During the last few years, several publications have reported the use of HDAC inhibitors to study histone acetylation and gene regulation. An important question to be addressed by the study of histone modification is how modifications affect not only chromatin dynamics but also various processes (e.g., DNA replication, RNA transcription) along the DNA-template. These processes can be influenced by a number of post-translational modifications of histones, including acetylation, methylation, phosphorylation, and ubiquitination. These modifications may not act alone, but in concert and in a context-dependent manner to facilitate or repress chromatin-mediated processes 
Our previous studies 
revealed that VFAs, especially butyrate, participate in metabolism, both as nutrients and as regulators of histone modification, thereby regulating the ‘epigenomic code.’ These findings implicate histone modifications induced by butyrate as determinants of bovine phenotype and in bovine ruminal development.
Epigenomics is an emerging area of scientific investigation that is confirming the complexity of the mechanisms used to determine the how, when, and where of gene expression in order to ensure the normal development, health, and homeostasis of the animal. The recently completed profiling of global gene expression used a high-density oligonucleotide microarray 
to identify 450 genes in bovine kidney epithelial cells that were significantly regulated by sodium butyrate at a very stringent false discovery rate (FDR) of 0%. The functional category and pathway analyses of the microarray data revealed that four canonical pathways (cell cycles: G2/M DNA damage checkpoint, pyrimidine metabolism, G1/S checkpoint regulation, and purine metabolism) were significantly perturbed. The biologically relevant networks and pathways of these genes were also identified, including genes such as IGF2, TGFB1, TP53, E2F4,
, which were established as central to these networks. However, because they are restricted to probes designed to target the genes in a given species' genome, hybridization-based microarray technologies offer a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes over a wide range of levels 
, and they often fail to capture the full catalogue of transcripts and their variations.
The development of the next-generation sequencing (NGS) has provided novel tools for expression profiling and genome analysis [17,18,19]. As a vital step towards a comprehensive understanding of the molecular mechanism of butyrate-induced acetylation, as well as its biological effects, the present study was designed to utilize next-generation sequencing technology in order to provide a more complete characterization of the RNA transcripts of MDBK cells. This study also focused on the comparison between the control group (without butyrate treatment) and the cells treated with 10 mM butyrate for 24 hours. With technical replicates (four lanes for controls and four lanes for butyrate-treated samples), the samples were deep-sequenced, with an average of more than 67 million reads per sample, and the results were used to estimate the differences induced by butyrate treatment. Therefore, our results show a very reliable and detailed profiling of the changes in gene expression induced by butyrate.
This study has generated comprehensive information on an experimental system that can be used in many functional genomics studies of bovine cells. To the best of our knowledge, this is the first study that has used NGS and IPA to identify the influences of butyrate on transcriptomic characterization in a normal bovine cell line. IPA analysis revealed that butyrate exerts a very broad range of effects on many biological pathways through its inhibitory action on HDACs in the MDBK cell line. Our NGS results, with a comparative transcriptomic profiling approach, extended far beyond the findings reported using microarray technologies 
. The phenomenal number of genes we identified that fall within a broad range of functional categories appear to provide a very detailed molecular basis for the butyrate-induced biological effects.
The stable propagation of genetic information requires that the entire genome of an organism be faithfully replicated only once in each cell cycle. In eukaryotes, this replication is initiated at hundreds to thousands of replication origins distributed over the genome, each of which must be prohibited from re-initiating DNA replication within a single cell cycle 
. Initiation of DNA replication is a two-step process: First, initiation proteins are assembled onto the replication origin in a stepwise fashion to develop a pre-replication complex. Second, the initiation complex is activated by protein kinases, resulting in the establishment of replication forks. This process is tightly regulated, such that initiation at a given replication origin occurs only once per cell cycle. In addition, initiation is down-regulated in response to agents that damage DNA or block DNA replication.
