GC content strongly influences the role of poly(dA) in the intrinsic nucleosome positioning in Saccharomyces cerevisiae.

The nucleosome is the basic structural element of genomic DNA packaging and plays a role in transcription, replication, and recombination. Poly(dA) tracts are considered major sequence determinants of nucleosome positioning, although their role is not well understood. Here, we show that the homopolymeric character and the low GC content of poly(dA)s play different roles in nucleosome formation. We found that the inherent low GC content of poly(dA) alone can account for the deep and anisotropic nucleosome depletion at structurally and functionally important regions of promoters and origins of replication. We also show that the level of nucleosome occupancy at poly(dA) is strongly related to the local nucleotide background and its high frequency of occurrence in Saccharomyces cerevisiae does not appear merely to be associated with its intrinsic nucleosome-excluding properties. In addition, we show that the GC content alone can predict more than 60% of the in vitro nucleosome map, providing further evidence that the intrinsic nucleosome positioning is more greatly determined by GC content than poly(dA) stretches. Our results are consistent with a model in which poly(dA) stretches act at two distinct levels: first, by its low GC content, which intrinsically contributes to hinder nucleosome formation, and second, by its contiguous runs of dA that selectively drive the recruitment of non-histone proteins with structural and functional roles.

RNA polymerase II (RNAP II)-associated factors are recruited to tRNA loci, revealing that RNAP II- and RNAP III-mediated transcriptions overlap in yeast.

In yeast (Saccharomyces cerevisiae), the synthesis of tRNAs by RNA polymerase III (RNAP III) down-regulates the transcription of the nearby RNAP II-transcribed genes by a mechanism that is poorly understood. To clarify the basis of this tRNA gene-mediated (TGM) silencing, here, conducting a bioinformatics analysis of available ChIP-chip and ChIP-sequencing genomic data from yeast, we investigated whether the RNAP III transcriptional machinery can recruit protein factors required for RNAP II transcription. An analysis of 46 genome-wide protein-density profiles revealed that 12 factors normally implicated in RNAP II-mediated gene transcription are more enriched at tRNA than at mRNA loci. These 12 factors typically have RNA-binding properties, participate in the termination stage of the RNAP II transcription, and preferentially localize to the tRNA loci by a mechanism that apparently is based on the RNAP III transcription level. The factors included two kinases of RNAP II (Bur1 and Ctk1), a histone demethylase (Jhd2), and a mutated form of a nucleosome-remodeling factor (Spt6) that have never been reported to be recruited to tRNA loci. Moreover, we show that the expression levels of RNAP II-transcribed genes downstream of tRNA loci correlate with the distance from the tRNA gene by a mechanism that depends on their orientation. These results are consistent with the notion that pre-tRNAs recruit RNAP II-associated factors, thereby reducing the availability of these factors for RNAP II transcription and contributing, at least in part, to the TGM-silencing mechanism.

Human NF-κB repressing factor acts as a stress-regulated switch for ribosomal RNA processing and nucleolar homeostasis surveillance.

The nucleolus, a dynamic nuclear compartment long regarded as the cell ribosome factory, is emerging as an important player in the regulation of cell survival and recovery from stress. In larger eukaryotes, the stress-induced transcriptional response is mediated by a family of heat-shock transcription factors. Among these, HSF1, considered the master regulator of stress-induced transcriptional responses, controls the expression of cytoprotective heat shock proteins (HSPs), molecular chaperones/cochaperones constituting a major component of the cell protein quality control machinery essential to circumvent stress-induced degradation and aggregation of misfolded proteins. Herein we identify human NF-κB repressing factor (NKRF) as a nucleolar HSP essential for nucleolus homeostasis and cell survival under proteotoxic stress. NKRF acts as a thermosensor translocating from the nucleolus to the nucleoplasm during heat stress; nucleolar pools are replenished during recovery upon HSF1-mediated NKRF resynthesis. Silencing experiments demonstrate that NKRF is an unconventional HSP crucial for correct ribosomal RNA (rRNA) processing and preventing aberrant rRNA precursors and discarded fragment accumulation. These effects are mediated by NKRF interaction with the 5'-to-3' exoribonuclease XRN2, a key coordinator of multiple pre-rRNA cleavages, driving mature rRNA formation and discarded rRNA decay. Under stress conditions, NKRF directs XRN2 nucleolus/nucleoplasm trafficking, controlling 5'-to-3' exoribonuclease nucleolar levels and regulating rRNA processing. Our study reveals a different aspect of rRNA biogenesis control in human cells and sheds light on a sophisticated mechanism of nucleolar homeostasis surveillance during stress.

Selective forces and mutational biases drive stop codon usage in the human genome: a comparison with sense codon usage.

