Differentiation of Expression Profiles of SREBF1 and SREBF2 Genes in Chicken

Mohan QIU Zengrong ZHANG Chunlin YU Xiaosong JIANG Huarui DU Qingyun LI Hengyong XU Huadong YIN Xiaoling ZHAO Qing ZHU Yan WANG Chaowu YANG

Abstract Sterol regulatory element-binding factor-1 and -2 （SREBF1 and SREBF2） are important transcription factors involved in the regulating lipid homeostasis. Based on the essential role of SREBF1 and SREBF2， we measured the mRNA expression levels of the two genes in six various tissues at different growth points. Our results showed that the SREBF1 and SREBF2 were expressed in all six tissues examined in Erlang mountainous chicken （SD02） at 42 d， and were expressed abundantly in the uropygial gland and liver， with relatively lowest levels of expression in the abdominal fat， sebum cutaneum and leg muscle. The expression ratio of SREBF1 and SREBF2 in breast muscle， leg muscle， sebum cutaneum and uropygial gland exhibited a "decline-rise" trend. However， in liver， the expression ratio of these two genes exhibited a "decline-rise-decline" trend. Meanwhile， the expression level of SREBF1 gene of all tissues was lower than that of SREBF2 except for uropygial gland. The findings will provide important references for further function investigation of the two genes involved in fat deposition in chickens.

Key words Chicken; SREBF1; SREBF2; Differential expression

Sterol regulatory element binding proteins （SREBPs）， as a family of membrane-bound transcription factors， control the metabolism of cholesterol and fatty acid[1-3]. In mammals， SREBP comprises three main proteins， namely SREBP-1a， SREBP-1c， and SREBP-2. Among them， SREBP-1a and SREBP-1c are both encoded by the SREBF1 gene， whereas SREBP-2 is encoded by the SREBF2 gene[4]. Several studies suggest that SREBP-1a and -1c preferentially activate the transcription of genes involved in the fatty acid synthesis， whereas SREBF2 is involved in cholesterol biosynthesis.

For now， studies of SREBF1 and SREBF2 have mainly conducted on human disease and other mammals. For example， Eberlé et al.[5] found that the 54G/C mutation of SREBF1 associated with morbid obesity， and also related with type 2 diabetes. Meanwhile， Lee et al.[6] reported that the IVS7+117A>G polymorphism of the SREBF1 gene maybe be a vital genetic factor in osteonecrosis of the femoral head （ONFH） susceptibility in the Korean population. Chen and his research team[7] think that SREBF1 might play a significant role in the regulation of muscle fat deposition， due to the SREBF1 mRNA level was related to IMF deposition in a muscle of suckling pig （2008）. Also， the polymorphisms of SREBF1 gene still significantly associated with milk productionl. Therefore， SREBF1 also can be used to develop genetic tools for the selection of animals producing milk with healthier fatty acid composition[8]. Meanwhile， studies of SREBF2 have mainly conducted human disease. For instance， some researchers reported that the polymorphism of SREBF2 associated with cholesterol metabolism， cardiometabolic abnormalities and glucose and lipid dysmetabolism[9-10]. Yang et al.[11-12] found that genetic variants in SREBF1 and SREBF2 also have an impact on the risk of metabolic syndrome and the treatment of psychoactive drugs for schizophrenia.

However， the expression pattern of these two genes in avian species is unclear. Therefore， we studied the distribution of mRNA of the SREBF1 and SREBF2 genes in some lipogenic and non-fat tissues in chicken to analyze the relationship between these two genes expression and the lipogenic tissue capacity. This work may give some clues for understanding the role of the SREBF1 and SREBF2 in chicken lipid metabolism and fat deposition.

Materials and Methods

Chicken populations and sample collection

The Laboratory Animals of the State-level Animal Experimental Teaching Demonstration Center of Sichuan Agricultural University approved that this study was conducted in accordance with ethical procedures and policies （DKY-S20143136）. The chicken breeds used for the experiment were Erlang mountainous chicken （SD02 strains）， an indigenous chicken breed distributed in Tianquan of the Sichuan Province， with superior performance traits and juicy meat characteristics. SD02 chickens with yellow partridge plumage， blue shanks， and white skin， and have a good meat quality and body weights. These strains were developed on an experimental farm for poultry breeding at the Sichuan Agricultural University （Ya'an， China）[13]. All chickens involved in this study bred under the same standard conditions. On days 14， 28， 42， 56， 70 and 91， five female chickens were randomly sampled. After weighing， the chickens were slaughtered， and six tissues of breast muscle， leg muscle， abdominal fat， sebum， uropygial gland， and liver collected， and all fresh tissues were separated， frozen immediately in liquid nitrogen and stored at -80 ℃ before RNA isolation.

