Genome reference and sequence variation in the large repetitive central exon of human MUC5AC.

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin.

The identification of protein kinase C iota as a regulator of the Mammalian heat shock response using functional genomic screens.

BACKGROUND: The heat shock response is widely used as a surrogate of the general protein quality control system within the cell. This system plays a significant role in aging and many protein folding diseases as well as the responses to other physical and chemical stressors. METHODS/PRINCIPAL FINDINGS: In this study, a broad-based functional genomics approach was taken to identify potential regulators of the mammalian heat shock response. In the primary screen, a total of 13724 full-length genes in mammalian expression vectors were individually co-transfected into human embryonic kidney cells together with a human HSP70B promoter driving firefly luciferase. A subset of the full-length genes that showed significant activation in the primary screen were then evaluated for their ability to hyper-activate the HSP70B under heat shock conditions. Based on the results from the secondary assay and gene expression microarray analyses, eight genes were chosen for validation using siRNA knockdown. Of the eight genes, only PRKCI showed a statistically significant reduction in the heat shock response in two independent siRNA duplexes compared to scrambled controls. Knockdown of the PRKCI mRNA was confirmed using quantitative RT-PCR. Additional studies did not show a direct physical interaction between PRKCI and HSF1. CONCLUSIONS/SIGNIFICANCE: The results suggest that PRKCI is an indirect co-regulator of HSF1 activity and the heat shock response. Given the underlying role of HSF1 in many human diseases and the response to environmental stressors, PRKCI represents a potentially new candidate for gene-environment interactions and therapeutic intervention.

Genome-wide analysis of DNA methylation and gene expression changes in the mouse lung following subchronic arsenate exposure.

Alterations in DNA methylation have been proposed as a mechanism for the complex toxicological effects of arsenic. In this study, whole-genome DNA methylation and gene expression changes were evaluated in lungs from female mice exposed for 90 days to 50 ppm arsenate (As) in drinking water. DNA methylation changes were measured using reduced representation bisulfite deep sequencing. Differential methylation was observed in approximately 700 and 1900 start and transcribed regions, respectively. The start regions showed bias toward decreased methylation. No bias was observed in the transcribed region. A comparison of absolute methylation levels in the control animals with treatment-related changes in methylation showed that baseline methylation levels play a role in determining which genes are methylated. Genes with low absolute methylation levels in the start region showed a trend toward increased As-related methylation and decreased expression. Genes with high levels of methylation in the transcribed region showed a trend toward decreased As-related methylation, but no change in expression. No overall correlation between treatment-related changes in methylation and expression was identified. Among genes showing differential methylation in the start region and differential expression, only 57% showed an inverse correlation. The results suggest that differential methylation following As treatment may only play a permissive role in regulating expression. Despite the low correlation, the subset of 17 genes that showed an inverse relationship between As-related methylation and expression included a substantial number that has been demonstrated to play a functional role in cancer-related processes and other effects consistent with arsenic exposure.

Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening.

Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multiple cellular pathways. In this study, metabolic clearance and plasma protein binding were experimentally measured for 35 ToxCast phase I chemicals. The experimental data were used to parameterize a population-based in vitro-to-in vivo extrapolation model for estimating the human oral equivalent dose necessary to produce a steady-state in vivo concentration equivalent to in vitro AC(50) (concentration at 50% of maximum activity) and LEC (lowest effective concentration) values from the ToxCast data. For 23 of the 35 chemicals, the range of oral equivalent doses for up to 398 ToxCast assays was compared with chronic aggregate human oral exposure estimates in order to assess whether significant in vitro bioactivity occurred within the range of maximum expected human oral exposure. Only 2 of the 35 chemicals, triclosan and pyrithiobac-sodium, had overlapping oral equivalent doses and estimated human oral exposures. Ranking by the potencies of the AC(50) and LEC values, these two chemicals would not have been at the top of a prioritization list. Integrating both dosimetry and human exposure information with the high-throughput toxicity screening efforts provides a better basis for making informed decisions on chemical testing priorities and regulatory attention. Importantly, these tools are necessary to move beyond hazard rankings to estimates of possible in vivo responses based on in vitro screens.

Chaperone regulation of the heat shock protein response.

The heat shock protein response appears to be triggered primarily by nonnative proteins accumulating in a stressed cell and results in increased expression of heat shock proteins (HSPs). Many heat shock proteins prevent protein aggregation and participate in refolding or elimination of misfolded proteins in their capacity as chaperones. Even though several mechanisms exist to regulate the abundance of cytosolic and nuclear chaperones, activation of heat shock transcription factor 1 (HSF1) is an essential aspect of the heat shock protein response. HSPs and co-chaperones that are assembled into multichaperone complexes regulate HSF1 activity at different levels. HSP90-containing multichaperone complexes appear to be the most relevant repressors of HSF1 activity. Because HSP90-containing multichaperone complexes interact not only specifically with client proteins including HSF1 but also generically with nonnative proteins, the concentration of nonnative proteins influences assembly on HSF1 of HSP90-containing complexes that repress activation, and may play a role in inactivation, of the transcription factor. Proteins that are unable to achieve stable tertiary structures and remain chaperone substrates are targeted for proteasomal degradation through polyubiquitination by co-chaperone CHIP. CHIP can activate HSF1 to regulate the protein quality control system that balances protection and degradation of chaperone substrates.

CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70.

Exposure of cells to various stresses often leads to the induction of a group of proteins called heat shock proteins (HSPs, molecular chaperones). Hsp70 is one of the most highly inducible molecular chaperones, but its expression must be maintained at low levels under physiological conditions to permit constitutive cellular activities to proceed. Heat shock transcription factor 1 (HSF1) is the transcriptional regulator of HSP gene expression, but it remains poorly understood how newly synthesized HSPs return to basal levels when HSF1 activity is attenuated. CHIP (carboxy terminus of Hsp70-binding protein), a dual-function co-chaperone/ubiquitin ligase, targets a broad range of chaperone substrates for proteasomal degradation. Here we show that CHIP not only enhances Hsp70 induction during acute stress but also mediates its turnover during the stress recovery process. Central to this dual-phase regulation is its substrate dependence: CHIP preferentially ubiquitinates chaperone-bound substrates, whereas degradation of Hsp70 by CHIP-dependent targeting to the ubiquitin-proteasome system occurs when misfolded substrates have been depleted. The sequential catalysis of the CHIP-associated chaperone adaptor and its bound substrate provides an elegant mechanism for maintaining homeostasis by tuning chaperone levels appropriately to reflect the status of protein folding within the cytoplasm.

Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress.

BACKGROUND: Heat shock factor (HSF/HSF1) not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs). HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser303, Ser307 and Ser363, which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser230 contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells. RESULTS: An alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser121, Ser230, Ser292, Ser303, Ser307, Ser314, Ser319, Ser326, Ser344, Ser363, Ser419, and Ser444. Phosphorylation of Ser326 but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser326 increased rapidly during heat stress as shown by experiments using a pSer326 phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type factor. CONCLUSION: Twelve Ser residues but no Thr or Tyr residues were identified that were phosphorylated in heat-activated HSF1. Mutagenesis experiments and functional studies suggested that phosphorylation of HSF1 residue Ser326 plays a critical role in the induction of the factor's transcriptional competence by heat stress. PhosphoSer326 also contributes to activation of HSF1 by chemical stress. To date, no functional role could be ascribed to any of the other newly identified phosphoSer residues.

DAXX interacts with heat shock factor 1 during stress activation and enhances its transcriptional activity.

DAXX, a modulator of apoptosis and a repressor of basal transcription, was identified in a two-hybrid screen as a protein capable of interacting with a trimeric form of human heat shock factor 1 (HSF1). In human cells, DAXX interacted with HSF1 essentially only during stress, i.e., when factor trimerization occurred. Several lines of experimentation suggested that DAXX is an important mediator of HSF1 activation: (i) overexpression of DAXX enhanced basal transactivation competence of HSF1 in the absence of a stress; (ii) a DAXX fragment exerted dominant-negative effects on HSF1 activation by different types of stress; (iii) induction of heat shock or stress protein (HSP)70 by heat stress was defective in a cell line lacking functional DAXX; and (iv) RNA interference depletion of DAXX also substantially reduced heat induction of HSF1 activity and HSP70 expression. HSF1 transactivation competence is repressed by an HSP90-containing multichaperone complex that interacts with trimeric factor. Overexpressed HSF1, known to be largely trimeric, only marginally increased HSF1 activity on its own but potentiated the activating effect of DAXX overexpression. Expression of a nonnative protein capable of competing for multichaperone complex also synergistically enhanced activation of HSF1 by DAXX. These observations suggest a model in which DAXX released from its nuclear stores during stress opposes repression of HSF1 transactivation competence by multichaperone complex through its interaction with trimerized HSF1. Our identification of DAXX as a mediator of HSF1 activation raises the question whether DAXX produces some of its pleiotropic effects through modulation of HSP levels.