BACKGROUND: The three stop codons UAA, UAG, and UGA signal the termination of mRNA translation. As a result of a mechanism that is not adequately understood, they are normally used with unequal frequencies. RESULTS: In this work, we showed that selective forces and mutational biases drive stop codon usage in the human genome. We found that, in respect to sense codons, stop codon usage was affected by stronger selective forces but was less influenced by neutral mutational biases. UGA is the most frequent termination codon in human genome. However, UAA was the preferred stop codon in genes with high breadth of expression, high level of expression, AT-rich coding sequences, housekeeping functions, and in gene ontology categories with the largest deviation from expected stop codon usage. Selective forces associated with the breadth and the level of expression favoured AT-rich sequences in the mRNA region including the stop site and its proximal 3'-UTR, but acted with scarce effects on sense codons, generating two regions, upstream and downstream of the stop codon, with strongly different base composition. By favouring low levels of GC-content, selection promoted labile local secondary structures at the stop site and its proximal 3'-UTR. The compositional and structural context favoured by selection was surprisingly emphasized in the class of ribosomal proteins and was consistent with sequence elements that increase the efficiency of translational termination. Stop codons were also heterogeneously distributed among chromosomes by a mechanism that was strongly correlated with the GC-content of coding sequences. CONCLUSIONS: In human genome, the nucleotide composition and the thermodynamic stability of stop codon site and its proximal 3'-UTR are correlated with the GC-content of coding sequences and with the breadth and the level of gene expression. In highly expressed genes stop codon usage is compositionally and structurally consistent with highly efficient translation termination signals.

The proteasome inhibitor bortezomib is a potent inducer of zinc finger AN1-type domain 2a gene expression: role of heat shock factor 1 (HSF1)-heat shock factor 2 (HSF2) heterocomplexes.

The zinc finger AN1-type domain 2a gene, also known as arsenite-inducible RNA-associated protein (AIRAP), was recently identified as a novel human canonical heat shock gene strictly controlled by heat shock factor (HSF) 1. Little is known about AIRAP gene regulation in human cells. Here we report that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, is a potent inducer of AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated protein accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of HSF2, a factor implicated in differentiation and development regulation. Different from heat-mediated induction, in bortezomib-treated cells, HSF1 and HSF2 interact directly, forming HSF1-HSF2 heterotrimeric complexes recruited to a specific heat shock element in the AIRAP promoter. Interestingly, whereas HSF1 has been confirmed to be critical for AIRAP gene transcription, HSF2 was found to negatively regulate AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor under stress conditions. AIRAP function is still not defined. However, the fact that AIRAP is expressed abundantly in primary human cells at bortezomib concentrations comparable with plasma levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment.

Selection on codon bias in yeast: a transcriptional hypothesis.

Codons that code for the same amino acid are often used with unequal frequencies. This phenomenon is termed codon bias. Here, we report a computational analysis of codon bias in yeast using experimental and theoretical genome-wide data. We show that the most used codons in highly expressed genes can be predicted by mRNA structural data and that the codon choice at each synonymous site within an mRNA is not random with respect to the local secondary structure. Because we also found that the folding stability of intron sequences is strongly correlated with codon bias and mRNA level, our results suggest that codon bias is linked to mRNA folding structure through a mechanism that, at least partially, operates before pre-mRNA splicing. Consistent with this, we report evidence supporting the adaptation of the tRNA pool to the codon profile of the most expressed genes rather than vice versa. We show that the correlation of codon usage with the gene expression level also includes the stop codons that are normally not decoded by aminoacyl-tRNAs. The results reported here are consistent with a role for transcriptional forces in driving codon usage bias via a mechanism that improves gene expression by optimizing mRNA folding structures.

AIRAP, a new human heat shock gene regulated by heat shock factor 1.

Heat shock factor-1 (HSF1) is the central regulator of heat-induced transcriptional responses leading to rapid expression of molecular chaperones that protect mammalian cells against proteotoxic stress. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat shock genes encoding a variety of heat shock proteins, including HSP70, HSP90, HSP27, and other proteins of the network. Herein we report that the zinc finger AN1-type domain-2a gene, also known as AIRAP, behaves as a canonical heat shock gene, whose expression is temperature-dependent and strictly controlled by HSF1. Transcription is triggered at temperatures above 40 degrees C in different types of human cancer and primary cells, including peripheral blood monocytes. As shown by ChIP analysis, HSF1 is recruited to the AIRAP promoter rapidly after heat treatment, with a kinetics that parallels HSP70 promoter HSF1-recruitment. In transfection experiments HSF1-silencing abolished heat-induced AIRAP promoter-driven transcription, which could be rescued by exogenous Flag-HSF1 expression. The HSF1 binding HSE sequence in the AIRAP promoter critical for heat-induced transcription was identified. Because its expression is induced at febrile temperatures in human cells, AIRAP may represent a new potential component of the protective response during fever in humans.

Sequence-specific recognition of DNA by the C-terminal domain of nucleoid-associated protein H-NS.

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage lambda CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::lambdaCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::lambdaCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr(109), Arg(113), Thr(114), and Ala(116)) belonging to the same protein loop and Glu(101). The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.

