Beyond gene sequences: Pan-transcriptome links transcriptional complexity to functional diversity

Transcription initiation is a complex, energy consuming process that requires the massive and coordinated activity of many proteins often organized in big multi-subunit complexes. The General Transcription Factors (GTFs) and the TATA-binding protein (TBP) orchestrate the formation of the so-called pre-initiation complex (PIC), a key step of transcription initiation required for RNA Polymerase II (RNAPII) association to promoters. Although the PIC is made by the GTFs, many other complexes generally referred to as transcriptional co-activators participate in the formation of the PIC. Transcriptional co-activator complexes include chromatin remodelers, Histone AcetylTransferases (HATs) and the Mediator complex. In yeast, it is has been reported that two main HAT complexes, NuA4 and SAGA, and the Mediator complex have a global role in transcription. However, though, it is well-known that histone acetylation is associated to high transcription and that Mediator plays a central function in PIC assembly, the deployment of NuA4, SAGA and Mediator at yeast genes is still elusive. Here, we investigate the contribution of NuA4, SAGA and Mediator in RNAPII association at all yeast genes through rapid nuclear depletion of key complex subunits. We reveal that Gcn5, the HAT of the SAGA complex, is modestly but equally involved in transcription of all yeast genes, while Esa1, the HAT of the NuA4 complex, is most strongly required for transcription of certain groups of genes. Curiously, we show that nuclear depletion of Med17, an essential subunit of the Mediator complex, strongly affects transcription of a small subset of genes characterized by a well-conserved TATA box, highlighting a previously underappreciated connection between Mediator complex and TBP. Our analysis reveals that three combinations of co-activator deployment are used to generate high transcription levels and that transcription of two groups of so called “house-keeping” or “growth-promoting genes” is particularly dependent on Tra1, a shared component of NuA4 and SAGA. Our work on Ribosomal Protein (RP) gene and Ribosome Biogenesis (RiBi) gene transcription discloses the importance of the transcription factors Ifh1 and Sfp1 in recruiting Tra1 at these promoters. 10 Furthermore, a deep investigation of transcription factors binding at RP gene promoters highlights the existence of a hierarchy, with Rap1 being required for the association of the other TFs. Our study also reveals that RP gene promoters, as well as the promoters of many other highly transcribed genes, are characterized by the presence of one or more unusually unstable MNase-sensitive nucleosomes referred to as fragile nucleosomes. Furthermore, our work discovers the main role of Rap1 and of other pioneer transcription factors in establishing nucleosome fragility and promoter architecture. Finally, we investigate the biophysical properties of fragile nucleosomes and provide evidence of their nucleosomal nature. Altogether the work presented in this thesis elucidates important features of yeast promoters and provides new insights into the mechanisms that drive eukaryotic transcription.

Mating type in the yeast Saccharomyces cerevisiae can be one of three types: a, α, and aα. These mating types reflect information present at MAT,which is normally MAT a or MATα in haploid cells, and MAT a /MATα in diploid cells. MATα and MAT a each have two open reading frames, but functions have only been identified for three gene products, Mata1p, Matα1p, and Matα2p. The MAT gene products are key regulators of the different stages of the yeast life cycle (reviewed in refs. 26,28,30). The Mata1p/Matα2p complex in diploid cells represses transcription of haploid-specific genes, including the repressor of meiosis, RME1,and HO. In MATα haploid cells, Matα1p complexes with Mcm1p to activate α-specific genes. Matα2p complexes with Mcm1p (as well as Tup1p and Ssn6p) to repress a-specific genes. MAT controls mating: a cells mate with α cells but not with a cells, and vice versa, and aα cells do not mate with any of the three cell types. The mating behavior of a and α cells reflects their expression of the haploid-specific mating pheromones, a -and α-factor and the cognate transmembrane receptors. MAT also controls meiosis and sporulation; MAT a /MATα diploids can carry out meiosis whereas diploids expressing only one of the two alleles do not. The ability of aα diploids to enter meiosis depends on repression of the regulatory gene RME11,which is turned off by the action of the Mata1p-Matα2p repressor complex. This leads to the expression of many meiosis-specific genes. Although natural aa or αα diploids are rare, they can be easily produced by using a regulated source of HO endonuclease (29) or by deleting one MAT allele (i.e. αΔ or aΔ) (44).

