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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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UNC-62 binds to and activates expression of all six C. elegans yolk protein genes.(A) The thick and thin blue lines indicate the exon and intron structure of each of the six vitellogenin loci. Red boxes indicate the position of regions significantly enriched in UNC-62 ChIP-seq of day 4 adult (YA) worms. (*) indicates q-value<0.001 and (**) indicates q<10−5 binding sites. Below each gene, RNA-seq results are displayed for worms fed either unc-62 RNAi or control bacteria. Bars indicate the mean, and error bars the standard deviation, of sequencing read density (reads per million mapped reads) for vitellogenin genes in triplicate RNA-seq experiments. (B) Bars indicate mean, and error bars indicate standard deviation, of measured fluorescence of a vit-2:GFP reporter under various RNAi conditions. Expression in ∼15 worms was quantified using ImageJ. (C) (top) A de novo motif search in UNC-62 young adult binding sites with RSAT [58] identifies a TGATTGACA motif as the prominent sequence motif. (bottom) This motif is similar to the ATTGACA VPE-1 vitellogenin regulatory motif previously described [30]. (D) All six vitellogenin genes decrease expression with age (as assayed by whole-worm microarrays of 2, 5, 8, and 11 day old adult worms [10]). Bars indicate mean expression observed across replicated arrays, with error bars indicating standard deviation. (E) UNC-62 activates vitellogenin expression in old adults. Bars indicate expression of two biological replicates of vit-2 (black) and vit-6 (grey), determined by qRT-PCR of RNA isolated from ∼100 dissected intestines of day 1 adults and day 15 adults fed either control or unc-62 RNAi bacteria. Error bars indicate standard deviation of triplicate qPCR technical replicates. Statistical significance is not indicated as this experiment included only two biological replicates.
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pgen-1003325-g005: UNC-62 binds to and activates expression of all six C. elegans yolk protein genes.(A) The thick and thin blue lines indicate the exon and intron structure of each of the six vitellogenin loci. Red boxes indicate the position of regions significantly enriched in UNC-62 ChIP-seq of day 4 adult (YA) worms. (*) indicates q-value<0.001 and (**) indicates q<10−5 binding sites. Below each gene, RNA-seq results are displayed for worms fed either unc-62 RNAi or control bacteria. Bars indicate the mean, and error bars the standard deviation, of sequencing read density (reads per million mapped reads) for vitellogenin genes in triplicate RNA-seq experiments. (B) Bars indicate mean, and error bars indicate standard deviation, of measured fluorescence of a vit-2:GFP reporter under various RNAi conditions. Expression in ∼15 worms was quantified using ImageJ. (C) (top) A de novo motif search in UNC-62 young adult binding sites with RSAT [58] identifies a TGATTGACA motif as the prominent sequence motif. (bottom) This motif is similar to the ATTGACA VPE-1 vitellogenin regulatory motif previously described [30]. (D) All six vitellogenin genes decrease expression with age (as assayed by whole-worm microarrays of 2, 5, 8, and 11 day old adult worms [10]). Bars indicate mean expression observed across replicated arrays, with error bars indicating standard deviation. (E) UNC-62 activates vitellogenin expression in old adults. Bars indicate expression of two biological replicates of vit-2 (black) and vit-6 (grey), determined by qRT-PCR of RNA isolated from ∼100 dissected intestines of day 1 adults and day 15 adults fed either control or unc-62 RNAi bacteria. Error bars indicate standard deviation of triplicate qPCR technical replicates. Statistical significance is not indicated as this experiment included only two biological replicates.

Mentions: We found that three of the eight genes that are bound and activated by UNC-62 are vitellogenin genes. C. elegans contains 6 vitellogenin transcripts (vit-1 through vit-6) that encode the major yolk proteins [28]. These transcripts are specifically expressed in the intestine during the reproductive period, and vitellogenin proteins are exported out of the intestine into oocytes [29]. RNA-seq experiments indicated that expression of all six vitellogenin transcripts is strongly dependent on unc-62 activity, as there is a 4–10-fold decline in expression for vit-2, vit-3, vit-4, vit-5 and vit-6 and a ∼100-fold decline for vit-1 in unc-62 RNAi worms as compared to controls (Figure 5A). unc-62 RNAi also results in a strong decrease in the level of expression of a VIT-2:GFP translational reporter, indicating that protein as well as mRNA levels of vit-2 are dependent upon UNC-62 activity (Figure 5B). RNAi targeting exon 7a of unc-62 results in a decrease of VIT-2:GFP expression, whereas RNAi targeting exon 7b does not, indicating that the unc-62(7a) isoform activates vitellogenin expression (Figure 5B).