Metal-binding and nuclease activity of an antimicrobial peptide analogue of the salivary histatin 5.

The salivary antimicrobial peptide histatin 5 is characterized by its cationic nature, structural flexibility, and the presence of two metal-binding sites (the ATCUN motif and a Zn-binding motif). These properties make this peptide a good model for the design of new drugs of low molecular weight. In this work, we have synthesized and studied a new peptide, an analogue of the histatin 5 named ATCUN-C16, which contains both metal-binding centers. The results show that our 20-residue-derived peptide preserves anticandidal activity and exhibits a higher propensity to assume a stable conformation in a hydrophobic environment than do histatin 5 and the C16 peptide that contains the 16 residues of the C-terminal part of histatin 5, although overall our peptide remains a flexible molecule. ACTUN-C16 was found to bind DNA in a gel retardation assay and to have a nuclease activity in the presence of copper and zinc ions and ascorbate. Its nuclease activity can be attributed to the synergistic action of oxidative and hydrolytic activities due to the Cu-ATCUN complex and to the zinc ion coordination, respectively. The results show a new property of this family of salivary peptides and suggest a novel use of this peptide as a small nuclease and biotechnological tool.

On the normalization of the minimum free energy of RNAs by sequence length.

The minimum free energy (MFE) of ribonucleic acids (RNAs) increases at an apparent linear rate with sequence length. Simple indices, obtained by dividing the MFE by the number of nucleotides, have been used for a direct comparison of the folding stability of RNAs of various sizes. Although this normalization procedure has been used in several studies, the relationship between normalized MFE and length has not yet been investigated in detail. Here, we demonstrate that the variation of MFE with sequence length is not linear and is significantly biased by the mathematical formula used for the normalization procedure. For this reason, the normalized MFEs strongly decrease as hyperbolic functions of length and produce unreliable results when applied for the comparison of sequences with different sizes. We also propose a simple modification of the normalization formula that corrects the bias enabling the use of the normalized MFE for RNAs longer than 40 nt. Using the new corrected normalized index, we analyzed the folding free energies of different human RNA families showing that most of them present an average MFE density more negative than expected for a typical genomic sequence. Furthermore, we found that a well-defined and restricted range of MFE density characterizes each RNA family, suggesting the use of our corrected normalized index to improve RNA prediction algorithms. Finally, in coding and functional human RNAs the MFE density appears scarcely correlated with sequence length, consistent with a negligible role of thermodynamic stability demands in determining RNA size.

Regulation of cyclooxygenase-2 expression by heat: a novel aspect of heat shock factor 1 function in human cells.

The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat-shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non-canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E(2) in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion-mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position -2495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function.

The 3-base periodicity and codon usage of coding sequences are correlated with gene expression at the level of transcription elongation.

BACKGROUND: Gene transcription is regulated by DNA transcriptional regulatory elements, promoters and enhancers that are located outside the coding regions. Here, we examine the characteristic 3-base periodicity of the coding sequences and analyse its correlation with the genome-wide transcriptional profile of yeast. PRINCIPAL FINDINGS: The analysis of coding sequences by a new class of indices proposed here identified two different sources of 3-base periodicity: the codon frequency and the codon sequence. In exponentially growing yeast cells, the codon-frequency component of periodicity accounts for 71.9% of the variability of the cellular mRNA by a strong association with the density of elongating mRNA polymerase II complexes. The mRNA abundance explains most of the correlation between the codon-frequency component of periodicity and protein levels. Furthermore, pyrimidine-ending codons of the four-fold degenerate small amino acids alanine, glycine and valine are associated with genes with double the transcription rate of those associated with purine-ending codons. CONCLUSIONS: We demonstrate that the 3-base periodicity of coding sequences is higher than expected by the codon usage frequency (CUF) and that its components, associated with codon bias and amino acid composition, are correlated with gene expression, principally at the level of transcription elongation. This indicates a role of codon sequences in maximising the transcription efficiency in exponentially growing yeast cells. Moreover, the results contrast with the common darwinian explanation that attributes the codon bias to translational selection by an adjustment of synonymous codon frequencies to the most abundant isoaccepting tRNA. Here, we show that selection on codon bias likely acts at both the transcriptional and translational level and that codon usage and the relative abundance of tRNA could drive each other in order to synergistically optimize the efficiency of gene expression.

Interaction of DAPI with individual strands of trinucleotide repeats. Effects of replication in vitro of the AAT x ATT triplet.

The structural changes produced by the minor-groove binding ligand DAPI (4',6-diamidine-2-phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band-shift analysis and related to their effects on DNA replication in vitro. Among the 20 possible single-stranded trinucleotide repeats, only the T-rich strand of the AAT.ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast-migrating hairpin structure by a minor-groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T.T with respect to A.A, C.C and G.G mismatch, in favouring the binding properties and the structural features of the ATT-DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC-rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI.