Sirtuins (sir2-like proteins) belong to the family of class III
NAD+-dependent histone deacetylases. Among them, SIRT1 is the most
studied sirtuin. It plays a key role in many biological processes in the
liver, adipose tissue, muscle, pancreas, testis, ovary and mammary gland. It
has an important function in endocrine signaling, with a specific role in
glucose and fat metabolism. To date, in bovine species, only few SIRT1 single
nucleotide polymorphisms (SNPs) have been reported in the literature. Thus,
the aims of the present study were to estimate the allele and genotype
frequencies at the g.-274C > G locus in the promoter region of the SIRT1
gene and to investigate, for the first time, the relationship among
different genotypes and milk production and some reproduction traits in a
sample of cows belonging to the Agerolese breed. All the animals were
genotyped at the abovementioned locus using the PCR-RFLP technique. The investigated population was found to be polymorphic
at the investigated locus. Concerning milk production performances,
significant differences between genotypes were found in daily milk yield
(DMY), fat percentage (FC), lactation length (LL), peak daily milk yield (PY)
and 305-day milk yield (305MY). Moreover, the effect of the g.-274C > G
genotype on age at first calving and calving period was significant. In
conclusion, our findings are promising and should encourage scientists to
further investigate the effect of genetic polymorphism of sirtuins on milk
performance and reproductive traits.
Introduction
Sirtuins (sir2-like proteins) are mammalian homologs of the silent
information regulator 2 (sir2) gene of Saccharomyces cerevisiae and they belong to the family of
class III NAD+-dependent histone deacetylases (HDAC III) (Frye, 1999;
North and Verdin, 2004).
The mammalian sirtuin family consists of seven members (SIRT1-7). Each
sirtuin is characterized by a conserved 275 amino acid catalytic core domain
and peculiar N-terminal and/or C-terminal sequences of variable length
(Michan and Sinclair, 2007). Mammalian sirtuins also differ in their
subcellular localization. SIRT1, SIRT6 and SIRT7 are predominately in the
nucleus, but SIRT1 also shows important cytoplasmic functions. SIRT2 resides
most prominently in the cytoplasm, while SIRT3-5 are mitochondrial sirtuins
(Michan and Sinclair, 2007; Michishita et al., 2005; North et al., 2003;
Vaziri et al., 2001).
Sirtuins shows a gene silencing activity by deacetylating nucleosomes to
form heterochromatin; moreover, they act as sensors of the cellular energy
status, establishing a link between gene silencing and metabolism in vivo
(Braunstein et al., 1993; Denu, 2003).
SIRT1, the most investigated sirtuin, shows a key role in many biological
processes in the liver, adipose tissue, muscle, pancreas, testis, ovary and
mammary gland to regulate gene transcription, DNA repair, genome stability,
cell proliferation, cell survival and apoptosis, and energy metabolism. SIRT1
interacts with several transcription factors in accordance with its role in
gene silencing and heterochromatin formation (Vaquero et al., 2004; Shoba et
al., 2009; Revollo and Li, 2013). It has an important function in endocrine
signaling, specifically in the glucose and fat metabolism inducing the
transcription of several genes involved in metabolism (Li et al., 2013a). In
fact, SIRT1 promotes the expression of gluconeogenic genes in the liver,
fatty acid oxidation in muscle and fat mobilization in adipose tissue (Picard
et al., 2004; Gerhart-Hines et al., 2007; Rodgers and Puigserver, 2007).
Moreover, SIRT1 is expressed in the mammary gland where it modulates the
efficacy of estrogen-IGF1 signaling and regulates the timing of ductal
morphogenesis during mammary gland development in mice (Li et al., 2007).
In the bovine species, the SIRT1 gene has been assigned to chromosome 28; its
coding region consists of nine exons (Ghinis-Hozumi et al., 2011). To date,
in bovine species, only few SIRT1 polymorphisms have been reported in the
literature. In particular, Li et al. (2013a, b) revealed the presence of five
single nucleotide polymorphisms (SNPs), namely g.-382G > A,
g.17324T > C, g.17379A > G, g.17491G > A and g.-274C > G. All
these SNPs were located in noncoding regions of the SIRT1 gene. In
particular, SNPs g.-382G > A and g.-274C > G were located in the
promoter region of the gene. Later, Gui et al. (2014, 2015) reported many
noncoding mutations in the 3′UTR of the SIRT1 gene (g.25764G > A,
g.25846A > G, g.25868T > C, g.25751A > C). Some of the
abovementioned SNPs were found to be associated with various growth and
carcass traits in Nanyang, Qinchuan and Luxi cattle breeds (Li et al.,
2013a, b; Gui et al., 2014, 2015; Liu et al., 2017).
The transversion g.-274C > G may modulate SIRT1 promoter activity,
causing the disruption of several transcription factor binding sites. In
detail, in the presence of the C allele, the binding site for the CDE
(cell-cycle-dependent element) was generated; this binding site was abolished
when the G allele occurred (Li et al., 2013a, b). Based on the biological
role of SIRT1 in metabolism regulation and mammary gland development, it is
possible to suppose an association between SIRT1 polymorphisms and milk
production traits. Thus, the aims of the present study were to estimate the
allele and genotype frequencies at the SIRT1 g.-274C > G locus and to
investigate the possible relationship among different genotypes and some milk
production traits in a sample of cows belonging to the Agerolese breed.
Moreover, the potential relationship between different genotypes and some
reproduction traits was explored.
Agerolese cattle are a dual-purpose (milk and meat) autochthonous Italian
breed reared in the Campania region. They ere established on the Lattari mountains
during the 19th century starting from an autochthonous nucleus of
Podolica cows crossed with Brown Swiss, Dutch Friesian and Jersey bulls.
After the Second World War, the Agerolese breed underwent genetic
contamination with Italian Brown and Holstein Friesian cattle (Felius, 1995).
The current Agerolese cattle population is very small: to date there are
208 animals, 181 of which (147 females and 34 males) are enrolled in the
birth register managed by the Italian Breeders Association (AIA, 2014). The
milk produced by Agerolese cows has excellent organoleptic properties.
Presently, this breed is used almost exclusively for milk production which is
mainly used for butter and cheese production such as “fior di latte”
(stretched curd) and “Provolone del Monaco”. a cheese recognized by the
Protected Designation of Origin (reg. CE no. 121/2010) and the Slow Food
Foundation for Biodiversity (Peretti et al., 2013; Selvaggi et al., 2017).
Material and methodsAnimals
A total of 90 cows belonging to the Agerolese breed were included in this
study. They were the progeny of 15 Agerolese sires with the number of half-sibs
ranging from 3 to 6. Animals were maintained at a single farm located in
the Campania region, in southern Italy. All the cows were fed with the same
lactation diet, according to the energy recommendations for lactating cows,
being able to graze for about 8–10 h after the morning milking. They had
free access to water. The animals were milked twice a day. Data concerning
milk production traits during all of lactation for each animal in the first,
second and third lactation were obtained from monthly records provided by
the National Breeders Association. In particular, data concerning the daily
milk yield (DMY, kg), the fat and protein percentage (FC, PC), the peak daily
milk yield (PY, defined as the highest daily milk volume produced for each
animal, kg), the lactation length (LL, days), the 305-day milk yield (305MY,
kg), and the 305-day fat and protein percentage (305FC, 305PC) were used in
the present study. Moreover, the 305 fat/protein ratio (305FC/PC) was
calculated. Thus, our dataset contained information concerning whole
lactations and standard (305 days) lactations measured in 4 consecutive
years (2010–2014) for each cow. Detailed information was available for some
reproductive traits and was used to determine age at first calving (days) and
calving intervals (days) for each animal.
Polymorphism determination
Individual blood samples for DNA genotyping were collected from all the cows
on K3-EDTA tubes and stored at -25∘C. Genomic DNA was isolated
from whole blood using ZR Genomic DNA II KitTM (Zymo Research). After
genomic DNA isolation, all the samples were genotyped for the g.-274C > G gene polymorphism using the PCR–RFLP technique. The
g.-274C > G polymorphism, located in the promoter region of the bovine
SIRT1 gene was determined as previously described by Li et al. (2013a). The
g.-274C > G polymorphism is detectable by enzymatic digestion with
SmaI (Li et al., 2013a, b).
Observed and expected numbers and percentages (in brackets) of
genotypes at the g.-274C < G locus, allele frequencies and population genetic
indices in the sample of Agerolese cows.
