Roles of HSB-1 in Regulation of Heat Shock Factor Activity, Histone Levels, Mitochondrial Function and Longevity

Signaling pathways that are involved in stress resistance have often been implicated in the regulation of the rate of aging in normal physiological conditions. The evolutionarily conserved transcription factor heat shock factor 1 (HSF-1) provides protection to animals from a multitude of environmental stresses. In addition, overexpression of HSF 1 promotes longevity in the nematode worm Caenorhabditis elegans also in non stressed conditions. Previous studies have shown that a negative regulator of HSF 1, termed as heat shock factor binding protein 1 (HSB-1), physically binds to HSF 1 and limits its transactivation potential. Genetic ablation of hsb-1 induces a robust hsf-1-dependent life span extension in worms via mechanisms that are less understood. In this study, we show that ablation of hsb-1 results in an altered transactivation status of the HSF-1 protein. In hsb-1 null animals, HSF-1 shows increased binding to its genomic target sequences. However, the transcriptome of hsb-1 null animals is largely distinct from that of HSF-1 overexpressing worms. While HSF-1 overexpression induces large-scale transcriptional upregulation in C. elegans, HSB-1 inhibition alters the expression of a much smaller number of genes, but still produces a similar life span extension as in HSF-1 overexpressing worms. Roughly half of the differentially regulated transcriptome in hsb-1 null animals overlaps with that of worms overexpressing HSF-1, and these genes show a strongly correlated expression pattern in the two long-lived strains. Genes that are upregulated via both HSB-1 inhibition and HSF-1 overexpression include many longevity-promoting transcriptional targets of the C. elegans FOXO homolog DAF-16. Overall, this suggests that HSB-1 acts as a selective regulator of HSF-1 transactivation potential and hence, inhibition of HSB-1 results in change in expression of a subset of HSF-1 target genes that are potentially involved in longevity determination in animals. In addition to the characterization of transcriptional changes associated with HSB-1 inhibition in worms, we also performed an unbiased RNAi-based screen to identify genetic suppressors of HSB-1 associated longevity. We found that knockdown of several histone H4-coding genes can completely suppress the life span extension phenotype in hsb-1 null animals. Worms lacking HSB-1 have elevated H4 protein levels in somatic tissues during development, while H4 overexpression in wild-type worms is sufficient to extend their life span. In hsb-1 null animals, elevated H4 levels induce reduced MNase-accessibility of both nuclear chromatin and mitochondrial DNA (mtDNA). This results in an H4-dependent reduction in expression of mtDNA-encoded genes and lower respiratory capacity in hsb-1 null worms, which leads to a mitochondrial unfolded protein response (UPRmt)-dependent life span extension. We further show that extranuclear histone H4 is present in mitochondria of intestinal tissue in C. elegans. This suggests a novel and unexpected role of histone H4 in regulating mitochondrial gene expression via directly modulating the accessibility of mtDNA. Moreover, our findings show interplay between three distinct cellular processes – stress resistance, chromatin dynamics and mitochondrial function – that were previously known to promote longevity in animals via seemingly independent mechanisms. In summary, this study has identified several novel HSF-1-mediated signaling interactions in C. elegans that have significantly broadened our understanding of how HSB-1/HSF-1 pathway regulates organismal life span in normal physiological conditions. Our findings will potentially guide the design of targeted therapies to selectively modulate the function of HSF-1 in order to delay the progression of aging and age-related diseases in metazoans.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/151726/1/ssural_1.pdfDescription of ssural_1.pdf : Restricted to UM users only

