Our results demonstrated that rGH and cyadox affected DNA methylation and histone modification of IGF-1 gene promoter. In addition, these two drugs can also affect the state of chromatin loosening in BRL cells. These findings shed new light on the relationships between epigenetics and body’s growth.
Currently, quite many studies have suggested that cyadox can improve the growth performance of animals including pigs and poultry [6, 7, 37–40]. Antimicrobials including cyadox might influence growth via an endocrine axis such as IGF-1, GH and epidermal growth factor (EGF) . One study has found that pigs given 50 mg/kg cyadox diet showed the greater average daily gain (ADG) and the better food efficiency compared with given 0 mg/kg cyadox diet . Interestingly, pigs given 50 mg/kg cyadox diet had higher EGF concentrations than pigs given control diet throughout the experiment . EGF is known to exert different biological actions in vivo and in vitro influencing the proliferation [42, 43], differentiation [44, 45]. Additionally, the insulin concentrations increased after the beginning of cyadox treatment and reached a peak at the level of 50 mg/kg in the weeks . Insulin also stimulates hepatic IGF-1 release [46, 47]. As known, the increase of IGF-1 expression can promote cell proliferation and differentiation in cells [48, 49]. In present study, cyadox also increased cell viability and enhanced the expression of IGF-1 in BRL cells, which suggests that cyadox may promote cell proliferation partially through high expression of IGF-1 gene.
DNA methylation is an important growth-promoting mechanism of cyadox and rGH. Although, there are many studies suggested that cyadox as growth promoting agent, however, those were focused on the changes of intestinal microorganisms and apparent digestibility of nutrients . In recent years, the role of DNA methylation in animal growth has gained extensive attention. DNA methylation mainly takes place in CpG dinucleotides, adding a methyl group to the cytosine to form 5-mC . DNA methylation is closely related to increased gene expression, whereas hypermethylation usually leads to inhibition of genes expression . DNA methylation can regulate several biological events, including X-chromosome inactivation, embryonic development, genomic imprinting, transcriptional regulation, and chromatin modification . However, the role of DNA methylation in cyadox-induced growth is unknown. The present study demonstrated that cyadox and rGH induced genome-wide DNA hypermethylation, which is regulated by DNMT1. Hypermethylation is usually not harmful, whereas hypomethylation typically leads to growth arrest or apoptosis in mammalian cells . Thus, the proper hypermethylation in cells is thought to be beneficial for growth and development. To our knowledge, this is the first study to demonstrate the effects of rGH and Cyadox on whole genome 5-mC level. Importantly, we found that DNA methylation level of IGF-1 promoter was reduced in different extent at ten CpG sites after rGH and cyadox treatments, which facilitates the transcription of IGF-1, increasing IGF-1 expression. These findings indicated that IGF-1 expression would be increased due to local hypomethylation under rGH and cyadox, which is in line with the findings of a report .
DNA methylation is regulated by DNMTs mainly DNMT1, DNMT 3A, DNMT 3B. DNMTs use S-adenosylmethionine (SAM) as a methyl donor to catalyze a methyl group to add to the cytosine ring to form methylcytosine . DNMT1 is responsible for the restoration of hemimethylated to full methylation (maintenance methylation) and is the predominant mammalian DNA methylating enzyme. DNMT 3A and DNMT 3B create new DNA methylation sites (de novo methylation) . In present study, the gene expression of DNMTs (DNMT1, DNMT 3A) decreased first and then increased in the time effect of cyadox and rGH. This may be responsible for maintaining cell homeostasis through demethylation during the compensatory process; and then with the continuous stimulation of drugs, drugs may downregulate certain genes through hypermethylation, and thus play its own pharmacological role. Although the mRNA lever of DNMT1 and DNMT 3A genes significantly increased than control group after BRL cells were exposed to 1 µM cyadox for 4 h, it only reflects genome-wide methylation level in cells. After the drug acts on the cells, some genes may be hypermethylated and others may be hypomethylated or unchanged.
H3K4me3 and H3K27ac can induce gene activation and play an important role in the growth . However, little known about how rGH and cyadox affect H3K27ac and H3K4me3 levels. Present study found that the H3K27ac and H3K4me3 levels are reduced by cyadox and rGH treatments. Modified histones can interact with other proteins to affect gene expression . Histone acetylation can activate gene transcription and mainly occurs in lysines . Histone methylation can regulate the chromatin structure and growth of the cells, and mainly occurs in lysine and arginine residues . The decreased level of total H3K27ac and H3K4me3 in BRL cells exposed to cyadox and rGH may be the result of the combination of different genomic protein modifications. Cyadox and rGH can increase the levels of H3K4me3 and H3K27ac around promoter 1 and promoter 2 of IGF-1, inducing IGF-1 expression. Some active enhancers are usually marked by H3K27ac, which is around transcriptional start sites (TSSs) [25, 26, 61, 62]. Some active enhancers are also associated with H3K4me3, which is related to RNA polymerase II activity . Thus, modification of H3K4me3 might enable the activity of RNA polymerase II, thus increasing IGF-1 transcription initiation. In addition, the increased level of H3K27ac was first to be found in promoter 1 and promoter 2 of IGF-1 when the BRL cells were treated with rGH and cyadox, respectively.
Histone methylation can be regulated Kdm 5a, Kdm 5b  and Kmt 2a . Kdm 5a and Kdm 5b can inhibit H3K4me3 , however, Kmt 2a, a methyltransferase, can activate H3K4me3 . We found that cyadox and rGH increased the level of Kdm 5a and Kdm 5b expression. The high expression of Kdm 5a and Kdm 5b and low expression of Kmt 2a caused the low level of H3K4me3 in BRL cells under cyadox and rGH. Additionally, Histone acetylation can be regulated by genes of Crebbp , Kat 2a , Kat 2b  and HDAC1 . Crebbp, Kat 2a, and Kat 2b are histone acetyltransferase, and HDAC1 is a histone deacetylase. Our present results suggested that cyadox and rGH downregulated the level of Kat 2a and Crebbp, and increased HDAC1 expression, which could lower the H3K27ac level in cells.
Chromatin structure is closely related to gene activation and inhibition in cells . The current results displayed that the visible space in the nucleus was smaller and gaps among nucleosomes were wider after BRL cells were treated with rGH and cyadox, which may be caused by DNA methylation and histone modification changes . These results indicated that rGH and cyadox loosens the chromatin of BRL cells, facilitating transcriptional activation of growth-related genes.