H1 Linker Histone Gene Regulates Lifespan via Dietary Restriction Pathways in Caenorhabditis elegans

doi:10.12300/j.issn.1674-5817.2022.183

Abstract

Abstract:

Objective To reveal the physiological function of H1 linker histone gene (hil-1) and its molecular mechanism for regulating the lifespan in Caenorhabditis elegans (C. elegans). Methods C. elegans was used as a model organism and hil-1 gene was knock-down, knock-out and over-expressed via RNA interference technology, hil-1(gk229) mutants backcross purification and microinjection technology. Then the survival and oviposition of C. elegans were observed. Physiological tests including heat shock test, paraquat stress test and heavy metal Cr⁶⁺ stress test were conducted to evaluate the stress resistance of hil-1 mutants. After constructing a dual mutant nematode, real-time fluorescence quantitative PCR (RT-qPCR) was used to further identify the signaling pathways and target sites associated with hil-1 gene regulatory lifespan. Results Compared with wild-type N2 worms, the lifespan of C. elegans of RNA interference and hil-1(gk229) mutants were significantly shortened (P<0.001), while overexpression of hil-1 in the whole body increased lifespan (P<0.05). The tolerance of hil-1(gk229) mutants to heat stress and oxidative stress was significantly decreased (P<0.001, P<0.05), but the tolerance to heavy metals was not different compared to wild-type N2 worms (P>0.05). In addition, the developmental cycle of hil-1(gk229) mutants was shortened and the time of oviposition was advanced (P<0.001), but there was no significant change in total number of oviposition (P>0.05). After feeding hil-1 RNA interference bacteria to eat-2(ad465) mutants, the down-regulation of hil-1 expression did not affect the lifespan of eat-2(ad465) mutants (P>0.05). Compared with wild-type N2 worms, the expression level of daf-16 in hil-1(gk229) mutants was significantly down-regulated (P<0.001), and the expressions of downstream genes, mtl-1 and ctl-1, were also down-regulated (P<0.05, P<0.001). Compared with daf-2(e1370) mutants, the lifespan of daf-2 (e1370); hil-1(gk229) mutants did not shortened (P>0.05). Compared with daf-16(mu86) mutants, the lifespan of daf-16(mu86); hil-1(gk229) mutants was significantly shortened (P<0.001). The knockdown of hil-1via RNA interference technology, specifically in epidermis and intestine, was sufficient for lifespan reduction (P<0.001). Conclusion The deletion of hil-1 gene significantly shortened the lifespan of C. elegans and decreased the tolerance to heat and oxidative stress. The hil-1 gene regulates the lifespan of C. elegans via dietary restriction pathway and acts mostly in epidermis and intestine.

Key words: Caenorhabditis elegans, H1 linker histone gene (hil-1), Aging, Lifespan, Dietary restriction pathway

CLC Number:

Q95-33

Hui CHENG, Fei FANG, Jiahao SHI, Hua YANG, Mengjie ZHANG, Ping YANG, Jian FEI. H1 Linker Histone Gene Regulates Lifespan via Dietary Restriction Pathways in Caenorhabditis elegans[J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 271-281.

Figures/Tables 8

Table 1 Prime sequences for PCR

基因

Gene

引物序列

Sequences of primers

hil-1

Forward: 5’-AATGGAACCGGAAGCAGCAA-3’

Reverse: 5’-TCCGCGAGTCTGTTCAATGT-3’

daf-16

Forward: 5’- GCTCCCTTCACTCGACACTT-3’

Reverse: 5’- TTCGATTGAGTTCGGGGACG-3’

mtl-1

Forward: 5’- GCTTGCAAGTGTGACTGCAA-3’

Reverse: 5’- TTGATGGGTCTTGTCTCCGC-3’

ctl-1

Forward: 5’- TACCGAGGAGGGTAACTGGG-3’

Reverse: 5’- AGTCTGTGGATTGCGCTTCA-3’

act-4

Forward: 5’- GCCACCGCTGCCTCCTCATC-3’

Reverse: 5’- CCGGCAGACTCCATACCCAAGAAG-3’

Figure 1 hil-1 is involved in regulation of the lifespan in C. elegansNote：A, Survival curves of wild-type N2 worms fed hil-1 RNA interference (RNAi) bacteria [compared with control group (fed L4440), P<0.001]; B, Survival curves of wild-type N2 worms and hil-1(gk229) mutants (compared with N2, P<0.001); C, Survival curves between wild-type N2 nematode and hil-1 systemic overexpression worms (compared with N2 nematode, P<0.05).

Table 2 Analysis of experimental data of lifespan in C. elegans

线虫种类 C. elegans strains	线虫数量 Number	平均寿命 Mean lifespan /d
N2	94	22±0.23
hil-1(gk229)	95	18±0.67^***
Whole body hil-1 OE	101	22±0.22^*
daf-2(e1370)	101	47±0.46
daf-2(e1370);hil-1(gk229)	97	47±0.57
daf-16(mu86)	98	15±0.24
daf-16(mu86);hil-1(gk229)	82	14±0.26^▲▲▲
N2-L4440	85	17±0.23
N2-hil-1 RNAi	95	13±0.60^△△△
eat-2(ad465)-L4440	91	26±0.41
eat-2(ad465)-hil-1 RNAi	103	26±0.42
NR350-L4440	90	16±0.56
NR350-hil-1 RNAi	95	16±0.57
TU3311-L4440	101	13±0.28
TU3311-hil-1 RNAi	105	13±0.33
NR222-L4440	105	22±0.27
NR222-hil-1 RNAi	91	17±0.34^aaa
VP303-L4440	91	18±0.62
VP303-hil-1 RNAi	96	13±0.44^bbb
AMJ345-L4440	99	15±0.21
AMJ345-hil-1 RNAi	99	15±0.23^cc

Figure 2 The deletion of hil-1 reduces the tolerance to heat shock and oxidative stress of C. elegansNote：A， Survival curves of wild-type N2 worms and hil-1(gk229) mutant worms transferred from 20 °C to 35 °C, P<0.001; B, Survival curves of wild-type N2 worms and hil-1(gk229) mutant worms exposed to 50 mmol/L paraquat, *P<0.05; C, Survival curves of wild-type N2 worms and hil-1(gk229) mutant worms exposed to 10 mmol/L K2Cr2O7, P>0.05.

Figure 3 The deletion of hil-1 affects the development of C. elegansNote：A, Observing the morphology of eggs of wild-type N2 worms and hil-1(gk229) mutant worms at 65 h and 72 h of development under microscope, with magnification of 100X; B, The duration of wild-type N2 worms and hil-1(gk229) mutant worms from egg to the laying of the first egg, n =10 in each group, ***P<0.001; C, The average number of eggs production every 12 h from egg development in wild-type N2 worms and hil-1(gk229) mutant worms, n > 10 in each group; D, The total number of eggs in wild-type N2 worms and hil-1(gk229) mutant worms. n > 10 in each group, nsP>0.05.

Figure 4 The knockdown of hil-1 has no significant effect on the lifespan of eat-2（ad465）Note： Survival curves of eat-2（ad465） mutants fed hil-1 RNA interference （RNAi） bacteria. Compared with control group （fed L4440）， P>0.05.

Figure 5 hil-1 is not completely dependent on insulin signaling pathway to regulate lifespan of C. elegansNote：A, Survival curves of daf-2(e1370) mutant worms and daf-2(e1370);hil-1(gk229) mutant worms, P>0.05; B, Analysis of the expression levels of daf-16 gene in wild-type N2 worms and hil-1(gk229) mutant worms（Compared with wild-type N2 worms, ***P<0.001）; C, Analysis of the expression levels of daf-16 target genes mtl-1 and ctl-1 in wild-type N2 and hil-1(gk229) mutant worms（compared with wild-type N2 worms *P<0.05，***P<0.001）; D, Survival curves of daf-16(mu86) mutant worms and daf-16 (mu86); hil-1(gk229) mutant worms, P<0.001.

Figure 6 hil-1 acts in the hypodermis or intestine to regulate lifespanNote：A-D， Survival curves of worms with tissue-specific knockdown of hil-1in muscle（A）， neuron（B）， hypodermis（C） and intestine（D）， respectively. Lifespan is shortened only in hypodermis-specific RNA interference （RNAi） or intestine-specific RNAi worms.

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