RNA extraction and cDNA synthesis

The total RNA was isolated for six different tissues using TRIzol Reagent （Invitrogen， Carlsbad， CA， USA） following the manufacturer's instructions and then dissolved in RNase-free water. The integrity of the RNA was evaluated via electrophoresis on 1.0% agarose gels， and the concentration and purity of the RNA detected with a NanoVue PlusTM spectrophotometer at a 260/280 nm absorbance ratio （Thermo Scientific， USA）. cDNA was synthesized using about 200 or 300 ng total RNA （in 20 μl final volume） according to the instructions of the PrimeScriptTM RT reagent kit protocol （Takara， Dalian， China） and cDNA was stored at -80 ℃ until quantitative real-time PCR analysis.

Quantitative analysis of mRNA expression

The primers for amplification of SREBF1 and SREBF2 gene were designed using Primer Primer 5.0 software based on the chicken SREBF1 and SREBF2 gene sequence （GenBank accession number： NM_204126.2 and XM_416222.2） and the β-actin （reference gene， GenBank accession number NM_205518）. All primers synthesized by Invitrogen Biotechnology （Shanghai） Co.， Ltd （Table 1）.

The mRNA levels of SREBF1 and SREBF2 genes were detected using the CFX-96 real-time PCR detection system （Bio-Rad， USA）. Each reaction （total volume 15 μl） contained 7.5 μl SYBR-Premix Ex TaqTM （2×）， 0.5 μl each gene specific primer （Table 1）， 1 μl normalized template cDNA from each tissue and 9.5 μl of sterile water. Besides， the procedures for qRT-PCR were as follows： 95 ℃ for 30 s， followed by 40 cycles of 95 ℃ for 5 s， 58 ℃ for 25 s and 72 ℃ for 30 s. An 80-cycle melting curve analysis was performed after each PCR run to confirm product specificity. The expression of chicken SREBF2 mRNA was calculated relative to the amount of β-actin present. All samples were amplified in triplicate as technical replicates， and the negative control was within the same 96-well microplate.

Statistical analysis

The real-time PCR data were analyzed using the 2-ΔΔCT method[14]. All data were described as the means±SE. The differential expression of SREBP1 and SREBF2 among six tissues analyzed with the one-way analysis of variance （ANOVA） followed by Duncan's multiple-range test with the SAS 6.12 software package （SAS Inc.， USA）. Comparisons were considered significant at P<0.05.

Results and Analysis

Tissue distribution of SREBF1 and SREBF2 expression in SD02 chicken

We used Erlang mountainous chicken SD02 to establish patterns of SREBF1 and SREBF2 expression in different tissues. For SD02 chicken， 42 d is the key time of the growth and development， so we analyzed expression at this growth point. As shown in Table 2， SREBF1 and SREBF2 were expressed in all tissues investigated and were expressed abundantly in the uropygial gland and liver， with relatively lowest levels of expression in the abdominal fat， sebum， and leg muscle. In all tissues， SREBF2 expression was higher than that of SREBF1 except for uropygial gland.

Ontogenic expression of SREBF1 in chicken

To determine whether the SREBF1 gene expression shows a specific developmental expression pattern in certain tissues， we investigated its expression in six tissues at six ages （days 14， 28， 42， 56， 70， and 91）. As shown in Fig.1， the SREBF1 mRNA expression level in all tissues except for liver varied significantly at different age （P<0.05）. The expression of the SREBF1 gene of abdominal fat first decreased significantly （P<0.01） from day 14 to 56， and finally increased to day 91， however， there was no significant （P>0.05） difference among days 28， 42， 56， 70 and 91. In contrast， the SREBF1 expression in sebum first decreased significantly （P<0.05） from day 14 to 42， then increased to day 56， decreased to day 70 and finally increased significantly （P<0.05） by day 91. The level of SREBF1 gene expression of the breast muscle first declined significantly （P<0.05） from day 14 to 56， then increased to day 70， and finally lowered to day 91. However， the highest expression level of the SREBF1 gene in leg muscle observed on day 14 which the expression level was significantly higher （P<0.01） than on the other days， and the lowest level was on day 70. In uropygial gland， the SREBF1 expression levels first increased significantly （P<0.01） from day 14 to 42， then decreased to day 56， increased to day 70 and finally reduced significantly （P<0.05） by day 91. For liver， on day 70， the expression level of the gene was higher than in the other stages. However， there were no significant （P>0.05） differences among all growth points.

Ontogenic expression of SREBF2 in chicken

Changes in SREBF2 expression in various tissues in the SD02 chicken of different ages were also analyzed. As shown in Fig.2， the expression level of the gene in breast muscle on day 28 was significantly higher （P<0.05） than in the other stages， however， there were no significant （P>0.05） difference among days 56， 70 and 91. On the contrary， the expression of the SREBF2 gene of the leg muscle decreased gradually from day 14 to 42， increased significantly （P<0.05） to day 56 and then declined to the lowest level on day 70. The highest expression level of the gene occurred on day 14 which the expression level was significantly higher （P<0.05） than on the other days. The pattern of SREBF2 gene expression of the abdominal fat and uropygial gland tissues were similar， and both increased from day 14 to 42， followed by a significant decline （P<0.05） to day 56， and then increased to day 70 and finally decreased significantly （P<0.05） by day 91. The expression of the SREBF2 gene of the sebum declined significantly from day 56 to 70 （P<0.05） and increased gradually to day 91. For liver， there was a slight increase （P<0.05） from day 28 to 70， followed by a decline to day 91.

The multiple proportions between expression of SREBF1 and SREBF2

The ratio of the relative quantity of SREBF1 gene to relative quantity of SREBF2 gene at different growth points is shown in Table 3. The expression ratio of SREBF1 and SREBF2 in breast muscle， leg muscle， sebum and uropygial gland exhibit "decline-rise" trend， however， in the liver， the expression ratio of these two genes display "decline-rise-decline" change. Meanwhile， the expression level of a SREBF1 gene of all tissues was lower than that of SREBF2 except for uropygial gland.

Conclusions and Discussion

According to our knowledge， it is the first study to investigate the mRNA expression profile of SREBF1 and SREBF2 in chicken. Knowledge of the expression pattern of the chicken SREBF1 and SREBF2 genes in specific tissues during different developmental stages may contribute to a more comprehensive understanding of how these genes are regulated and their role in regulating lipid homeostasis and fatty acids synthesis in the animal.

SREBP transcription factors are pivotal activators of critical enzymes involved in cholesterol synthesis， low-density lipoprotein endocytosis， fatty acids synthesis and glucose metabolism[1，15]. For SREBF1， our data showed that SREBF1 was expressed in a wide variety of tissues in chicken and preferentially expressed in two tissues， the uropygial gland， and liver. It has reported that fatty acids in uropygial gland play a crucial role in protecting the birds feathers are synthesized and stored， and the liver which is the main site of fatty acid synthesis in chicken. In addition， similar to Gondret et al.[16]， this study found that the SREBF1 expression level of liver was about three times of the abdominal fat in chicken but different from the data of Gondret et al.[16] who found that the SREBF1 expression level in liver was about 3.5 times lower than abdominal fat in pig. In contrast， SREBF1 was weakly expressed in tissues where lipogenesis is very small， including sebum， abdominal fat and leg muscle， which was similar to what was found in broiler male chicken by Assaf et al.[17]. Based on the above evidence， we infer that SREBF1 is critical for regulating lipogenesis， which has until now mainly described in mammals[18-21].

Different from SREBF1， the SREBF2 expression level seems to be roughly similar in all tissues examined except for uropygial gland. Interesting， the SD02 chicken SREBF2 gene was also highly expressed in breast muscle， suggesting that SREBF2 might play a vital role in meat quality. It is unclear how this protein regulates muscle fiber growth in chicken. Therefore， in future studies， we need to study its regulation effect in SD02 chicken to determine the function of SREBF2 protein in muscle fiber growth. Therefore， we could deduce that SREBF2 expression is independent of the lipogenic ability of the skeletal muscle tissues investigated in this study.

To sum up， we evaluated the ontogenic expression of SREBF1 and SREBF2 genes in chickens and found that SREBF1 and SREBF2 expression was tissue-specific and showed age-dependent changes. Our findings will provide a major reference for further function investigation of these two transcription factors in the regulation of cholesterol and fatty acid metabolism in broiler chicken.

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