Eukaryotic Transcriptional Regulation Research Articles

NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I

DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.

TRF4, the novel TBP-related protein of Drosophila melanogaster, is concentrated at the endoplasmic reticulum and copurifies with proteins participating in the processes associated with endoplasmic reticulum.

Understanding the functions of TBP-related factors is essential for studying chromatin assembly and transcription regulation in higher eukaryotes. The novel TBP-related protein-coding gene, trf4, was described in Drosophila melanogaster. trf4 is found only in Drosophila and has likely originated in Drosophila common ancestor. TRF4 protein has a distant homology with TBP and TRF2 in the region of TBP-like domain and is evolutionarily conserved among distinct Drosophila species, which indicates its functional significance. TRF4 is widely expressed in D. melanogaster with high levels of its expression being observed in testes. Interestingly enough, TRF4 has become a cytoplasmic protein having lost nuclear localization signal sequence. TRF4 is concentrated at the endoplasmic reticulum (ER) and copurifies with the proteins participating in the ER-associated processes. We suggest that trf4 gene is an example of homolog neofunctionalization by protein subcellular relocalization pathway, where the subcellular relocalization of gene product of duplicated gene leads to the new functions in ER-associated processes.

INuc-ext-PseTNC: an efficient ensemble model for identification of nucleosome positioning by extending the concept of Chou's PseAAC to pseudo-tri-nucleotide composition.

Nucleosome is a central element of eukaryotic chromatin, which composes of histone proteins and DNA molecules. It performs vital roles in many eukaryotic intra-nuclear processes, for instance, chromatin structure and transcriptional regulation formation. Identification of nucleosome positioning via wet lab is difficult; so, the attention is diverted towards the accurate intelligent automated prediction. In this regard, a novel intelligent automated model "iNuc-ext-PseTNC" is developed to identify the nucleosome positioning in genomes accurately. In this predictor, the sequences of DNA are mathematically represented by two different discrete feature extraction techniques, namely pseudo-tri-nucleotide composition (PseTNC) and pseudo-di-nucleotide composition. Several contemporary machine learning algorithms were examined. Further, the predictions of individual classifiers were integrated through an evolutionary genetic algorithm. The success rates of the ensemble model are higher than individual classifiers. After analyzing the prediction results, it is noticed that iNuc-ext-PseTNC model has achieved better performance in combination with PseTNC feature space, which are 94.3%, 93.14%, and 88.60% of accuracies using six-fold cross-validation test for the three benchmark datasets S1, S2, and S3, respectively. The achieved outcomes exposed that the results of iNuc-ext-PseTNC model are prominent compared to the existing methods so far notifiable in the literature. It is ascertained that the proposed model might be more fruitful and a practical tool for rudimentary academia and research.

Transcriptome Analysis of Four Arabidopsis thaliana Mediator Tail Mutants Reveals Overlapping and Unique Functions in Gene Regulation.

The Mediator complex is a central component of transcriptional regulation in Eukaryotes. The complex is structurally divided into four modules known as the head, middle, tail and kinase modules, and in Arabidopsis thaliana, comprises 28-34 subunits. Here, we explore the functions of four Arabidopsis Mediator tail subunits, MED2, MED5a/b, MED16, and MED23, by comparing the impact of mutations in each on the Arabidopsis transcriptome. We find that these subunits affect both unique and overlapping sets of genes, providing insight into the functional and structural relationships between them. The mutants primarily exhibit changes in the expression of genes related to biotic and abiotic stress. We find evidence for a tissue specific role for MED23, as well as in the production of alternative transcripts. Together, our data help disentangle the individual contributions of these MED subunits to global gene expression and suggest new avenues for future research into their functions.

Structural-Functional Organization of the Eukaryotic Cell Nucleus and Transcription Regulation: Introduction to This Special Issue of Biochemistry (Moscow).