N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells

Abstract Background The DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations. Methods Immunoblot and comet assays were performed to detect DNA breaks and activation of the canonical checkpoint signalling kinases following NEU damage upto 2 hours. To investigate whether mismatch repair played a role in checkpoint activation, knock-down studies were performed while flow cytometry analysis was done to understand whether the activation of the checkpoint kinases was cell cycle phase specific. Finally, breast epithelial cells were grown as 3-dimensional spheroid cultures to study whether NEU can induce upregulation of vimentin as well as disrupt cell polarity of the breast acini, thus causing transformation of epithelial cells in culture. Results We report a novel finding that NEU causes activation of major checkpoint signalling kinases, Chk1 and Chk2. This activation is temporally controlled with Chk2 activation preceding Chk1 phosphorylation, and absence of cross talk between the two parallel signalling pathways, ATM and ATR. Damage caused by NEU leads to the temporal formation of both double strand and single strand breaks. Activation of checkpoints following NEU damage is cell cycle phase dependent wherein Chk2 is primarily activated during G2-M phase whilst in S phase, there is immediate Chk1 phosphorylation and delayed Chk2 response. Surprisingly, the mismatch repair system does not play a role in checkpoint activation, at doses and duration of NEU used in the experiments. Interestingly, NEU caused disruption of the well-formed polarised spheroid archithecture and upregulation of vimentin in three-dimensional breast acini cultures of non-malignant breast epithelial cells upon NEU treatment indicating NEU to have the potential to cause early transformation in the cells. Conclusion NEU causes damage in mammalian cells in the form of double strand and single strand breaks that temporally activate the major checkpoint signalling kinases without the occurrence of cross-talk between the pathways. NEU also appear to cause transformation in three-dimensional spheroid cultures.http://deepblue.lib.umich.edu/bitstream/2027.42/109493/1/12885_2013_Article_4466.pd

Co-chaperone p23 Regulates C. elegans Lifespan in Response to Temperature

Temperature potently modulates various physiologic processes including organismal motility, growth rate, reproduction, and ageing. In ectotherms, longevity varies inversely with temperature, with animals living shorter at higher temperatures. Thermal effects on lifespan and other processes are ascribed to passive changes in metabolic rate, but recent evidence also suggests a regulated process. Here, we demonstrate that in response to temperature, daf-41/ZC395.10, the C. elegans homolog of p23 co-chaperone/prostaglandin E synthase-3, governs entry into the long-lived dauer diapause and regulates adult lifespan. daf-41 deletion triggers constitutive entry into the dauer diapause at elevated temperature dependent on neurosensory machinery (daf-10/IFT122), insulin/IGF-1 signaling (daf-16/FOXO), and steroidal signaling (daf-12/FXR). Surprisingly, daf-41 mutation alters the longevity response to temperature, living longer than wild-type at 25 degrees C but shorter than wild-type at 15 degrees C. Longevity phenotypes at 25 degrees C work through daf-16/FOXO and heat shock factor hsf-1, while short lived phenotypes converge on daf-16/FOXO and depend on the daf-12/FXR steroid receptor. Correlatively daf-41 affected expression of DAF-16 and HSF-1 target genes at high temperature, and nuclear extracts from daf-41 animals showed increased occupancy of the heat shock response element. Our studies suggest that daf-41/p23 modulates key transcriptional changes in longevity pathways in response to temperature

HSB-1 Inhibition and HSF-1 Overexpression Trigger Overlapping Transcriptional Changes To Promote Longevity in Caenorhabditis elegans

Heat shock factor 1 (HSF-1) is a component of the heat shock response pathway that is induced by cytoplasmic proteotoxic stress. In addition to its role in stress response, HSF-1 also acts as a key regulator of the rate of organismal aging. Overexpression of HSF-1 promotes longevity in C. elegans via mechanisms that remain less understood. Moreover, genetic ablation of a negative regulator of HSF-1, termed as heat shock factor binding protein 1 (HSB-1), results in hsf-1-dependent life span extension in animals. Here we show that in the absence of HSB-1, HSF-1 acquires increased DNA binding activity to its genomic target sequence. Using RNA-Seq to compare the gene expression profiles of the hsb-1 mutant and hsf-1 overexpression strains, we found that while more than 1,500 transcripts show ≥1.5-fold upregulation due to HSF-1 overexpression, HSB-1 inhibition alters the expression of less than 500 genes in C. elegans. Roughly half of the differentially regulated transcripts in the hsb-1 mutant have altered expression also in hsf-1 overexpressing animals, with a strongly correlated fold-expression pattern between the two strains. In addition, genes that are upregulated via both HSB-1 inhibition and HSF-1 overexpression include numerous DAF-16 targets that have known functions in longevity regulation. This study identifies how HSB-1 acts as a specific regulator of the transactivation potential of HSF-1 in non-stressed conditions, thus providing a detailed understanding of the role of HSB-1/HSF-1 signaling pathway in transcriptional regulation and longevity in C. elegans

<i>daf-41</i> longevity is dependent on <i>daf-16</i>/FOXO.