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures the rapid degradation of mRNAs containing premature translation termination codons in all studied eukaryotes. Originally, NMD was thought of as a quality control pathway that targets non-functional mRNAs arising from mutations and splicing errors. More recently, NMD has been shown to also regulate normal gene expression and NMD thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Despite the progress in the understanding of the role and mechanism of this pathway, the physiological impact of NMD on humans is not yet fully uncovered. To explore the functions of NMD in humans, I combined RNAi against the essential NMD factor UPF1 with genome-wide microarray analysis. My research indicate that NMD affects the expression of a large number of genes implicated in a wide diversity of functions although a majority seems to be affected indirectly and – consequently – do not represent legitimate NMD targets. The validation of five bona fide NMD transcripts allowed me to develop an assay to quantitate differences in NMD efficiency. Using three different strains of HeLa cells as a simple model, I have systematically analysed the molecular mechanism underlying quantitative differences in NMD efficiency. The results of this analysis show that the quantitative differences in NMD efficiency represent a stable characteristic of the investigated strains. Low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1 in one of the analysed HeLa strains. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in the RNPS1 deficient cell line. I conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that the cell type specific co-factor availability represents a novel principle that quantitatively controls NMD. I also tested the hypothesis of NMD as a genetic modifier in the phenotypic expression of disease. A specific -thalassemia – common in Mediterranean Asia – was assayed as a model. My results do not support a role of NMD for the variable severity of this specific mutation leading to anemia.

Extensive intragenic divergences amongst ancient WRKY transcription factor gene family is largely associated with their functional diversity in plants

OVERVIEW Viral immediate-early proteins are the first viral proteins to be synthesized following infection (or upon reactivation from latency) and are required for the transcription, or the enhancement of transcription, of many viral genes. More than 20 viral immediate-early proteins have been identified. Until recently, it has been generally assumed that these viral proteins function in a manner fundamentally different from that of typical cellular transcriptional activators. In this review, we summarize current evidence indicating that many viral transcriptional activators function through mechanisms that are analogous to those of cellular transcriptional activators: The viral proteins directly participate in the transcription complex to stimulate transcription. Variations on the theme of the typical sequence-specific cellular activators can account for the idiosyncratic features of viral activators, which have been difficult to reconcile with notions of cellular activators. Such characteristics of some viral activators include the lack of specific DNA-binding activity, promiscuous activation, and requirements for RNA elements. INTRODUCTION Typical Cellular Activators Transcriptional initiation by RNA polymerase II is usually regulated by cellular factors that bind to the DNA located upstream of the gene, termed a promoter. Promoters consist of a variety of DNA sequence elements, or regions of about 8–20 bp that conform to one of a number of consensus motifs, which are specifically recognized and bound by DNA-binding proteins. On the basis of the functional characteristics of the proteins that bind to these sequence elements, the promoter can be divided into two distinct regions. The first region, located in the vicinity...

Functional sites define the diversity of protein functions and are the central object of research of the structural and functional organization of proteins. The mechanisms underlying protein functional sites emergence and their variability during evolution are distinguished by duplication, shuffling, insertion and deletion of the exons in genes. The study of the correlation between a site structure and exon structure serves as the basis for the in-depth understanding of sites organization. In this regard, the development of programming resources that allow the realization of the mutual projection of exon structure of genes and primary and tertiary structures of encoded proteins is still the actual problem. Previously, we developed the SitEx system that provides information about protein and gene sequences with mapped exon borders and protein functional sites amino acid positions. The database included information on proteins with known 3D structure. However, data with respect to orthologs was not available. Therefore, we added the projection of sites positions to the exon structures of orthologs in SitEx 2.0. We implemented a search through database using site conservation variability and site discontinuity through exon structure. Inclusion of the information on orthologs allowed to expand the possibilities of SitEx usage for solving problems regarding the analysis of the structural and functional organization of proteins. Database URL: http://www-bionet.sscc.ru/sitex/ .

The huge diversity of fungi may reflect both the heterogeneity of the niches they occupy and the diverse stresses they must cope with. In order to investigate the genetic and functional diversity in the ericoid mycorrhizal fungus Oidiodendron maius subjected to heavy metal stress, we isolated O. maius strains from a serpentine site naturally enriched by heavy metals. Despite the high Cr and Ni soil concentrations, a high level of diversity was found in the serpentine fungal community. The growth of these isolates in the presence of different metal contaminants identified some tolerant strains, suggesting a site-specific adaptation. To investigate within-species gene divergence in stressful environments, we then compared the sequence polymorphism of a neutral (internal transcribed spacer) and a functional (Cu,ZnSOD) gene in O. maius isolates derived from the serpentine site, from a site heavily polluted with industrial wastes and from unpolluted sites. For all isolates tested, the polymorphism was higher in the nucleotide sequence of the functional gene. However, when compared with isolates from the serpentine area, isolates from industrially polluted sites showed a significantly higher polymorphism in the Cu,ZnSOD promoter region, suggesting that environmental stress may influence the rate of mutations in specific regions of the Sod1 locus.

ObjectiveThe coiled coil is a superhelical structural protein motif that has been thoroughly investigated in recent years and coiled-coil domain-containing proteins have exhibited a large diversity of function in biological systems (e.g., gene regulation, cell division, membrane fusion, drug extrusion). The aim of this study was to investigate the critical role of coiled-coil domain-containing protein 34 (CCDC34) in bladder carcinogenesis, which has never been reported to date.MethodsImmunohistochemical staining and western blot were used to detect CCDC34 expression in bladder cancers specimens and cell lines. Lentivirus-mediated small interfering RNA (siRNA) against CCDC34 was designed and the silencing effect was examined by quantitative RT-PCR in bladder cancer cell lines T24 and 5637. The biological functions of CCDC34 knockdown on bladder cancer cells were investigated by examining cell proliferation using a high content screening (HCS) assay, BrdU incorporation assay and colony formation assay, as well as cell migration by in vitro wound healing assay. Cell cycle distribution and apoptosis were detected by flow cytometric analysis. The expressions of c-Raf and c-Jun as well as the phosphorylation of MEK, ERK1/2, JNK, P38 and AKT were also measured using Western blot. We further investigated the effect of therapeutic siRNA targeting CCDC34 on T24 xenograft tumor growth in nude mice.ResultsCCDC34 level was significantly increased in human bladder cancer tissues and cell lines. CCDC34 knockdown significantly suppressed bladder cancer cells proliferation and migration, and induced cell cycle arrest at G2/M phase and increased apoptosis in vitro. In addition, intratumor delivery of therapeutic siRNA targeting CCDC34 elicited delayed tumor growth of bladder cancer xenograft in nude mice. Moreover, CCDC34 knockdown decreased the phosphorylation of MEK, ERK1/2, JNK, p38 and Akt, and the expressions of c-Raf and c-Jun, indicating MAPK and AKT pathways (ERK/MAPK, p38/MAPK, JNK/MAPK and PI3K/Akt) might be involved in CCDC34 modulation of bladder cancer cell proliferation and migration.ConclusionsOur findings revealed for the first time a potential oncogenic role for CCDC34 in bladder carcinoma pathogenesis and it may serve as a biomarker or even a therapeutic target for bladder cancer.

Multiple Transcription Initiation Sites, Alternative Splicing, and Differential Polyadenylation Contribute to the Complexity of Human Neurofibromatosis 2 Transcripts

WRKY is one of the largest transcription factor families in plants and plays an important role in plant growth and development as well as in abiotic and biological stresses. However, there is little information about the WRKY family in Melastoma dodecandrum. In this study, 126 WRKY members were identified in M. dodecandrum. According to phylogenetic analysis, they were divided into three major groups, and group II was further divided into five subgroups. MedWRKY genes were unevenly distributed on 12 chromosomes. Additionally, the gene structure and sequence composition were similar within the same group and differed between groups, suggesting their functional diversity. The promoter sequence analysis identified a number of cis-acting elements related to plant growth and development, stress response, and secondary metabolite synthesis in the WRKY gene family. The collinearity analysis showed that gene replication events were the main driving force of MedWRKY gene evolution. The transcriptome data and RT-qPCR analysis suggested that MedWRKY genes had higher expression in the roots and ripe fruit of M. dodecandrum. In short, this paper lays a foundation for further study of the functions and molecular mechanism of M. dodecandrum WRKY gene family.