The following primers were used to amplify a fragment of 273 bp; SIRT1F
(forward): 5′ – GTA TAG TCC ACG GGG TTA CAG – 3′; SIRT1R (reverse): 5′ – CCA
AAC TTG TCT TTC AGA GTC – 3′. The SIRT1 gene fragment was amplified using 34
amplification cycles of 94 ∘C for 1 min, 56 ∘C for
1 min and 72 ∘C for 1 min. The 273 bp product was digested with SmaI
restriction endonuclease (CCC↓GGG) and then analyzed on a 3 %
agarose gel stained with ethidium bromide in a TBE buffer.
Statistical analysis
The allele frequencies were calculated by simple allele counting (Falconer
and Mackay, 1996). The possible deviations of genotypic frequencies from
expectation under Hardy–Weinberg equilibrium were tested by a chi-square
test. Population genetic indices, such as gene heterozygosity (He), gene
homozygosity (Ho), effective allele numbers (Ne) and fixation
index (FIS), were performed by POPGENE32 software version 1.32 (Yeh et al.,
2000). Moreover, polymorphism information content (PIC) was calculated
according to Botstein et al. (1980).
A mixed model for repeated measures implemented with
the SAS software (SAS 9.2 Institute, Inc., Cary, NC; SAS, 1999) was used to detect a
possible relationship between the SIRT1 genotypes and performance traits under
study. Data were considered as repeated measures and the correlations between
the measures in the same individual were considered in the statistical
model. The model included SIRT1 genotype and lactation number as fixed
effects: the random effect of the sire, the random animal effect and the
residual error term. The values were considered significant at P<0.05 and
presented as least squares means ± standard errors.
Frequencies of C and G alleles and population genetic indices in
Agerolese and in different cattle breeds as observed by other authors. Allele
frequencies are shown in increasing order for the C allele.
Allelic Breedfrequencies HeNePICReferencesCGChinese Red Steppe0.4470.5530.4941.9780.372Li et al. (2013a, b)Agerolese0.5220.4780.4001.9960.375Present workQinchuan0.5310.4690.4981.9920.374Li et al. (2013a, b)Nanyang0.6510.3490.4541.8330.351Li et al. (2013a, b)Jiaxian0.6530.3470.4531.8290.350Li et al. (2013a, b)Luxi0.7440.2560.3811.6150.308Li et al. (2013a, b)Luxi0.7690.2310.3551.5500.292Liu et al. (2017)
Means and standard error of productive and reproductive traits in
Agerolese cows with different genotypes at g.-274C > G.
TraitsGenotypes CCCGGGDMY (kg)15.04b±0.6916.58±0.6117.19a±0.65FC (%)3.84a±0.053.62±0.033.55b±0.06PC (%)3.31±0.033.28±0.02.28±0.03LL (days)337.33A±9.90304.60A±8.31259.87B±11.15PY (kg)22.49b±0.6525.29a±0.5625.79a±0.64305MY4165.67b±198.155018.30a±166.184378.83±187.27305FC (%)3.81±0.053.60±0.033.55±0.06305PC (%)3.26±0.033.26±0.023.27±0.03305FC/PC1.17±0.011.11±0.011.09±0.01Age at first calving (days)848.23a±28.65786.82±21.79772.50b±26.42Calving period (days)454.85Aa±15.18401.30b±12.42398.97B±13.99
The nuclease cuts the 273 bp amplification product into 235 and 38 bp
fragments for allele G, while allele C remains uncut. The following DNA
restriction fragments were expected: 235 and 38 bp for the GG genotype; 273,
235 and 38 bp for the CG genotype; and 273 bp for the CC genotype. As
shown in Table 1, the investigated population was found to be polymorphic
at the g.-274C > G locus: in particular, 29 individuals out of 90 were
genotyped as CC, 25 as GG and 36 were heterozygotes. Thus, the frequencies of
C and G alleles were 0.522 and 0.478, respectively. The expected genotype
frequencies, calculated according to the Hardy–Weinberg equilibrium, were
27.28 % (CC), 49.90 % (GC), and 22.82 % (GG). The calculated
chi-square value was 3.54 (degree of freedom = 1), indicating a Hardy–Weinberg equilibrium
in the population (P=0.06). Table 1 also illustrates the calculated values
of the genetic indices. FIS is a measure of the deviation of genotypic
frequencies from panmictic frequencies in terms of heterozygous deficiency
or excess. Negative FIS values indicate heterozygote excess and
positive values indicate heterozygote deficiency when compared with
Hardy–Weinberg equilibrium expectations. In the Agerolese population a slight
excess of homozygosity at the considered locus was found (FIS=0.198).
PIC is a parameter indicative of the degree of informativeness of a
marker. The PIC value may range from 0 to 1. In the studied population, the PIC
value was 0.375. According to the classification of PIC (low polymorphism if
PIC value < 0.25, median if 0.25 < PIC value < 0.50
and high if PIC value > 0.50), the examined population possesses
an intermediate polymorphism level at the considered locus. Moreover, the
calculated Ne indicates a good level of genetic variability in the Agerolese
cattle breed at the studied locus. Table 2 summarizes the previous findings
on allele frequencies and genetic indices at the SIRT1 c.-274C > G
locus in other breeds. This SNP was found to be polymorphic in all the
investigated breeds. The C allele is the most frequent in all these
populations, except for Chinese Red Steppe. Moreover, all the breeds possess
a high effective allele number (ranging from 1.550 to 1.996) and a moderate
value of PIC (ranging from 0.292 to 0.375).
Effects of polymorphism on productive and reproductive traits
Data reported in Table 3 show the effects of the g.-274C > G polymorphism
on milk production traits and some reproductive traits. Concerning milk
production performances, significant differences between genotypes were found
in DMY, FC, LL, PY and 305MY. In particular, when compared with the other
genotypes, GG individuals produced a greater quantity of milk in terms of DMY
(17.19 kg day-1 vs. 15.04 kg day-1 for GG and CC, respectively;
P<0.05); however the difference between GG and CG was found not to be
significant in statistical analysis. On the other hand, CC and CG animals
showed a longer lactation when compared to GG genotypes (337.33 and
304.60 days vs. 259.87 days, respectively; P<0.01). Moreover, PY of cows
carrying the CC genotype was lower when compared with the CG and GG ones
(22.49 kg vs. 25.29 and 25.79 kg, respectively; P<0.05). Milk quality was
significantly affected only in terms of FC (3.84 % vs. 3.55 % for CC and
GG cows, respectively; P<0.05), with no difference in protein content. When
production data during standard lactation are considered, no differences were
found concerning the quality of milk. Moreover, CG animals yielded a greater
amount of milk when compared to cows belonging to CC genotype
(5018.30 kg vs. 4165.67 kg; P<0.05).
Nevertheless, this last result needs more attention due to the short
lactation length of GG animals (less than 305 days). Although GG animals
produced less milk during the standard lactation when compared to CG animals,
this difference was not significant in statistical analysis.
The effect of the g.-274C > G genotype on age at first calving and
calving period was significant. In detail, GG cows had a younger age at first
calving than CC ones (772.50 days vs. 848.23 days; P<0.05). Animals having
a CG genotype showed an intermediate age at first calving, although the
differences to the other genotypes were not significant in statistical
analysis. Finally, cows carrying the GG genotype had a shorter calving
interval when compared with CC individuals (-55.88 days; P<0.01), and
also the difference between CC and CG genotype was found to be significant
(454.85 days vs. 401.30 days, respectively; P<0.05). No significant result
was obtained by comparing the length of the calving period of CG and
GG animals.
To the best of our knowledge, no other studies on the potential effect of
SIRT1 or SmaI polymorphism on milk performances and reproductive traits are
available in the literature. On the basis of our results, GG animals were
more productive in terms of DMY although they showed a shorter lactation
period that is the reason for the lower 305MY. Nevertheless, this aspect can
be balanced out considering the more advantageous reproductive traits of
GG cows.
To date, only few studies have been conducted in order to find a possible
relationship between SIRT1 gene polymorphisms and livestock performance
traits. Nevertheless, the SIRT1 gene seems to be a potentially useful genetic
marker for carcass and body measurement characteristics (Li et al., 2013a, b;
Gui et al., 2014; Liu et al., 2017). In particular, Li
et al. (2013a, b) found a positive association between the c.-274C > G
polymorphism and growth traits in Nanyang cattle suggesting that the presence
of the G allelic variant could be responsible for a decreased gene promoter
activity that, in turn, is responsible for the improved growth traits
observed in CG and GG animals. Considering this last aspect, it is possible
to suppose that CG and GG animals may reach the minimum liveweight at mating
early. Thus, the lower age at first calving observed in CG and GG individuals
in the present study may be explained by an improved somatic precocity.
Conclusion
Undoubtedly, sirtuins are a very interesting family of protein, playing a key
role in endocrine signaling. Considering the specific involvement of SIRT1 in
the glucose and fat metabolism, to study the variability of SIRT1 gene may
offer the possibility to contribute to improving livestock production. This
is the first report on the possible association between different genotypes
at the SIRT1 g.-274C > G locus and milk productive and reproductive
traits in bovine species. Although other studies should be carried out on
sirtuins and their genetic variability, our results are promising and should
encourage scientists to further investigate the effect of genetic
polymorphism of sirtuins on milk performance and reproductive traits.
Data availability
The data sets are available upon request from the
corresponding author.
Author contributions
All the authors contributed equally to this study.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
The authors thank their respective departments for support.
Review statement
This paper was edited by Steffen Maak and reviewed by
Francesco Vizzarri, Ibrahim Alhidary, and one anonymous referee.
ReferencesAIA: Quadro economico e Relazione attività 2013, available at: http://www.aia.it/aia-website/it/home
(last access: 26 October 2018), 2014.
Botstein, D., White, R. L., Skalnick, M. H., and Davies, R. W.: Construction
of a genetic linkage map in man using restriction fragment length polymorphisms,
Am. J. Hum. Genet., 32, 314–331, 1980.
Braunstein, M., Rose, A. B., Holmes, S. G., Allis, C. D., and Broach, J. R.:
Transcriptional silencing in yeast is associated with reduced nucleosome
acetylation, Gene Dev., 7, 592–604, 1993.Denu, J. M.: Linking chromatin function with metabolic networks: Sir2 family
of NAD+-dependent deacetylases, Trends Biochem. Sci., 28, 41–48, 2003.
Falconer, D. S., and Mackay, T. F. C.: Introduction to quantitative genetics,
in: 4th Edn., Longman Group Ltd, Essex, 1996.
Felius, M.: Cattle breeds: an encyclopedia, Misset, Doetinchem, 1995.
Frye, R. A.: Characterization of five human cDNAs with homology to the yeast
SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein
ADP-ribosyltransferase activity, Biochem. Biop. Res. Co., 260, 273–279, 1999.Gerhart-Hines, Z., Rodgers, J. T., Bare, O., Lerin, C., Kim, S.-H., Mostoslavsky,
R., Alt, F. W., Wu, Z., and Puigserver, P.: Metabolic control of muscle
mitochondrial function and fatty acid oxidation through SIRT1/PGC-1α,
EMBO J., 26, 1913–1923, 2007.
Ghinis-Hozumi, Y., González-Gallardo, A., González-Dávalos, L.,
Antaramian, A., Villarroya, F., Shimada, A., and Mora, O.: Bovine sirtuins:
initial characterization and expression of sirtuins 1 and 3 in liver, muscle,
and adipose tissue, J. Anim. Sci., 89, 2529–2536, 2011.
Gui, L., Wang, H., Wei, S., Zhang, Y., and Zan, L.: Molecular characterization,
expression profiles, and analysis of Qinchuan cattle SIRT1 gene association with
meat quality and body measurement traits (Bos taurus), Mol. Biol. Rep.,
41, 5237–5246, 2014.
Gui, L., Hao, R., Zhang, Y., Zhao, X., and Zan, L.: Haplotype distribution in
the class I sirtuin genes and their associations with ultrasound carcass
traits in Qinchuan cattle (Bos taurus), Mol. Cell. Probe, 29, 167–171, 2015.
Li, H., Rajendran, G. K., Liu, N., Ware, C., Rubin, B. P., and Gu, Y.: SirT1
modulates the estrogen–insulin-like growth factor-1 signaling for postnatal
development of mammary gland in mice, Breast Cancer Res., 9, 1, 2007.Li, M., Sun, X., Zhang, L., Wang, J., Huang, Y., Sun, Y., Hu, S., Lan, X., Lei,
C., and Chen, H.: A novel c.-274C > G polymorphism in bovine SIRT1 gene
contributes to diminished promoter activity and is associated with increased
body size, Anim. Genet., 44, 584–587, 2013a.
Li, M., Sun, X., Hua, L., Lai, X., Lan, X., Lei, C., Zhang, C., Qi, X., Wang,
J., and Chen, H.: SIRT1 gene polymorphisms are associated with growth traits
in Nanyang cattle, Mol. Cell. Probes, 27, 215–220, 2013b.Liu, G., Zhao, H., Tan, X., Cheng, H., You, W., Wan, F., Liu, Y., Song, E.,
and Liu, X.: SIRT1 gene polymorphisms associated with carcass traits
in Luxi cattle, Arch. Anim. Breed., 60, 27–32, 10.5194/aab-60-27-2017, 2017.
Michan, S. and Sinclair, D.: Sirtuins in mammals: insights into their biological
function, Biochem. J., 404, 1–13, 2007.
Michishita, E., Park, J. Y., Burneskis, J. M., Barrett, J. C., and Horikawa,
I.: Evolutionarily conserved and nonconserved cellular localizations and
functions of human SIRT proteins, Mol. Biol. Cell., 16, 4623–4635, 2005.
North, B. J. and Verdin, E.: Sirtuins: Sir2-related NAD-dependent protein
deacetylases, Genome Biol., 5, 224.1–224.12, 2004.North, B. J., Marshall, B. L., Borra, M. T., Denu, J. M., and Verdin, E.: The
human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylases, Mol.
Cell., 11, 437–444, 2003.Peretti, V., Ciotola, F., and Iannuzzi, L.: Characterization, conservation
and sustainability of endangered animal breeds in Campania (Southern Italy),
Nat. Sci., 5, 1–9, 2013.
Picard, F., Kurtev, M., Chung, N., Topark-Ngarm, A., Senawong, T., Machado de
Oliveira, R., Leid, M., McBurney, M. W., and Guarente, L.: Sirt1 promotes fat
mobilization in white adipocytes by repressing PPARγ, Nature, 429, 771–776, 2004.
Revollo, J. R. and Li, X.: The ways and means that fine tune Sirt1 activity,
Trends Biochem. Sci., 38, 160–167, 2013.
Rodgers, J. T. and Puigserver, P.: Fasting-dependent glucose and lipid metabolic
response through hepatic sirtuin, P. Natl. Acad. Sci. USA, 104, 12861–12866, 2017.
Sartore, S., Barbieri, V., Rasero, R., Sacchi, P., Di Stasio, L., and Sartore,
G.: Analysis of genetic variation in Agerolese cattle breed, Biochem. Genet.,
43, 485–490, 2005.
SAS: User's Guide Statistics, Version 8.0 Edition, SAS Inst. Inc., Cary, NC, 1999.
Selvaggi, M., Albarella, S., Dario, C., Peretti, V., and Ciotola, F.: Association
of STAT5A Gene Variants with Milk Production Traits in Agerolese Cattle, Biochem.
Genet., 55, 158–167, 2017.
Shoba, B., Lwin, Z. M., Ling, L. S., Bay, B. H., Yip, G. W., and Kumar, S. D.:
Function of sirtuins in biological tissues, Anat. Rec., 292, 536–543, 2009.
Vaquero, A., Scher, M., Lee, D., Erdjument-Bromage, H., Tempst, P., and Reinberg,
D.: Human SirT1 interacts with histone H1 and promotes formation of facultative
heterochromatin, Mol. Cell., 16, 93–105, 2004.
Vaziri, H., Dessain, S. K., Eaton, E. Ng, Imai, S. I., Frye, R. A., Pandita,
T. K., Guarente, L., and Weinberg, R. A.: hSIR2 (SIRT1) functions as an
NAD-dependent p53 deacetylase, Cell, 107, 149–159, 2001.
Yeh, F. C., Yang, R., Boyle, T. J., Ye, Z., and Xiyan, J. M.: PopGene32,
Microsoft Windows-based freeware for population genetic analysis, version 1.32,
Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton,
Alberta, Canada, 2000.