<p>(A) <i>daf-16(mgDf50)</i> equally reduced the lifespan of <i>daf-41(ok3052)</i> and N2 worms at 20°C. (B) <i>daf-16(mgDf50)</i> abolished <i>daf-41</i> longevity at 25°C. (C) <i>daf-16(mgDf50)</i> did not further reduce the short life span of <i>daf-41(ok3052)</i> worms at 15°C. (D) Mean lifespan from 3 individual experiments were plotted for the indicated genotypes. Error bars; S.D.; **,p<0.01 versus N2; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (E) DAF-16 target genes, <i>sod-3</i>, <i>dod-3</i> and <i>lipl-4</i>, were significantly upregulated in response to warm temperature in <i>daf-41(ok3052)</i> relative to N2. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus <i>daf-41(ok3052)</i> of 20°C by t-test.</p

Ageing data of daf-41 mutants.

<p>Mean, median and maximum lifespan are shown</p><p>*, p<0.05</p><p>**, p<0.01 versus N2,</p><p>^†, p<0.05</p><p>^††, p<0.01 versus daf-41(ok3052),</p><p>^¶, p<0.05</p><p>^¶¶, p<0.01 versus 20°C by t-test.</p><p>Ageing data of daf-41 mutants.</p

<i>daf-41</i> partially interacts with the chemosensory and thermosensory apparatus to regulate longevity.

<p>(A) Mean lifespan of 3 individual experiments were plotted. The triple mutant of <i>gcy-23(nj37) gcy-8(oy44) gcy-18(nj38) (gcy</i> triple) caused a parallel reduction of lifespan in N2 and <i>daf-41(ok3052)</i>, respectively at 25°C. The <i>gcy</i> triple mutant did not further shorten the life span of daf<i>-41(ok3052)</i> at 15°C. (B) <i>daf-10</i> mutation increased lifespan in parallel to <i>daf-41</i> at 15°C and 20°C. <i>daf-10</i> mutation did not further extend the life span of <i>daf-41(ok3052)</i> worms at 25°C. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (C) A schematic model describing the regulatory mechanism of longevity by <i>daf-41</i> at different temperatures. At 25°C, <i>daf-41</i> negatively regulates the transcriptional activities of DAF-16 and HSF-1 and their down-regulation results in normal life span. Thermotaxis and steroidal signaling may regulate longevity in parallel to <i>daf-41</i>. At 15°C, <i>daf-41</i> (+) contributes to longevity possibly via <i>daf-16/</i>FOXO. <i>daf-41(+)</i> may also prevent life shortening activities of <i>daf-12(+)</i>, while <i>hsf-1</i> may promote longevity in parallel. These are working models that we interpret with caution, and may reflect direct or indirect interactions.</p

<i>daf-41(ok3052)</i> longevity is <i>hsf-1</i> dependent.