Developments in molecular techniques have led to rapid and reliable tools to monitor microbial community structures and dynamics under in situ conditions. However, even though various functional genes are localized on mobile genetic elements such that metabolic potential/activity is not necessarily reflected by the community structure, there has been a lack of emphasis on monitoring functional diversity. A more detailed picture of the catabolic gene structure and sequence diversity in environmental samples will significantly increase our knowledge of the functional potential of microbial communities. We adapted suitable techniques to follow functional gene diversity and applied those to target catechol 2,3-dioxygenases as key genes in aromatic hydrocarbon degradation. Catabolic gene diversity in differentially BTEX contaminated environments was assessed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs. The PCR-SSCP technique could be shown to be a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the biodegradation functioning of microbial communities. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish pre-selection or enrichment bias and to assess the function of predominant gene polymorphs. To rapidly determine phylogenetic diversity of functional genes from strain collections or environmental DNA amplifications, a restriction enzyme, theoretically producing characteristic profiles, the similarities of which reassembled the main divergent branches of C23O gene phylogeny, was used to perform an amplified functional DNA restriction analysis (AFDRA) on C23O genes of reference strains and isolates. Sequences of PCR fragments from isolates were in close agreement with the phylogenetic correlations predicted with the AFDRA approach. AFDRA thus provided a quick assessment of C23O diversity in a strain collection and insights of its gene phylogeny affiliation. AFDRA was also used to determine the predominant polymorphism of the C23O gene present in environmental DNA extracts and in combination with a most-probable-number-PCR approach, its abundance. This approach may be useful to differentiate functional genes also for many other gene families. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralising phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralise benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. Complete C23O encoding open reading frames were cloned, sequenced and overexpressed by using conserved regions in operon neighbouring genes. Such strategy also allows the direct access to complete genes from environmental DNA. A single amino acid substitution at position 218 had severe influence on enzyme kinetics, and the Tyr218 variant differed from the His218 variant by lower turnover number but higher affinity. The information in this work underlines the importance to analyze catabolic gene diversity at different scales, from global views of diversity and fitness of genes in the ecosystem, to detailed understanding of sequence variations effects on catalytic activities.

Nested-primer polymerase chain reaction (PCR) has been applied to the molecular cloning of 4.6-kb half-genome fragments of human immunodeficiency virus type 1 (HIV-1) taken directly from the peripheral blood mononuclear cells (PBMC) of an individual with neurological symptoms of HIV-1 infection. In a similar manner, gp120-coding portions of the envelope gene were cloned after PBMC from the same blood sample were cocultivated with uninfected PBMC for 28 days. The complete 1.6-kb nucleotide sequence of the gp120 gene was determined from each of 35 clones examined. Two of 13 (15%) PBMC-derived gp120 genes and 3 of 22 (14%) coculture-derived gp120 genes were defective as a result of frameshifts and an in-frame stop codon(s). Mean diversity between individual gp120-coding sequences in PBMC was fivefold greater (3.24%) than after coculture (0.65%). A predominant sequence of "strain" was found after coculture that was distinct from the diverse viral genotypes detected in vivo and therefore was selectively amplified during in vitro propagation. Multiple distinct third variable (V3) regions encoding the principal neutralizing domain of the envelope protein were detected in PBMC-derived genes, suggesting the presence of immunologic diversity of HIV env genes in vivo not reflected in the cocultured virus sample. The large size of the HIV fragments generated in this study will permit analysis of the diversity of immunologic reactivity, gene function, and pathogenicity of HIV genomes present within infected individuals, including the functional significance of the loss of diversity that occurs upon coculture.

Soil pH determines arsenic-related functional gene and bacterial diversity in natural forests on the Taibai Mountain

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.