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)

UNC-62 binds to and activates expression of all six C. elegans yolk protein genes.(A) The thick and thin blue lines indicate the exon and intron structure of each of the six vitellogenin loci. Red boxes indicate the position of regions significantly enriched in UNC-62 ChIP-seq of day 4 adult (YA) worms. (*) indicates q-value<0.001 and (**) indicates q<10−5 binding sites. Below each gene, RNA-seq results are displayed for worms fed either unc-62 RNAi or control bacteria. Bars indicate the mean, and error bars the standard deviation, of sequencing read density (reads per million mapped reads) for vitellogenin genes in triplicate RNA-seq experiments. (B) Bars indicate mean, and error bars indicate standard deviation, of measured fluorescence of a vit-2:GFP reporter under various RNAi conditions. Expression in ∼15 worms was quantified using ImageJ. (C) (top) A de novo motif search in UNC-62 young adult binding sites with RSAT [58] identifies a TGATTGACA motif as the prominent sequence motif. (bottom) This motif is similar to the ATTGACA VPE-1 vitellogenin regulatory motif previously described [30]. (D) All six vitellogenin genes decrease expression with age (as assayed by whole-worm microarrays of 2, 5, 8, and 11 day old adult worms [10]). Bars indicate mean expression observed across replicated arrays, with error bars indicating standard deviation. (E) UNC-62 activates vitellogenin expression in old adults. Bars indicate expression of two biological replicates of vit-2 (black) and vit-6 (grey), determined by qRT-PCR of RNA isolated from ∼100 dissected intestines of day 1 adults and day 15 adults fed either control or unc-62 RNAi bacteria. Error bars indicate standard deviation of triplicate qPCR technical replicates. Statistical significance is not indicated as this experiment included only two biological replicates.
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pgen-1003325-g005: UNC-62 binds to and activates expression of all six C. elegans yolk protein genes.(A) The thick and thin blue lines indicate the exon and intron structure of each of the six vitellogenin loci. Red boxes indicate the position of regions significantly enriched in UNC-62 ChIP-seq of day 4 adult (YA) worms. (*) indicates q-value<0.001 and (**) indicates q<10−5 binding sites. Below each gene, RNA-seq results are displayed for worms fed either unc-62 RNAi or control bacteria. Bars indicate the mean, and error bars the standard deviation, of sequencing read density (reads per million mapped reads) for vitellogenin genes in triplicate RNA-seq experiments. (B) Bars indicate mean, and error bars indicate standard deviation, of measured fluorescence of a vit-2:GFP reporter under various RNAi conditions. Expression in ∼15 worms was quantified using ImageJ. (C) (top) A de novo motif search in UNC-62 young adult binding sites with RSAT [58] identifies a TGATTGACA motif as the prominent sequence motif. (bottom) This motif is similar to the ATTGACA VPE-1 vitellogenin regulatory motif previously described [30]. (D) All six vitellogenin genes decrease expression with age (as assayed by whole-worm microarrays of 2, 5, 8, and 11 day old adult worms [10]). Bars indicate mean expression observed across replicated arrays, with error bars indicating standard deviation. (E) UNC-62 activates vitellogenin expression in old adults. Bars indicate expression of two biological replicates of vit-2 (black) and vit-6 (grey), determined by qRT-PCR of RNA isolated from ∼100 dissected intestines of day 1 adults and day 15 adults fed either control or unc-62 RNAi bacteria. Error bars indicate standard deviation of triplicate qPCR technical replicates. Statistical significance is not indicated as this experiment included only two biological replicates.
Mentions: We found that three of the eight genes that are bound and activated by UNC-62 are vitellogenin genes. C. elegans contains 6 vitellogenin transcripts (vit-1 through vit-6) that encode the major yolk proteins [28]. These transcripts are specifically expressed in the intestine during the reproductive period, and vitellogenin proteins are exported out of the intestine into oocytes [29]. RNA-seq experiments indicated that expression of all six vitellogenin transcripts is strongly dependent on unc-62 activity, as there is a 4–10-fold decline in expression for vit-2, vit-3, vit-4, vit-5 and vit-6 and a ∼100-fold decline for vit-1 in unc-62 RNAi worms as compared to controls (Figure 5A). unc-62 RNAi also results in a strong decrease in the level of expression of a VIT-2:GFP translational reporter, indicating that protein as well as mRNA levels of vit-2 are dependent upon UNC-62 activity (Figure 5B). RNAi targeting exon 7a of unc-62 results in a decrease of VIT-2:GFP expression, whereas RNAi targeting exon 7b does not, indicating that the unc-62(7a) isoform activates vitellogenin expression (Figure 5B).

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
Related in: MedlinePlus