This issue of Biochemistry (Moscow) is devoted to the cell nucleus and mechanisms of transcription regulation. Over the years, biochemical processes in the cell nucleus have been studied in isolation, outside the context of their spatial organization. Now it is clear that segregation of functional processes within a compartmentalized cell nucleus is very important for the implementation of basic genetic processes. The functional compartmentalization of the cell nucleus is closely related to the spatial organization of the genome, which in turn plays a key role in the operation of epigenetic mechanisms. In this issue of Biochemistry (Moscow), we present a selection of review articles covering the functional architecture of the eukaryotic cell nucleus, the mechanisms of genome folding, the role of stochastic processes in establishing 3D architecture of the genome, and the impact of genome spatial organization on transcription regulation.

Cryo-electron microscopy structure of a human PRMT5:MEP50 complex.

Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry. The catalytic PRMT5 subunits form a core tetramer and the MEP50 subunits are arranged peripherally in complex with the PRMT5 N-terminal domain. The cryo-EM reconstruction shows good side chain definition and shows a well-resolved peak for a bound dehydrosinefungin inhibitor molecule. These results demonstrate the applicability of cryo-EM in determining structures of human protein complexes of biomedical significance and suggests cryo-EM could be further utilized to understand PRMT5 interactions with other biologically important binding proteins and ligands.

Nuclear microenvironments modulate transcription from low-affinity enhancers.

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

Histone Hypervariants H2A.Z.1 and H2A.Z.2 Play Independent and Context-Specific Roles in Neuronal Activity-Induced Transcription of Arc/Arg3.1 and Other Immediate Early Genes.

The histone variant H2A.Z is an essential and conserved regulator of eukaryotic gene transcription. However, the exact role of this histone in the transcriptional process remains perplexing. In vertebrates, H2A.Z has two hypervariants, H2A.Z.1 and H2A.Z.2, that have almost identical sequences except for three amino acid residues. Due to such similarity, functional specificity of these hypervariants in neurobiological processes, if any, remain largely unknown. In this study with dissociated rat cortical neurons, we asked if H2A.Z hypervariants have distinct functions in regulating basal and activity-induced gene transcription. Hypervariant-specific RNAi and microarray analyses revealed that H2A.Z.1 and H2A.Z.2 regulate basal expression of largely nonoverlapping gene sets, including genes that code for several synaptic proteins. In response to neuronal activity, rapid transcription of our model gene Arc is impaired by depletion of H2A.Z.2, but not H2A.Z.1. This impairment is partially rescued by codepletion of the H2A.Z chaperone, ANP32E. In contrast, under a different context (after 48 h of tetrodotoxin, TTX), rapid transcription of Arc is impaired by depletion of either hypervariant. Such context-dependent roles of H2A.Z hypervariants, as revealed by our multiplexed gene expression assays, are also evident with several other immediate early genes, where regulatory roles of these hypervariants vary from gene to gene under different conditions. Together, our data suggest that H2A.Z hypervariants have context-specific roles that complement each other to mediate activity-induced neuronal gene transcription.

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

BackgroundThe molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized.ResultsTo identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation. We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription.ConclusionsOur results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.

Recent advances in high-throughput approaches to dissect enhancer function.

The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We highlight how these approaches contribute toward a better understanding of enhancer function, eventually leading to the discovery of new types of regulatory sequences, and how the alteration of enhancers can affect transcriptional regulation.

Cell scientist to watch – Lidia Vasilieva

ABSTRACT Lidia received a bachelor's degree from Krasnoyarsk State University in Russia and a master's degree from the Institute of Protein Research in Moscow. She then moved to Finland to work with Leevi Kääriäinen on the replication of RNA viruses for her PhD. Her postdoctoral studies led her to Harvard Medical School with a fellowship from the Leukemia and Lymphoma Society. Under the supervision of Steve Buratowski, Lidia focused on transcriptional regulation in eukaryotes. In 2009, Lidia obtained a Wellcome Trust Career Development Fellowship and started her own research group at the Department of Biochemistry in Oxford. She has been named a Wellcome Trust Senior Research fellow in 2015, and was awarded the British Society for Cell Biology Women in Cell Biology Early Career Medal the following year. Her group investigates the molecular transcriptional and post-transcriptional mechanisms that control RNA levels of protein-coding and non-coding transcripts in response to cellular or developmental programmes.

