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PubMed2006

A role for TFIIIC transcription factor complex in genome organization.

Eukaryotic genome complexity necessitates boundary and insulator elements to partition genomic content into distinct domains. We show that inverted repeat (IR) boundary elements flanking the fission yeast mating-type heterochromatin domain contain B-box sequences, which prevent heterochromatin from spreading into neighboring euchromatic regions by recruiting transcription factor TFIIIC complex without RNA polymerase III (Pol III). Genome-wide analysis reveals TFIIIC with Pol III at all tRNA genes, many of which cluster at pericentromeric heterochromatin domain boundaries. However, a single tRNA(phe) gene with modest TFIIIC enrichment is insufficient to serve as boundary and requires RNAi-associated element to restrain heterochromatin spreading. Remarkably, we found TFIIIC localization without Pol III at many sites located between divergent promoters. These sites appear to act as chromosome-organizing clamps by tethering distant loci to the nuclear periphery, at which TFIIIC is concentrated into several distinct bodies. Our analyses uncover a general genome organization mechanism involving conserved TFIIIC complex.

Chromosomes↗

PubMed2006

An integrative approach for causal gene identification and gene regulatory pathway inference.

MOTIVATION: Gene expression variation can often be linked to certain chromosomal regions and are tightly associated with phenotypic variation such as disease conditions. Inferring the causal genes for the expression variation is of great importance but rather challenging as the linked region generally contains multiple genes. Even when a single candidate gene is proposed, the underlying biological mechanism by which the regulation is enforced remains unknown. Novel approaches are needed to both infer the causal genes and generate hypothesis on the underlying regulatory mechanisms. RESULTS: We propose a new approach which aims at achieving the above objectives by integrating genotype information, gene expression, protein-protein interaction, protein phosphorylation, and transcription factor (TF)-DNA binding information. A network based stochastic algorithm is designed to infer the causal genes and identify the underlying regulatory pathways. We first quantitatively verified our method by a test using data generated by yeast knock-out experiments. Over 40% of inferred causal genes are correct, which is significantly better than 10% by random guess. We then applied our method to a recent genome-wide expression variation study in yeast. We show that our method can correctly identify the causal genes and effectively output experimentally verified pathways. New potential gene regulatory pathways are generated and presented as a global network. AVAILABILITY: Source code is available upon request.

Chromosome Mapping↗

PubMed2006

Forces shaping the fastest evolving regions in the human genome.

Comparative genomics allow us to search the human genome for segments that were extensively changed in the last approximately 5 million years since divergence from our common ancestor with chimpanzee, but are highly conserved in other species and thus are likely to be functional. We found 202 genomic elements that are highly conserved in vertebrates but show evidence of significantly accelerated substitution rates in human. These are mostly in non-coding DNA, often near genes associated with transcription and DNA binding. Resequencing confirmed that the five most accelerated elements are dramatically changed in human but not in other primates, with seven times more substitutions in human than in chimp. The accelerated elements, and in particular the top five, show a strong bias for adenine and thymine to guanine and cytosine nucleotide changes and are disproportionately located in high recombination and high guanine and cytosine content environments near telomeres, suggesting either biased gene conversion or isochore selection. In addition, there is some evidence of directional selection in the regions containing the two most accelerated regions. A combination of evolutionary forces has contributed to accelerated evolution of the fastest evolving elements in the human genome.

Animals↗

PubMed2005

Cloning and characterization of cDNAs expressed during chick development and encoding different isoforms of a putative zinc finger transcriptional regulator.

Development proceeds through successive activation of different sets of genes by specific transcription factors as a consequence of cell interactions and signaling. It is thus of primary interest to identify new putative transcriptional regulators. We report here the isolation of chicken clones bearing sequences coding for a chicken zinc finger protein (chZFp) which contains four pairs of zinc fingers of mixed type C2-H-C/C2-H2. At least five chZFp isoforms are produced through differential splicing of four small exons. The amino acid domains encoded by these four exons are highly conserved across species. Northern blot analysis and RNase-protection assays showed that chZFp transcripts are present in brain, heart, skin and liver during chick development. Reverse transcription mediated polymerase chain reaction (RT-PCR) experiments suggested that the relative amount of some chZFp isoforms increases at critical stages of development and skin morphogenesis. Finally, the main chZFp isoforms are able to directly interact in vitro with the scaffold attachment factor-A (SAF-A, also known as heterogenous nuclear ribonucleoprotein U) through both their aminoterminal and carboxyterminal domains.

Alternative Splicing↗

PubMed2006

Novel gene members in the Pho regulon of Escherichia coli.

The transcriptome profiles of the wild-type and the phoB mutant strains were compared at the time point showing the highest expression levels of the phoB and phoR genes under a P-limiting condition. Among the 18 new putative genes that were found to be under the control of the PhoB transcriptional regulator, five genes that contain the consensus Pho box were identified by sequence analysis. A reporter gene assay was carried out by fusing the upstream regions of these genes to the promoterless enhanced green fluorescent protein gene, followed by expression. It was found that the expressions of the amn (AMP nucleosidase), yibD (metal ion stress response gene) and ytfK (hypothetical protein) genes were activated by PhoB. These results indicate the additional roles of PhoB as a global regulator.

Bacterial Proteins↗

PubMed2006

Juxtaposed Polycomb complexes co-regulate vertebral identity.

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 and EED, pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity.

Animals↗

PubMed2006

Long-term inducible expression in striatal neurons from helper virus-free HSV-1 vectors that contain the tetracycline-inducible promoter system.

Direct gene transfer into neurons in the brain via a virus vector system has potential for both examining neuronal physiology and for developing gene therapy treatments for neurological diseases. Many of these applications require precise control of the levels of recombinant gene expression. The preferred method for controlling the levels of expression is by use of an inducible promoter system, and the tetracycline (tet)-inducible promoter system is the preferred system. Helper virus-free Herpes Simplex Virus (HSV-1) vectors have a number of the advantages, including their large size and efficient gene transfer. Also, we have reported long-term (14 months) expression from HSV-1 vectors that contain a modified neurofilament heavy gene promoter. A number of studies have reported short-term, inducible expression from helper virus-containing HSV-1 vector systems. However, long-term, inducible expression has not been reported using HSV-1 vectors. The goal of this study was to obtain long-term, inducible expression from helper virus-free HSV-1 vectors. We examined two different vector designs for adapting the tet promoter system to HSV-1 vectors. One design was an autoregulatory design; one transcription unit used a tet-regulated promoter to express the tet-regulated transcription factor tet-off, and another transcription unit used a tet-regulated promoter to express the Lac Z gene. In the other vector design, one transcription unit used the modified neurofilament heavy gene promoter to express tet-off, and another transcription unit used a tet-regulated promoter to express the Lac Z gene. The results showed that both vector designs supported inducible expression in cultured fibroblast or neuronal cell lines and for a short time (4 days) in the rat striatum. Of note, only the vector design that used the modified neurofilament promoter to express tet-off supported long-term (2 months) inducible expression in striatal neurons.

Animals↗

PubMed2005

Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest gene in Drosophila melanogaster.

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, we previously identified several cis-regulatory modules that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, we used the Gal4/UAS system to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method we identified the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16. Furthermore, we showed that these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, we could show that it regulates rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolished rst expression; indicating that the regulation of rst by Sim is direct.

Animals↗

PubMed2005

Analysis of a non-canonical NtcA-dependent promoter in Synechococcus elongatus and its regulation by NtcA and PII.

In this communication, we present a genetic analysis of the glnN promoter region of Synechococcus elongatus PCC 7942. luxAB reporter fusions were used to characterize the glnN promoter by deletion and site-directed mutational analysis. Reporter gene expression analysis was performed in S. elongatus wild-type and mutant strains to reveal the role of the global nitrogen responsive transcription factor NtcA and of the P(II) signalling protein on regulation of glnN gene expression. A non-canonical NtcA-binding motif is responsible for NtcA-dependent, nitrogen-responsive regulation of glnN. The P(II) signalling protein has opposing effects on NtcA-dependent glnN expression. Under conditions of nitrate-growth, it depresses expression, whereas under conditions of combined nitrogen starvation, it is required for full induction. Furthermore, sequences upstream of the NtcA-binding site have repressive effect on glnN promoter activity.

Bacterial Proteins↗

PubMed2006

Role of ubiquitin-like proteins in transcriptional regulation.

Conjugation of ubiquitin-like proteins (Ubls) to components of the transcriptional machinery represents an important mechanism to allow switching between different activity states. While ubiquitin modification of transcription factors is associated with transcriptional activation, SUMO modification of transcription factors is most often associated with transcriptional repression. Recent experiments indicate that another Ubl, NEDD8, can also influence transcription. One of the characteristics of Ubl modification is that the biological consequences of conjugation do not appear proportionate to the small fraction of substrate that is modified. The low steady state levels of Ubl-modified substrates can be attributed to a highly dynamic situation in which proteins are conjugated to a particular Ubl only for the modification to be removed by Ubl-specific proteases. It therefore appears that an unmodified protein with a history of Ubl modification may have different properties from a protein that never has been modified. Here the diverse effects of Ubl modification are discussed and models proposed to explain Ubl actions.

