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Roles of the developmental regulator unc-62/Homothorax in limiting longevity in Caenorhabditis elegans.

Van Nostrand EL, Sánchez-Blanco A, Wu B, Nguyen A, Kim SK - PLoS Genet. (2013)

Bottom Line: Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging.Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other.These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University Medical Center, Stanford, California, USA.

ABSTRACT
The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.

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ins-7 and other unc-62-dependent class N targets are activated in the old intestine.(A) Class N unc-62 targets tend to increase with age in the intestine. Eight class N targets (bound by UNC-62 in the young adult with neuronal-enriched expression in development (dark grey bars)), as well as ten control targets that have neuronal-enriched expression and are bound by UNC-62 in L3 stage but not the adult stages (light grey bars), were assayed for age-related changes in the intestine (between day 1 and day 15 adults). Expression in dissected intestines was quantified by qPCR as described in Methods using a beta-tubulin control (C36E8.5). Bars indicate the ratio of the expression between young and old as measured by the ΔΔCt method, and error bars indicate standard deviation of technical triplicate qPCR measurements. + indicates transcripts that were quantified in young but not detected in old intestines; for these transcripts, age-downregulation was calculated using a Ct value of 40 as an upper bound of old intestinal expression. (B) unc-62 activates four of five class N targets in old intestines. For the five UNC-62 adult targets that increased with age by more than two-fold, we analyzed expression in day 1, day 15 control, and day 15 unc-62 RNAi micro-dissected intestines (two biological replicate samples each). (Left) Schematics for these five genes indicates gene structures (in blue), as well as associated UNC-62 binding sites in young adult (red) ChIP-seq. All binding sites shown were factor-specific and significant at q-value 10−5 (**). (Right) Expression fold-change was calculated using a beta-tubulin control as before, and are shown relative to the average expression between young intestine replicates. For four of the five (tsp-1, max-1, ins-7, and T05B11.1), expression in old intestines was diminished when unc-62 was knocked down. unc-62 RNAi had no effect on stdh-1 expression in old intestines. Although the binding site overlapping the 3′ end of tsp-1 was associated with tsp-1 using our analysis pipeline, we found that tsp-2 showed a similar expression pattern in dissected intestines (Figure S10). (C) unc-62 RNAi activates activity of main insulin pathway target DAF-16/FOXO. We quantified expression of sod-3:GFP, which is a reporter of DAF-16 activity [40]. Approximately 25 worms were imaged for each aging timepoint (x-axis), and fluorescence in the head (y-axis) was quantified using ImageJ. Error bars indicate standard error of the mean. (D) unc-62 RNAi extends lifespan in wild-type worms (37% increase in mean lifespan, p<10−5), but not in daf-16(m26) mutants (3% increase, p>0.1). X-axis indicates days of adulthood, and the y-axis indicates percent of worms remaining alive. Similar results were observed in a replicate experiment using daf-16 RNAi (lifespan data in Table S2).
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pgen-1003325-g007: ins-7 and other unc-62-dependent class N targets are activated in the old intestine.(A) Class N unc-62 targets tend to increase with age in the intestine. Eight class N targets (bound by UNC-62 in the young adult with neuronal-enriched expression in development (dark grey bars)), as well as ten control targets that have neuronal-enriched expression and are bound by UNC-62 in L3 stage but not the adult stages (light grey bars), were assayed for age-related changes in the intestine (between day 1 and day 15 adults). Expression in dissected intestines was quantified by qPCR as described in Methods using a beta-tubulin control (C36E8.5). Bars indicate the ratio of the expression between young and old as measured by the ΔΔCt method, and error bars indicate standard deviation of technical triplicate qPCR measurements. + indicates transcripts that were quantified in young but not detected in old intestines; for these transcripts, age-downregulation was calculated using a Ct value of 40 as an upper bound of old intestinal expression. (B) unc-62 activates four of five class N targets in old intestines. For the five UNC-62 adult targets that increased with age by more than two-fold, we analyzed expression in day 1, day 15 control, and day 15 unc-62 RNAi micro-dissected intestines (two biological replicate samples each). (Left) Schematics for these five genes indicates gene structures (in blue), as well as associated UNC-62 binding sites in young adult (red) ChIP-seq. All binding sites shown were factor-specific and significant at q-value 10−5 (**). (Right) Expression fold-change was calculated using a beta-tubulin control as before, and are shown relative to the average expression between young intestine replicates. For four of the five (tsp-1, max-1, ins-7, and T05B11.1), expression in old intestines was diminished when unc-62 was knocked down. unc-62 RNAi had no effect on stdh-1 expression in old intestines. Although the binding site overlapping the 3′ end of tsp-1 was associated with tsp-1 using our analysis pipeline, we found that tsp-2 showed a similar expression pattern in dissected intestines (Figure S10). (C) unc-62 RNAi activates activity of main insulin pathway target DAF-16/FOXO. We quantified expression of sod-3:GFP, which is a reporter of DAF-16 activity [40]. Approximately 25 worms were imaged for each aging timepoint (x-axis), and fluorescence in the head (y-axis) was quantified using ImageJ. Error bars indicate standard error of the mean. (D) unc-62 RNAi extends lifespan in wild-type worms (37% increase in mean lifespan, p<10−5), but not in daf-16(m26) mutants (3% increase, p>0.1). X-axis indicates days of adulthood, and the y-axis indicates percent of worms remaining alive. Similar results were observed in a replicate experiment using daf-16 RNAi (lifespan data in Table S2).

