Heat shock factor binding in Alu repeats expands its involvement in stress through an antisense mechanism

Background: Alu RNAs are present at elevated levels in stress conditions and, consequently, Alu repeats are increasingly being associated with the physiological stress response. Alu repeats are known to harbor transcription factor binding sites that modulate RNA pol II transcription and Alu RNAs act as transcriptional co-repressors through pol II binding in the promoter regions of heat shock responsive genes. An observation of a putative heat shock factor (HSF) binding site in Alu led us to explore whether, through HSF binding, these elements could further contribute to the heat shock response repertoire. Results: Alu density was significantly enriched in transcripts that are down-regulated following heat shock recovery in HeLa cells. ChIP analysis confirmed HSF binding to a consensus motif exhibiting positional conservation across various Alu subfamilies, and reporter constructs demonstrated a sequence-specific two-fold induction of these sites in response to heat shock. These motifs were over-represented in the genic regions of down-regulated transcripts in antisense oriented Alus. Affymetrix Exon arrays detected antisense signals in a significant fraction of the down-regulated transcripts, 50% of which harbored HSF sites within 5 kb. siRNA knockdown of the selected antisense transcripts led to the over-expression, following heat shock, of their corresponding down-regulated transcripts. The antisense transcripts were significantly enriched in processes related to RNA pol III transcription and the TFIIIC complex. Conclusions: We demonstrate a non-random presence of Alu repeats harboring HSF sites in heat shock responsive transcripts. This presence underlies an antisense-mediated mechanism that represents a novel component of Alu and HSF involvement in the heat shock response

Evolution and distribution of RNA polymerase II regulatory sites from RNA polymerase III dependant mobile Alu elements

BACKGROUND: The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting. RESULTS: We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes. CONCLUSIONS: Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of regulatory elements in the preexisting genes through Alus could thus have contributed to evolution of novel regulatory networks in the primate genomes. With such a wide spectrum of regulatory sites present in Alus, it also becomes imperative to screen for variations in these sites in candidate genes, which are otherwise repeat-masked in studies pertaining to identification of predisposition markers

Alu repeat analysis in the complete human genome: trends and variations with respect to genomic composition

Motivation: Transposon-derived Alu repeats are exclusively associated with primate genomes. They have gained considerable importance in the recent times with evidence of their involvement in various aspects of gene regulation, e.g. alternative splicing, nucleosome positioning, CpG methylation, binding sites for transcription factors and hormone receptors, etc. The objective of this study is to investigate the factors that influence the distribution of Alu repeat elements in the human genome. Such analysis is expected to yield insights into various aspects of gene regulation in primates. Results: Analysis of Alu repeat distribution for the human genome build 32 (released in January 2003) reveals that they occupy nearly one-tenth portion of the sequenced regions. Huge variations in Alu frequencies were seen across the genome with chromosome 19 being the most and chromosome Y being the least Alu dense chromosomes. The highlights of the analysis are as follows: (1) three-fourth of the total genes in the genome are associated with Alus. (2) Alu density is higher in genes as compared with intergenic regions in all the chromosomes except 19 and 22. (3) Alu density in human genome is highly correlated with GC content, gene density and intron density with GC content being major deterministic factor compared with other two. (4) Alu densities were correlated more with gene density than intron density indicating the insertion of Alus in untranslated regions of exons

Withaferin A, a Cytotoxic Steroid from Vassobia breWiflora, Induces Apoptosis in Human Head and Neck Squamous Cell Carcinoma

As part of a program to discover drug leads from plant biodiversity, the present investigation was undertaken to explore the anticancer potential of compounds derived from selected Latin American plants. Bioassay-guided fractionation of a crude extract of the aerial parts of Vassobia breviflora led to the isolation of the withanolide-type steroidal lactone withaferin A (1). This compound was tested for antiproliferative activity against the head and neck squamous cell carcinoma (HNSCC) cell lines, MDA1986, JMAR, UM-SCC-2, and JHU011. The inhibitory concentrations to reduce cell viability to 50% (IC50) were determined by the MTS cytotoxicity assay, and 1 reduced cell viability with IC50 values in the range 0.5−2.2 μM. A mechanistic study showed that 1 induces apoptosis and cell death in HNSCC cells as well as a cell-cycle shift from G0/G1 to G2/M. Cells treated with 1 exhibited inactivation of Akt and a reduction in total Akt concentration. This investigation constitutes the first report of the antiproliferative activity of withaferin A (1) against head and neck squamous carcinoma

Novel C-Terminal Hsp90 Inhibitor for Head and Neck Squamous Cell Cancer (HNSCC) with in vivo Efficacy and Improved Toxicity Profiles Compared with Standard Agents

The final publication is available at Springer via http://dx.doi.org/10.1245/s10434-011-1971-1.Background - Current therapies for HNSCC, especially platinum agents, are limited by their toxicities and drug resistance. This study evaluates a novel C-terminal Hsp90 inhibitor (CT-Hsp90-I) for efficacy and toxicity in vitro and in vivo in an orthotopic HNSCC model. Our hypothesis is that C-terminal inhibitors exhibit improved toxicity/efficacy profiles over standard therapies and may represent a novel group of anticancer agents. Methods - MDA-1986 HNSCC cells were treated with doses of 17-AAG or KU363 (a CT-Hsp90-I) and compared for antiproliferation by GLO-Titer and trypan blue exclusion and for apoptosis by PARP cleavage and caspase-3 inactivation by Western analysis. In vivo studies in Nu/Nu mice examined an orthotopic model of MDA-1986 cells followed by drug dosing intraperitoneally for a 21-day period (mg/kg/dose: cisplatin = 3.5, low-dose KU363 = 5, high-dose KU363 = 25, 17-AAG = 175). Tumor size, weight, and toxicity (body score) were measured 3×/week. Results - The IC50 levels for KU363 = 1.2–2 μM in MDA-1986. KU363 induces apoptosis at 1 μM with cleavage of PARP and inactivation of caspase-3 levels after 24 h. Client proteins Akt and Raf-1 were also downregulated at 1–3 μM of drug. In vivo, 100% of controls had progressive disease, while 100% of cisplatin animals showed some response, all with significant systemic toxicity. High-dose KU363 showed 88% of animals responding and low-dose KU363 showed 75% responding. KU363 animals showed significantly less toxicity (P < 0.01) than cisplatin or 17-AAG. Conclusion - This novel CT-Hsp90-I KU363 manifests potent anticancer activity against HNSCC, showing excellent in vivo efficacy and reduced toxicity compared with standard agents justifying future translational evaluation