Animals↗

PubMed2006

Antibacterial lysine analogs that target lysine riboswitches.

Lysine riboswitches are bacterial RNA structures that sense the concentration of lysine and regulate the expression of lysine biosynthesis and transport genes. Members of this riboswitch class are found in the 5' untranslated region of messenger RNAs, where they form highly selective receptors for lysine. Lysine binding to the receptor stabilizes an mRNA tertiary structure that, in most cases, causes transcription termination before the adjacent open reading frame can be expressed. A lysine riboswitch conceivably could be targeted for antibacterial therapy by designing new compounds that bind the riboswitch and suppress lysine biosynthesis and transport genes. As a test of this strategy, we have identified several lysine analogs that bind to riboswitches in vitro and inhibit Bacillus subtilis growth, probably through a mechanism of riboswitch-mediated repression of lysine biosynthesis. These results indicate that riboswitches could serve as new classes of antibacterial drug targets.

Anti-Bacterial Agents↗

PubMed2006

The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways.

Few things can be considered to be more important to a cell than its threshold for apoptotic cell death, which can be modulated up or down, but rarely in both directions, by a single enzyme. Therefore, it came as quite a surprise to find that one enzyme, glycogen synthase kinase-3 (GSK3), has the perplexing capacity to either increase or decrease the apoptotic threshold. These apparently paradoxical effects now are known to be due to GSK3 oppositely regulating the two major apoptotic signaling pathways. GSK3 promotes cell death caused by the mitochondrial intrinsic apoptotic pathway, but inhibits the death receptor-mediated extrinsic apoptotic signaling pathway. Intrinsic apoptotic signaling, activated by cell damage, is promoted by GSK3 by facilitation of signals that cause disruption of mitochondria and by regulation of transcription factors that control the expression of anti- or pro-apoptotic proteins. The extrinsic apoptotic pathway entails extracellular ligands stimulating cell-surface death receptors that initiate apoptosis by activating caspase-8, and this early step in extrinsic apoptotic signaling is inhibited by GSK3. Thus, GSK3 modulates key steps in each of the two major pathways of apoptosis, but in opposite directions. Consequently, inhibitors of GSK3 provide protection from intrinsic apoptosis signaling but potentiate extrinsic apoptosis signaling. Studies of this eccentric ability of GSK3 to oppositely influence two types of apoptotic signaling have shed light on important regulatory mechanisms in apoptosis and provide the foundation for designing the rational use of GSK3 inhibitors for therapeutic interventions.

Animals↗

PubMed2005

Gene activation and deactivation related changes in the three-dimensional structure of chromatin.

Chromatin in the interphase nucleus is dynamic, decondensing where genes are activated and condensing where they are silenced. Local chromatin remodelling to a more open structure during gene activation is followed by changes in nucleosome distribution through the action of the transcriptional machinery. This leads to chromatin expansion and looping out of whole genomic regions. Such chromatin loops can extend beyond the chromosome territory. As several studies point to the location of transcription sites inside chromosome territories as well as at their periphery, extraterritorial loops cannot simply be a mechanism for making transcribed genes accessible to the transcriptional machinery and must occur for other reasons. The level of decondensation within an activated region varies greatly and probably depends on the density of activated genes and the number of engaged RNA polymerases. Genes that are silenced during development form a more closed chromatin structure. Specific histone modifications are correlated with gene activation and silencing, and silenced genes may become associated with heterochromatin protein 1 homologues or with polycomb group complexes. Several levels of chromatin packaging are found in the nucleus relating to the different functions of and performed by active genes; euchromatic and heterochromatic regions and the models explaining higher-order chromatin structure are still disputed.

Animals↗

PubMed2006

cis- and trans-Acting determinants of transcription termination by yeast RNA polymerase II.

Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3'-end formation in the context of an mRNA 3' untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.

Amino Acid Sequence↗

PubMed1989

Structural organization of the human mitochondrial cytochrome c1 gene.

The structural organization of the entire human nuclear encoded gene for mitochondrial cytochrome c1 was determined by analyzing a clone obtained from an EMBL3 genomic DNA library. The gene spans 2.4-kilobase pairs and contains seven exons interrupted by six introns of relatively small sizes. All intron/exon splice junctions follow the GT/AG rule. The 5'-flanking region of the gene lacks typical transcriptional regulatory sequence elements such as TATA and CAAT boxes but contains seven putative GC boxes (Sp1 binding sites) and several sequences that resemble another type of the Sp1 responsive element, the enhancer core consensus sequence, the AP-1 responsive element, and the cAMP- and phorbol ester-inducible element. The region also contains a 15-nucleotide sequence highly homologous to the AP-4 consensus sequence and to those in the 5'-flanking regions of the genes for two enzymes associated with respiratory function, the beta subunit of human ATP synthase and chicken 5-aminolevulinate synthase. The presequence, which is essential for the transport of the cytochrome c1 precursor into mitochondria, is encoded in both the first and second exons, and the nucleotide sequence corresponding to the presequence is separated by the first intron. This is the first example of a leader sequence coding for a presequence clearly separated into two parts by an intron.

Amino Acid Sequence↗

PubMed2006

Probabilistic inference of transcription factor concentrations and gene-specific regulatory activities.

MOTIVATION: Quantitative estimation of the regulatory relationship between transcription factors and genes is a fundamental stepping stone when trying to develop models of cellular processes. Recent experimental high-throughput techniques, such as Chromatin Immunoprecipitation (ChIP) provide important information about the architecture of the regulatory networks in the cell. However, it is very difficult to measure the concentration levels of transcription factor proteins and determine their regulatory effect on gene transcription. It is therefore an important computational challenge to infer these quantities using gene expression data and network architecture data. RESULTS: We develop a probabilistic state space model that allows genome-wide inference of both transcription factor protein concentrations and their effect on the transcription rates of each target gene from microarray data. We use variational inference techniques to learn the model parameters and perform posterior inference of protein concentrations and regulatory strengths. The probabilistic nature of the model also means that we can associate credibility intervals to our estimates, as well as providing a tool to detect which binding events lead to significant regulation. We demonstrate our model on artificial data and on two yeast datasets in which the network structure has previously been obtained using ChIP data. Predictions from our model are consistent with the underlying biology and offer novel quantitative insights into the regulatory structure of the yeast cell. AVAILABILITY: MATLAB code is available from http://umber.sbs.man.ac.uk/resources/puma

Algorithms↗

PubMed2006

Alternative poly(A) site-selection regulates the production of alternatively spliced vesl-1/homer1 isoforms that encode postsynaptic scaffolding proteins.

The vesl-1/homer1 gene encodes a scaffold protein that interacts with several receptors to modulate synaptic functions. The gene also encodes two shorter forms that counteract the functions of the long form of Vesl. Expression of the shorter forms is driven by neural activities such as long-term potentiation. Here we analyzed the mechanism regulating vesl-1 alternative splicing. Each functional poly(A) site was in a different part of the 3'-terminal exon, with promoter-proximal and promoter-distal sites at the end of exons corresponding to the short and long form Vesl-1, respectively. 3'-End-processing at proximal poly(A) site, specifically at the vesl-1M poly(A) site, was enhanced by extracellular stimuli, thereby switching transcription termination from promoter-distal to -proximal poly(A) site. This switch was not specifically coupled to the vesl-1 promoter and was independent of de novo protein synthesis. Analysis of transcripts from mini-genes that mimic the structure of endogenous vesl-1 revealed that the vesl-1M poly(A) region plays a crucial role in switching to the alternative pre-mRNA splicing that is triggered by extracellular stimuli. Therefore, a 3'-end-processing event regulates the neural activity-dependent alternative splicing of vesl-1. This is the first report of a gene in which alternative poly(A) site-selection regulates alternative splicing in a protein synthesis-independent manner.

Alternative Splicing↗

PubMed2005

Natural antisense RNAs in the nervous system.

Natural antisense RNAs are endogenous molecules that are complementary to RNA transcripts of already established function. They were discovered first in prokaryotes in which they are now recognised as an important component of molecular mechanisms involved in the regulation of gene expression. Recently, through the cumulative efforts of molecular biologists and bioinformaticians, natural antisense RNAs have been demonstrated in significant numbers in eukaryotic systems also. Probably the most exciting outcome of these studies is that natural antisense RNAs are particularly prevalent in the nervous system. Here we discuss the major known types of natural antisense RNAs in eukaryotic systems and focus on their potential roles in the regulation of gene expression in the brain.

Animals↗