<p>(A) <i>hsf-1(sy441)</i> shortened both N2 and <i>daf-41(ok3052)</i> life span at 20°C. (B) <i>hsf-1(sy441)</i> abolished <i>daf-41(ok3052)</i> longevity at 25°C. (C) <i>hsf-1(sy441)</i> further reduced <i>daf-41(ok3052)</i> short life span at 15°C. (D) Mean lifespan from of 3 individual experiments were plotted for indicated genotypes and conditions. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (E) <i>daf-41(ok3052)</i> enhanced the upregulation of HSF-1 target genes, <i>hsp-16</i>.<i>2</i>, <i>hsp-4</i>, and <i>hsp-70</i>, in response to warm temperature. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus <i>daf-41(ok3052)</i> of 20°C by t-test. (F) HSF-1 binding activity to HSE was 1.5 fold increased in <i>daf-41(ok3052)</i> at 25°C. Error bars, S.E.M; **, p<0.01 versus N2. (G) At 15°C, <i>daf-21(p673)</i> mutation enhanced the longevity of N2 and <i>daf-41(ok3052)</i>. At 20°C, <i>daf-21</i> mutant animals lived slightly longer than N2. At 25°C, <i>daf-21(p673)</i> animals lived slightly longer than WT but the mutation reduced longevity in the <i>daf-41(ok3052)</i> background.</p

<i>daf-41</i>/ZC395.10 regulates dauer formation and stress resistance.

<p>(A) An alignment of protein sequences between <i>C</i>. <i>elegans</i> DAF-41, <i>D</i>. <i>melanogaster</i> CG16817 and <i>Homo sapiens</i> p23<i>/PTGES3</i>. The similarity between DAF-41 and p23<i>/PTGES3</i> is 44.6%. (B) Schematic illustration of the <i>daf-41</i>, and deletion alleles of <i>ok3015</i> and <i>ok3052</i>. Black arrows indicate the direction of transcription. Red area indicates HSP20-like co-chaperone domain. (C) <i>daf-41</i> mutants constitutively formed dauer larvae (Daf-c) weakly at 25°C and strongly at 27°C. (D) Dauer alae of <i>daf-41(ok3052)</i> animals grown at 27°C are indicated by the white arrows. (E) <i>daf-41(ok3052)</i> worms were resistant for oxidative stress (20mM of H₂O_2, 2.5 hrs) and heat stress (35°C, 8 hrs). <i>gst-4(ok2358)</i> worms were also slightly stress tolerant. (F) <i>daf-41p</i>::<i>gfp</i> (i.e. <i>dpy-5(e907); sEx10796 [rCes daf-41</i>::<i>gfp + pCeh361])</i> worms were labeled with DiI and photos taken at the young adult stage. Patterns of gene expression of <i>daf-41p</i>::<i>gfp</i> (green), DiI (red), and merged figures are shown, with arrows indicating individual neurons.</p

Genetic interactions of <i>daf-41</i> with dauer signaling pathways.

<p>(A) <i>daf-41(ok3052)</i> Daf-c phenotypes were partially suppressed in <i>af-16(mgDf50)</i> and completely suppressed in <i>daf-12(rh61rh411)</i> backgrounds. (B) <i>daf-41(ok3052)</i> Daf-c phenotypes were suppressed in various chemotaxis mutant backgrounds. (C-D) <i>daf-21(p673)</i> had no additive effect on Daf-c phenotypes at 25°C, and modestly reduced dauer formation at 27°C in the <i>daf-41(ok3052)</i> background. (E) <i>daf-11(m47)</i> had no additive effect on dauer formation in the <i>daf-41(ok3052)</i> background at 22.5°C. (F) <i>hsf-1(sy441)</i> strongly enhanced dauer formation of <i>daf-41(ok3052)</i> at 22.5°C. (G) Cultures of <i>daf-41(ok3052)</i>, <i>hsf-1(sy441) and daf-41;hsf-1</i> are shown grown at 22.5°C. White arrows indicate dauer larvae. All error bars indicate S.D. *, p<0.05; **, p<0.01 versus <i>daf-41(ok3052);</i> ††, p<0.01 versus <i>daf-21(p673)A</i> or <i>hsf-1(sy441)</i> by t-test. (H) <i>daf-41</i> regulates dauer formation via <i>daf-10</i>, <i>daf-12</i> and <i>daf-16</i> similar to <i>daf-21</i>. However, <i>hsf-1</i> suppresses dauer formation in <i>daf-21</i> but not <i>daf-41</i>. Note that the model reflects genetic interactions, not necessarily direct biochemical interactions.</p