Nuclear Receptor HNF4α Binding Sequences are Widespread in Alu Repeats

<p>Abstract</p> <p>Background</p> <p>Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression.</p> <p>Results</p> <p>Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4α binds Alu elements in the promoters of target genes (<it>ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR</it>) and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation <it>in vivo </it>(<it>APOM, PRODH2, TTR, APOA4</it>). HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites.</p> <p>Conclusions</p> <p>Our findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.</p

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 ± 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 ± 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms

Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

Background: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods and Findings: Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACiinduced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. Conclusions: These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples

Conserved Secondary Structures in Aspergillus

Background: Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs. Methodology/Principal Findings: We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted,60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 59 UTRs primarily contain groupings of short hairpins located near the start codon, and 39 UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene

Reducing the Cost of Conditional Transfers of Control by Using Comparison Specifications Abstract

A significant portion of a program’s execution cycles are typically dedicated to performing conditional transfers of control. Much of the research on reducing the costs of these operations has focused on the branch, while the comparison has been largely ignored. In this paper we investigate reducing the cost of comparisons in conditional transfers of control. We decouple the specification of the values to be compared from the actual comparison itself, which now occurs as part of the branch instruction. The specification of the register or immediate values involved in the comparison is accomplished via a new instruction called a comparison specification, which is loop invariant. Decoupling the specification of the comparison from the actual comparison performed before the branch reduces the number of instructions in the loop, which provides performance benefits not possible when using conventional comparison instructions. Results from applying this technique on the ARM processor show that both the number of instructions executed and execution cycles are reduced

Regulation of alternative splicing in Drosophila

Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in higher eukaryotes. It is estimated that over 70% of all genes are alternatively spliced as a means for producing functionally diverse proteins from a single gene. The changes in mRNA due to alternative splicing can function in a number of developmental processes, including sex-determination and neuronal development. Identifying and understanding the role of cis-acting sequences involved in regulating alternative splicing will help to elucidate the mechanisms that govern alternative splicing and protein diversity. ^ The Drosophila fruitless (fru) gene is a component of the sex-determination cascade and plays central role in determining male sexual behavior. The fru pre-mRNA is alternatively spliced in a sex-specific manner and involves the selection of alternative 5′ splice sites. The upstream male-specific splice site is used by default while the downstream female-specific 5′ splice site is activated by the splicing regulators Transformer (Tra) and Transformer 2 (Tra2). Tra and Tra2 function by binding to three repeats of a 13 nt element located immediately upstream of the female-specific splice site. It has previously been proposed that the splicing enhancer complex functions by recruiting UI snRNP to the female-specific 5′ splice site. However, we find that in the absence of Tra and Tra2 the female-specific 5′ splice site is actively repressed in a manner requiring an intact UI snRNP and the hnRNP protein, PSI. This suggests that the main function of Tra and Tra2 is to activate female-specific splicing by counteracting a repressor rather than by actively recruiting UI snRNP. ^ Through alternative splicing most genes produce transcripts that can generate a relatively modest number of isoforms, however, there are several examples of genes that can potentially generate thousands of alternatively spliced mRNAs. The Drosophila Dscam gene encodes an axon guidance receptor that potentially generates 38,016 different isoforms due to alternative splicing of 95 of its 115 exons. The alternative exons are organized into four clusters that are spliced in a mutually exclusive manner. Here we describe an RNA secondary structural element required for efficient inclusion of all twelve variable exons in the exon 4 cluster, term the inclusion stem (iStem). The iStem resides in the intron between exon 3 and the first exon 4 variant. Interestingly, though the iStem primarily functions as a master regulator that governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons. Taken together, our data suggest a new mechanism for the function of 5′ splice site enhancer-dependent alternative splicing of the Drosophila fru pre-mRNA and we have identified the iStem, a novel type of regulatory element that simultaneously controls whether or not any of several exons are included in the mRNA.

REDUCING THE COST OF COMPARISONS WITHIN CONDITIONAL TRANSFERS OF CONTROL By

The Office of Graduate Studies has verified and approved the above named committee members. This work is dedicated to Dr. Alice McRae, the one who started me down this road in the first place. And to my family, who supported me through the good times and the bad. It’s those bad ones that count. iii ACKNOWLEDGMENTS I would like to thank Steve Hines, Prasad Kulkarni and Clint Whaley, for all your support, technical and moral. I think you guys understand the heart attack! I would also like to thank my advisor, Dr. David Whalley. This work could not have been done without his help