Single-cell, single-mRNA analysis of Ccnb1 promoter regulation

Promoter activation drives gene transcriptional output. Here we report generating site-specifically integrated single-copy promoter transgenes and measuring their expression to indicate promoter activities at single-mRNA level. mRNA counts, Pol II density and Pol II firing rates of the Ccnb1 promoter transgene resembled those of the native Ccnb1 gene both among asynchronous cells and during the cell cycle. We observed distinct activation states of the Ccnb1 promoter among G1 and G2/M cells, suggesting cell cycle-independent origin of cell-to-cell variation in Ccnb1 promoter activation. Expressing a dominant-negative mutant of NF-YA, a key transcriptional activator of the Ccnb1 promoter, increased its “OFF”/“ON” time ratios but did not alter Pol II firing rates during the “ON” period. Furthermore, comparing H3K4me2 and H3K79me2 levels at the Ccnb1 promoter transgene and the native Ccnb1 gene indicated that the enrichment of these two active histone marks did not predispose higher transcriptional activities. In summary, this experimental system enables bridging transcription imaging with molecular analysis to provide novel insights into eukaryotic transcriptional regulation.

Post‐transcriptional Regulation and the Bacterial Response to Stress

The molecular understanding of how organisms regulate their gene expression began in 1961 with the ground‐breaking work of Jacob and Monod, using bacterial genetics to explore the circuits for regulation of the lac operon and bacteriophage lambda lysogeny. In the intervening 50+ years, it has become clear that the lessons learned there, with variations, provide the basic mechanism for understanding transcriptional regulation in eukaryotes as well as prokaryotes. It has also become clear that regulating the initiation of transcription is only step one, and that cells regulate every possible step for gene expression, in myriad ways. We have found that different mechanisms of post‐transcriptional regulation collaborate to allow bacteria to mount a general stress response. In many bacteria, cells respond to nutrient starvation and/or various types of damage by inducing a response that provides cross‐resistance to a wide range of stresses. In E. coli, this stress response is mediated by expression of an alternative sigma factor, RpoS. Post‐transcriptional regulation increases expression of RpoS, highlighting the ways in which rapid responses to changes in the environment can be mediated post‐transcriptionally. Translation of RpoS requires the action of one or more small non‐coding RNAs (sRNAs), each of which positively regulates translation by opening up an inhibitory hairpin. These sRNAs are examples of a wide‐spread network of RNA regulators, akin to microRNAs in eukaryotes, that modulate mRNA stability and translation and are each induced in response to a different stress signal. The discovery of these sRNAs, when they are expressed, and what genes they regulate has uncovered unexpected regulatory networks in most bacteria. RpoS, once synthesized, binds to the core RNA polymerase to turn on the general stress regulon. However, when cells are growing rapidly (are not stressed), RpoS is rapidly degraded by the ATP‐dependent ClpXP protease; RpoS becomes stable when cells are starved or stressed. This protease, like the eukaryotic proteasome, consists of an ATPase and unfoldase, ClpX, through which substrates must be fed to the proteolytic chamber, ClpP. However, unlike eukaryotes, recognition of substrates in most bacteria does not depend upon ubiquitin tagging. For RpoS, an adaptor protein, RssB, delivers RpoS to the ATPase component of the protease. We have found that a set of anti‐adaptor proteins regulate RpoS stability via titration of the adaptor. Each anti‐adaptor is made in response to a different stress or starvation condition. Thus, as with the sRNAs, multiple signals can converge to induce RpoS via different antiadaptors, each leading to stabilization and thus increased activity of RpoS. The ability of bacteria like E. coli to adapt rapidly to multiple environments continues to provide new insights into regulation many years after the initial insights of Jacob and Monod.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.”

Expression regulation by a methyl-CpG binding domain in an E. coli based, cell-free TX-TL system

Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.

Evolution of DNA Methylation across Insects.

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects.