In eukaryotic cells, cell cycle checkpoint regulation assures the fidelity of cell division. The G1 (first gap phase)/S cell cycle checkpoint controls the passage of eukaryotic cells from the G1 into the S phase. Mitogen-dependent progression through the G1 of the cell-division cycle is accurately regulated to ensure that normal cell division is synchronous with cell growth and that the initiation of DNA synthesis (the S phase) is timed precisely to avoid inappropriate DNA amplification. The G1/S checkpoint control is vital for normal cell division and involves the key components that include cell cycle kinases, CDK4/6-cyclin D and CDK2-cyclin E, and the transcription complex composed of the retinoblastoma protein (Rb) and transcription factor E2F. The activation of E2F is necessary for the G1-S transition. In the present report, CDK4/6 and cyclins E and E2F were significantly down-regulated by butyrate-induced histone acetylation. In contrast, p21, a cell cycle inhibitor protein, was significantly up-regulated. All of these perturbations of gene expression in the G1/S cell cycle checkpoint pathways are consistent with the observed biological effects of butyrate, which induces cell cycle arrest at the G1/S boundary 
Butyrate is able to inhibit all class I HDACs. It also seems to affect many other epigenetic-related enzymes by regulating the expression of genes. The missing link is why this inhibition of enzymatic activities, in turn, regulates their own expression at the mRNA level. In this report, we found a vastly complicated depiction of the expression of HDACs induced by butyrate treatment. Whereas the expression of HCACs 7, 8, and 9 are down-regulated, HDACs 5 and 11 are up-regulated, and HDACs 1, 2, 4, and 6 are unchanged (Table S1). HDAC inhibitors that affect the expression of the HDACs themselves have been observed in mouse neural cells 
. In that report, both TSA and SB indeed elevated the expression of HADC1, HDAC3, HDAC5, and HDAC6, whereas the mRNA levels for HDAC 2 and HDAC7 did not change. The mRNA levels of HDAC8 and HDAC10 were not detectable in these cells. The mechanism and biological relevance of HDAC inhibitors in the regulation of the expression of HDACs is not clear, but may possibly indicate the existence of an auto-regulatory feedback loop for the expression of several HDACs after their activities are inhibited.
Butyrate, as a histone deacetylase inhibitor, can also decrease histone methylation 
, suggesting an interplay between histone acetylation and histone methylation. An emerging possibility is that histone modifications can influence one another. In other words, there may be “crosstalk among histone modification” 
. Consistently, KDM5B, a specific histone demethylase (H3-trimethyl-K4), was significantly up-regulated by butyrate treatment (Table S1). However, JSRID2, which is directly related to histone methylation and responsible for maintaining the methylation level on histone H3 lysine 27 trimethylation (H3K27me3) 
, was also significantly up-regulated. JARID2 possesses an in vitro
methyl-protective activity, stabilizing Polycom Repressive Complex 2 (PRC2)-catalyzed H3K27me3 by protecting it from the activity of H3K27 demathylases 
. These data may indicate that different histone marks (modifications) are differentially regulated and that in turn, differentially regulated histone marks regulate different biological functions 
. On the other side, a reversal of DNA methylation by butyrate has also recently been reported to occur by the regulation of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) through ERK signaling 
. In this report, we found that three DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, were significantly down-regulated by the butyrate treatment (Table S1). While DNMT1 functions in the establishment and regulation of tissue-specific patterns of methylated cytosine residues, DNMT3A and DNMT3B function in the de novo methylation of DNA 
. These DNMTs are regulated by several mechanisms in terms of their expression and catalytic activity. However, for the first time, our data directly indicated that histone modification has a role in the regulation of the expression of DNMTs, thereby affecting the level of DNA methylation.
