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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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Alternative splicing of UNC-62 generates intestine-specific unc-62(7a) and neuronal/hypodermal-specific unc-62(7b) isoforms.(A) Strains expressing isoform-specific reporters for unc-62(7a) and unc-62(7b) show stage- and tissue-specific expression. (top) Beginning with the transgenic fosmid described in Figure 1C, stop codons were inserted into unc-62 exons 7a or 7b by site-directed mutagenesis to obtain isoform-specific reporters. During this process, a kanamycin resistance cassette was inserted into the eighth intron of unc-62. (bottom) Alternative isoforms of UNC-62 show tissue-specific expression in adults. (left) UNC-62:GFP is observed in intestinal, neuronal, and hypodermal cells. (center) UNC-62(7a):GFP is highly expressed in the intestine in the L4 larval stage and young adults, but is not visible in other tissues. (right) UNC-62(7b):GFP is not observed in the intestine, but is expressed in the hypodermis (not shown), the ventral nerve cord, and other neurons. Strains were imaged in a glo-4(ok623) background to limit gut autofluorescence. (B) UNC-62(7a):GFP expression decreases between day 1 and day 8 of adulthood. Strain contains glo-4(ok623) to reduce gut autofluorescence, and expression was quantified only in the first pair of intestinal nuclei (dotted red circles). (C) Quantification of UNC-62(7a):GFP as shown in (B). Bars indicate mean fluorescence (in arbitrary units) observed from populations of at least 32 worms measured at different days of adulthood, with error bars indicating standard error of the mean. Day 1 expression was significantly higher compared to expression in days 3, 8, or 12 (p<10−4 by Student's t-test) in two independent experiments (see Figure S2).
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pgen-1003325-g002: Alternative splicing of UNC-62 generates intestine-specific unc-62(7a) and neuronal/hypodermal-specific unc-62(7b) isoforms.(A) Strains expressing isoform-specific reporters for unc-62(7a) and unc-62(7b) show stage- and tissue-specific expression. (top) Beginning with the transgenic fosmid described in Figure 1C, stop codons were inserted into unc-62 exons 7a or 7b by site-directed mutagenesis to obtain isoform-specific reporters. During this process, a kanamycin resistance cassette was inserted into the eighth intron of unc-62. (bottom) Alternative isoforms of UNC-62 show tissue-specific expression in adults. (left) UNC-62:GFP is observed in intestinal, neuronal, and hypodermal cells. (center) UNC-62(7a):GFP is highly expressed in the intestine in the L4 larval stage and young adults, but is not visible in other tissues. (right) UNC-62(7b):GFP is not observed in the intestine, but is expressed in the hypodermis (not shown), the ventral nerve cord, and other neurons. Strains were imaged in a glo-4(ok623) background to limit gut autofluorescence. (B) UNC-62(7a):GFP expression decreases between day 1 and day 8 of adulthood. Strain contains glo-4(ok623) to reduce gut autofluorescence, and expression was quantified only in the first pair of intestinal nuclei (dotted red circles). (C) Quantification of UNC-62(7a):GFP as shown in (B). Bars indicate mean fluorescence (in arbitrary units) observed from populations of at least 32 worms measured at different days of adulthood, with error bars indicating standard error of the mean. Day 1 expression was significantly higher compared to expression in days 3, 8, or 12 (p<10−4 by Student's t-test) in two independent experiments (see Figure S2).

Mentions: Alternative RNA splicing of unc-62 produces isoforms that differ in usage of the seventh exon (7a and 7b), which encodes the N-terminal region of the UNC-62 TALE homeodomain [13]. Transcripts including exon 7a increase in abundance more than 100-fold between embryos and adults, whereas transcripts containing 7b are expressed roughly equivalently throughout development [13]. To query the tissue-specific expression of these isoforms, we generated UNC-62:GFP reporters that express only UNC-62(7a) or UNC-62(7b). These reporters were made by inserting a stop codon in exon 7b into the UNC-62:GFP translational reporter such that it can only express UNC-62(7a):GFP, or a stop codon in exon 7a such that the reporter can only express UNC-62(7b):GFP (Figure 2A). Strains stably expressing either of these isoform-specific GFP reporters were then generated by biolistic bombardment. Fluorescent imaging of these strains indicated that UNC-62(7a) was predominantly expressed in the intestine starting in L3 and continuing through adulthood. In contrast, UNC-62(7b) was expressed in neurons, the ventral nerve cord, vulval precursor cells, and hypodermis beginning in embryos and continuing through adulthood (Figure 2A).


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)

Alternative splicing of UNC-62 generates intestine-specific unc-62(7a) and neuronal/hypodermal-specific unc-62(7b) isoforms.(A) Strains expressing isoform-specific reporters for unc-62(7a) and unc-62(7b) show stage- and tissue-specific expression. (top) Beginning with the transgenic fosmid described in Figure 1C, stop codons were inserted into unc-62 exons 7a or 7b by site-directed mutagenesis to obtain isoform-specific reporters. During this process, a kanamycin resistance cassette was inserted into the eighth intron of unc-62. (bottom) Alternative isoforms of UNC-62 show tissue-specific expression in adults. (left) UNC-62:GFP is observed in intestinal, neuronal, and hypodermal cells. (center) UNC-62(7a):GFP is highly expressed in the intestine in the L4 larval stage and young adults, but is not visible in other tissues. (right) UNC-62(7b):GFP is not observed in the intestine, but is expressed in the hypodermis (not shown), the ventral nerve cord, and other neurons. Strains were imaged in a glo-4(ok623) background to limit gut autofluorescence. (B) UNC-62(7a):GFP expression decreases between day 1 and day 8 of adulthood. Strain contains glo-4(ok623) to reduce gut autofluorescence, and expression was quantified only in the first pair of intestinal nuclei (dotted red circles). (C) Quantification of UNC-62(7a):GFP as shown in (B). Bars indicate mean fluorescence (in arbitrary units) observed from populations of at least 32 worms measured at different days of adulthood, with error bars indicating standard error of the mean. Day 1 expression was significantly higher compared to expression in days 3, 8, or 12 (p<10−4 by Student's t-test) in two independent experiments (see Figure S2).
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pgen-1003325-g002: Alternative splicing of UNC-62 generates intestine-specific unc-62(7a) and neuronal/hypodermal-specific unc-62(7b) isoforms.(A) Strains expressing isoform-specific reporters for unc-62(7a) and unc-62(7b) show stage- and tissue-specific expression. (top) Beginning with the transgenic fosmid described in Figure 1C, stop codons were inserted into unc-62 exons 7a or 7b by site-directed mutagenesis to obtain isoform-specific reporters. During this process, a kanamycin resistance cassette was inserted into the eighth intron of unc-62. (bottom) Alternative isoforms of UNC-62 show tissue-specific expression in adults. (left) UNC-62:GFP is observed in intestinal, neuronal, and hypodermal cells. (center) UNC-62(7a):GFP is highly expressed in the intestine in the L4 larval stage and young adults, but is not visible in other tissues. (right) UNC-62(7b):GFP is not observed in the intestine, but is expressed in the hypodermis (not shown), the ventral nerve cord, and other neurons. Strains were imaged in a glo-4(ok623) background to limit gut autofluorescence. (B) UNC-62(7a):GFP expression decreases between day 1 and day 8 of adulthood. Strain contains glo-4(ok623) to reduce gut autofluorescence, and expression was quantified only in the first pair of intestinal nuclei (dotted red circles). (C) Quantification of UNC-62(7a):GFP as shown in (B). Bars indicate mean fluorescence (in arbitrary units) observed from populations of at least 32 worms measured at different days of adulthood, with error bars indicating standard error of the mean. Day 1 expression was significantly higher compared to expression in days 3, 8, or 12 (p<10−4 by Student's t-test) in two independent experiments (see Figure S2).
Mentions: Alternative RNA splicing of unc-62 produces isoforms that differ in usage of the seventh exon (7a and 7b), which encodes the N-terminal region of the UNC-62 TALE homeodomain [13]. Transcripts including exon 7a increase in abundance more than 100-fold between embryos and adults, whereas transcripts containing 7b are expressed roughly equivalently throughout development [13]. To query the tissue-specific expression of these isoforms, we generated UNC-62:GFP reporters that express only UNC-62(7a) or UNC-62(7b). These reporters were made by inserting a stop codon in exon 7b into the UNC-62:GFP translational reporter such that it can only express UNC-62(7a):GFP, or a stop codon in exon 7a such that the reporter can only express UNC-62(7b):GFP (Figure 2A). Strains stably expressing either of these isoform-specific GFP reporters were then generated by biolistic bombardment. Fluorescent imaging of these strains indicated that UNC-62(7a) was predominantly expressed in the intestine starting in L3 and continuing through adulthood. In contrast, UNC-62(7b) was expressed in neurons, the ventral nerve cord, vulval precursor cells, and hypodermis beginning in embryos and continuing through adulthood (Figure 2A).

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