Interactions of CCAAT/enhancer-binding protein β with transcriptional coregulators

CCAAT/enhancer-binding proteins (C/EBPs) are key regulators of numerous cellular processes, including cell proliferation, differentiation, and tumorigenesis. Their biological activities require interactions with several protein partners and the formation of functional multiprotein complexes involved in DNA repair and cell cycle control. Members of the family (C/EBPα, β, δ, ε, and γ) bind to common DNA sequence motifs as homo- or hetero-dimers and interact with other transcription factors to control transcription of a number of eukaryotic genes. Of particular interest is C/EBPβ, which binds to closed chromatin and acts as a pioneering factor for initiating tissue-specific gene expression at several promoters. This review focuses on intermolecular interactions that underlie C/EBPβ's ability to regulate chromatin accessibility and directs readers to general reviews describing transcription regulation in eukaryotes.

A Class 1 Histone Deacetylase with Potential as an Antifungal Target.

ABSTRACTHistone deacetylases (HDACs) remove acetyl moieties from lysine residues at histone tails and nuclear regulatory proteins and thus significantly impact chromatin remodeling and transcriptional regulation in eukaryotes. In recent years, HDACs of filamentous fungi were found to be decisive regulators of genes involved in pathogenicity and the production of important fungal metabolites such as antibiotics and toxins. Here we present proof that one of these enzymes, the class 1 type HDAC RpdA, is of vital importance for the opportunistic human pathogen Aspergillus fumigatus. Recombinant expression of inactivated RpdA shows that loss of catalytic activity is responsible for the lethal phenotype of Aspergillus RpdA null mutants. Furthermore, we demonstrate that a fungus-specific C-terminal region of only a few acidic amino acids is required for both the nuclear localization and catalytic activity of the enzyme in the model organism Aspergillus nidulans. Since strains with single or multiple deletions of other classical HDACs revealed no or only moderate growth deficiencies, it is highly probable that the significant delay of germination and the growth defects observed in strains growing under the HDAC inhibitor trichostatin A are caused primarily by inhibition of catalytic RpdA activity. Indeed, even at low nanomolar concentrations of the inhibitor, the catalytic activity of purified RpdA is considerably diminished. Considering these results, RpdA with its fungus-specific motif represents a promising target for novel HDAC inhibitors that, in addition to their increasing impact as anticancer drugs, might gain in importance as antifungals against life-threatening invasive infections, apart from or in combination with classical antifungal therapy regimes.

The SAGA complex in the rice pathogen Fusarium fujikuroi: structure and functional characterization

Post-translational modification of histones is a crucial mode of transcriptional regulation in eukaryotes. A well-described acetylation modifier of certain lysine residues is the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex assembled around the histone acetyltransferase Gcn5 in Saccharomyces cerevisiae. We identified and characterized the SAGA complex in the rice pathogen Fusarium fujikuroi, well-known for producing a large variety of secondary metabolites (SMs). By using a co-immunoprecipitation approach, almost all of the S. cerevisiae SAGA complex components have been identified, except for the ubiquitinating DUBm module and the chromodomain containing Chd1. Deletion of GCN5 led to impaired growth, loss of conidiation and alteration of SM biosynthesis. Furthermore, we show that Gcn5 is essential for the acetylation of several histone 3 lysines in F. fujikuroi, that is, H3K4, H3K9, H3K18 and H3K27. A genome-wide microarray analysis revealed differential expression of about 30% of the genome with an enrichment of genes involved in primary and secondary metabolism, transport and histone modification. HPLC-based analysis of known SMs revealed significant alterations in the Δgcn5 mutant. While most SM genes were activated by Gcn5 activity, the biosynthesis of the pigment bikaverin was strongly increased upon GCN5 deletion underlining the diverse roles of the SAGA complex in F. fujikuroi.

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon.

Eukaryotic Transcription Regulation: Getting to the Heart of the Matter: Commentary on Mediator Architecture and RNA Polymerase II Function by Plaschka et al

Eukaryotic Transcription Regulation: Getting to the Heart of the Matter: Commentary on Mediator Architecture and RNA Polymerase II Function by Plaschka et al