/Rpd3L histone deacetylase complex binds to nucleic acid G-quadruplexes

Chromatin-modifying complexes containing histone deacetylase (HDAC) activities play critical roles in the regulation of gene transcription in eukaryotes. These complexes are thought to lack intrinsic DNA-binding activity, but according to a well-established paradigm, they are recruited via protein–protein interactions by gene-specific transcription factors and posttranslational histone modifications to their sites of action on the genome. The mammalian Sin3L/Rpd3L complex, comprising more than a dozen different polypeptides, is an ancient HDAC complex found in diverse eukaryotes. The subunits of this complex harbor conserved domains and motifs of unknown structure and function. Here, we show that Sds3, a constitutively-associated subunit critical for the proper functioning of the Sin3L/Rpd3L complex, harbors a type of Tudor domain that we designate the capped Tudor domain. Unlike canonical Tudor domains that bind modified histones, the Sds3 capped Tudor domain binds to nucleic acids that can form higher-order structures such as G-quadruplexes and shares similarities with the knotted Tudor domain of the Esa1 histone acetyltransferase that was previously shown to bind single-stranded RNA. Our findings expand the range of macromolecules capable of recruiting the Sin3L/Rpd3L complex and draw attention to potentially new biological roles for this HDAC complex.

The Elongation Regulators and Architectural Proteins as New Participants of Eukaryotic Gene Transcription

The Elongation Regulators and Architectural Proteins as New Participants of Eukaryotic Gene Transcription

Repurposing the CRISPR‐Cas9 System for Targeted Chromatin O ‐linked β‐ N ‐acetylglucosamine Editing

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia have identified O-linked β-N-acetylglucosamine (O-GlcNAc) as an abundant post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, chromatin dynamics, and cofactor activity. Decoding the precise function of all the intracellular O-GlcNAc-regulated elements identified in these studies remains challenging. Technologies to manipulate O-GlcNAcylation at specific DNA loci for functional analysis without pleiotropic consequences were lacking, but the advent of CRISPR-Cas9-based technologies has enabled the precise perturbation of DNA sequences and delivery of proteins to specific cis-acting DNA regulatory elements to elucidate their role in transcription. We reasoned that a CRISPR-Cas9 system could be developed to study the function of O-GlcNAcylation in transcription, which would circumvent the problems inherent in conventional gene manipulation and pharmacological approaches. We developed a programmable CRISPR-Cas9-based system that allows for O-GlcNAc manipulation of transcriptional complex function at specific cis-regulatory elements. The tools consist of nuclease-null Cas9 (dCas9) fused to O-GlcNAc transferase (OGT), which adds the O-GlcNAc modification, or O-GlcNAcase (OGA), which removes the modification. Previously, we demonstrated that O-GlcNAc plays a role in regulating human □-globin gene transcription. Increasing fetal hemoglobin (HbF) via activation of □-globin chain synthesis is widely accepted as the most effective treatment for SCD and certain types of β-thalassemias. O-GlcNAcylation modulates the formation of a GATA-1-FOG-1-NuRD repressor complex that binds the -566 GATA site of the A□-globin promoter when □-globin gene expression is silent. OGT and OGA interact with GATA-1 and CHD4, a component of the NuRD complex. O-GlcNAcylation of CHD4 stimulates the formation of this repressor complex, whereas removing this PTM results in activation of A□-globin gene expression. As proof of principle to demonstrate the utility of our system, we targeted dCas9-OGT or dCas9-OGA fusion proteins to DNA sequences flanking the -566 GATA site of the A□-globin gene promoter. Increased □-globin gene transcription was measured by RT-qPCR when dCas9-OGA was targeted to this area, whereas □-globin gene expression decreased when dCas9-OGT was targeted to the identical sequences. These data support our published work, confirming that O-GlcNAcylation regulates A□-globin gene transcription. RT-qPCR data coupled with Cut and Run assays demonstrate the robust and highly specific nature of our targeting system, which can be employed as a generally applicable tool to study the role of O-GlcNAcylation in transcriptional regulation.