Mentions: We observed that expression of the eight UNC-62 Class N young adult targets in dissected intestines showed a significant shift towards increased expression in old age (p = 0.013 by Wilcoxon rank-sum test)(Figure 7A). Specifically, five of the eight adult targets increased more than two-fold in expression with age (including one gene (ins-7) which has previous been shown to increase with age in the intestine [36]), whereas none of the ten control targets did so. To determine whether expression of class N genes was dependent on UNC-62 activity, we obtained RNA from micro-dissected intestines from old adults grown on unc-62 RNAi. We found that four (tsp-1, max-1, ins-7, and T05B11.1) of the five class N genes that increase expression by two-fold in intestines with age showed decreased expression upon unc-62 RNAi (Figure 7B). In summary, class N genes are an intriguing set of unc-62-dependent genes that are expressed predominantly in neuronal tissues in development, but then become expressed in the intestine in old adults.


Roles of the developmental regulator unc-62/Homothorax in limiting longevity in Caenorhabditis elegans.

Van Nostrand EL, Sánchez-Blanco A, Wu B, Nguyen A, Kim SK - PLoS Genet. (2013)

ins-7 and other unc-62-dependent class N targets are activated in the old intestine.(A) Class N unc-62 targets tend to increase with age in the intestine. Eight class N targets (bound by UNC-62 in the young adult with neuronal-enriched expression in development (dark grey bars)), as well as ten control targets that have neuronal-enriched expression and are bound by UNC-62 in L3 stage but not the adult stages (light grey bars), were assayed for age-related changes in the intestine (between day 1 and day 15 adults). Expression in dissected intestines was quantified by qPCR as described in Methods using a beta-tubulin control (C36E8.5). Bars indicate the ratio of the expression between young and old as measured by the ΔΔCt method, and error bars indicate standard deviation of technical triplicate qPCR measurements. + indicates transcripts that were quantified in young but not detected in old intestines; for these transcripts, age-downregulation was calculated using a Ct value of 40 as an upper bound of old intestinal expression. (B) unc-62 activates four of five class N targets in old intestines. For the five UNC-62 adult targets that increased with age by more than two-fold, we analyzed expression in day 1, day 15 control, and day 15 unc-62 RNAi micro-dissected intestines (two biological replicate samples each). (Left) Schematics for these five genes indicates gene structures (in blue), as well as associated UNC-62 binding sites in young adult (red) ChIP-seq. All binding sites shown were factor-specific and significant at q-value 10−5 (**). (Right) Expression fold-change was calculated using a beta-tubulin control as before, and are shown relative to the average expression between young intestine replicates. For four of the five (tsp-1, max-1, ins-7, and T05B11.1), expression in old intestines was diminished when unc-62 was knocked down. unc-62 RNAi had no effect on stdh-1 expression in old intestines. Although the binding site overlapping the 3′ end of tsp-1 was associated with tsp-1 using our analysis pipeline, we found that tsp-2 showed a similar expression pattern in dissected intestines (Figure S10). (C) unc-62 RNAi activates activity of main insulin pathway target DAF-16/FOXO. We quantified expression of sod-3:GFP, which is a reporter of DAF-16 activity [40]. Approximately 25 worms were imaged for each aging timepoint (x-axis), and fluorescence in the head (y-axis) was quantified using ImageJ. Error bars indicate standard error of the mean. (D) unc-62 RNAi extends lifespan in wild-type worms (37% increase in mean lifespan, p<10−5), but not in daf-16(m26) mutants (3% increase, p>0.1). X-axis indicates days of adulthood, and the y-axis indicates percent of worms remaining alive. Similar results were observed in a replicate experiment using daf-16 RNAi (lifespan data in Table S2).
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pgen-1003325-g007: ins-7 and other unc-62-dependent class N targets are activated in the old intestine.(A) Class N unc-62 targets tend to increase with age in the intestine. Eight class N targets (bound by UNC-62 in the young adult with neuronal-enriched expression in development (dark grey bars)), as well as ten control targets that have neuronal-enriched expression and are bound by UNC-62 in L3 stage but not the adult stages (light grey bars), were assayed for age-related changes in the intestine (between day 1 and day 15 adults). Expression in dissected intestines was quantified by qPCR as described in Methods using a beta-tubulin control (C36E8.5). Bars indicate the ratio of the expression between young and old as measured by the ΔΔCt method, and error bars indicate standard deviation of technical triplicate qPCR measurements. + indicates transcripts that were quantified in young but not detected in old intestines; for these transcripts, age-downregulation was calculated using a Ct value of 40 as an upper bound of old intestinal expression. (B) unc-62 activates four of five class N targets in old intestines. For the five UNC-62 adult targets that increased with age by more than two-fold, we analyzed expression in day 1, day 15 control, and day 15 unc-62 RNAi micro-dissected intestines (two biological replicate samples each). (Left) Schematics for these five genes indicates gene structures (in blue), as well as associated UNC-62 binding sites in young adult (red) ChIP-seq. All binding sites shown were factor-specific and significant at q-value 10−5 (**). (Right) Expression fold-change was calculated using a beta-tubulin control as before, and are shown relative to the average expression between young intestine replicates. For four of the five (tsp-1, max-1, ins-7, and T05B11.1), expression in old intestines was diminished when unc-62 was knocked down. unc-62 RNAi had no effect on stdh-1 expression in old intestines. Although the binding site overlapping the 3′ end of tsp-1 was associated with tsp-1 using our analysis pipeline, we found that tsp-2 showed a similar expression pattern in dissected intestines (Figure S10). (C) unc-62 RNAi activates activity of main insulin pathway target DAF-16/FOXO. We quantified expression of sod-3:GFP, which is a reporter of DAF-16 activity [40]. Approximately 25 worms were imaged for each aging timepoint (x-axis), and fluorescence in the head (y-axis) was quantified using ImageJ. Error bars indicate standard error of the mean. (D) unc-62 RNAi extends lifespan in wild-type worms (37% increase in mean lifespan, p<10−5), but not in daf-16(m26) mutants (3% increase, p>0.1). X-axis indicates days of adulthood, and the y-axis indicates percent of worms remaining alive. Similar results were observed in a replicate experiment using daf-16 RNAi (lifespan data in Table S2).
Mentions: We observed that expression of the eight UNC-62 Class N young adult targets in dissected intestines showed a significant shift towards increased expression in old age (p = 0.013 by Wilcoxon rank-sum test)(Figure 7A). Specifically, five of the eight adult targets increased more than two-fold in expression with age (including one gene (ins-7) which has previous been shown to increase with age in the intestine [36]), whereas none of the ten control targets did so. To determine whether expression of class N genes was dependent on UNC-62 activity, we obtained RNA from micro-dissected intestines from old adults grown on unc-62 RNAi. We found that four (tsp-1, max-1, ins-7, and T05B11.1) of the five class N genes that increase expression by two-fold in intestines with age showed decreased expression upon unc-62 RNAi (Figure 7B). In summary, class N genes are an intriguing set of unc-62-dependent genes that are expressed predominantly in neuronal tissues in development, but then become expressed in the intestine in old adults.

Bottom Line: Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging.Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other.These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University Medical Center, Stanford, California, USA.

ABSTRACT
The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.

Show MeSH