The first clear evidence that a six-subunit “origin recognition complex's” (ORC) activity in mammalian cells is regulated by cell cycle–dependent changes in the affinity of the largest subunit (Orc1) for chromatin has been reported 
. Evidence has since confirmed these findings and extended them to show that mammalian Orc1 is selectively ubiquitinated and phosphorylated during the S-to-M–phase transition, while ORC subunits 2 to 5, which constitute a stable core complex, remain tightly bound to chromatin throughout cell division 
. In addition, a second mechanism prevents the assembly of a functional ORC until the completion of mitosis: the selective association of Orc1 with Cdk1 (Cdc2)/cyclin A during the G2/M phase of cell division. This association accounted for the appearance in M-phase cells with hyperphosphorylated Orc1 that was subsequently dephosphorylated during the M-to-G1 transition 
. The rebinding of Orc1 to chromatin follows the same time course as the degradation of cyclin B, suggesting that the exit from mitosis triggers Orc1 binding to chromatin. In fact, the inhibition of Cdk activity in metaphase cells resulted in the rapid binding of Orc1 to chromatin, and NGS profiling shows that all six subunits of ORC are down-regulated by butyrate-induced histone acetylation, adding yet another layer of regulation of ORC activities via the modified expression of those genes. In our previous microarray profiling 
, some of the components of this pathway were found to be perturbed by butyrate-induced gene regulation; however, ORC1 was the only one of the six ORC complex genes that was detected to be a down-regulated gene. In the present report, ORC1 is still the most significantly down-regulated gene, but the other ORC components (ORC2 to ORC6) are all also identified as down-regulated. This result certainly indicates the superb sensitivity of deep RNA sequencing.
We also found significant up-regulation of both BTG1 and BTG2. The BTG family member-2 (BTG2) has antiproliferative activity, and the expression of BTG2 in cycling cells induces the accumulation of hypophosphorylated, growth-inhibitory forms of retinoblastoma protein (Rb) and leads to G1 arrest through the impairment of DNA synthesis. These up-regulated antiproliferation activities are strengthened by the extensive repression of cyclin-dependent kinase and cell cycle-related genes that are clearly associated with the cell growth arrest induced by butyrate.
Tumor protein p53 (TP53, a nuclear protein), transcription factor E2F4, and many other transcription factors, were deregulated by butyrate treatment in the present study. TP53 plays an essential role in the regulation of the cell cycle, specifically in the transition from G0 to G1. It is found in very low levels in normal cells; however, in a variety of transformed cell lines, it is expressed in high amounts and is believed to contribute to transformation and malignancy. P53 is a DNA-binding protein that contains DNA-binding, oligomerization, and transcription activation domains. P53 is postulated to bind as a tetramer to a p53-binding site and activate the expression of downstream genes that inhibit growth and/or invasion, thereby functioning as a tumor suppressor.
P53 has been extensively studied for its function and involvement in butyrate-induced biological effects 
. Butyrate efficiently suppresses the growth of WT p53-containing cells. It leads to a major G2/M arrest of cells in the presence of p53, while cells without wild-type p53 accumulate mainly in the G1 phase of the cell cycle. Apoptosis induction by butyrate is also greatly reduced in the absence of p53, suggesting that a p53 pathway mediates, in part, growth suppression by butyrate and that p53 status may be an important determinant of chemosensitivity to butyrate 
. Our data also indicate that the TP53 genes may have different responses and different roles to play in normal and transformed cells. In our dataset, 518 genes were potential targets for TP53 regulation. Among these 518 genes, 238 genes showed expression directions consistent with the activation of TP53. However, one remaining question is why the expression of TP53 was down-regulated, even as its function was more active. As an extremely regulated gene, two major factors may contribute to this complexity of TP53. First, the expression of TP53 is subject to multiple regulations at transcriptional, post-transcriptional, and translational levels, with very complex expression patterns of alternative splicing, alternative promoter usage, and alternative translation. Secondly, the regulation of p53 function is extremely complex and occurs at many levels. Post-translational modifications of p53 (phosphorylation, methylation, acetylation, etc.) alter the functions of p53 (recognition of DNA sequences, interactions with transcription factors at promoters of target genes, etc.) 
. Indeed, deep RNA-seq and IPA analysis revealed significant changes in the expression of genes related to the molecular function of protein post-translational modification (). There are 333 genes related to the phosphorylation of proteins, 80 genes related to the tyrosine phosphorylation of proteins, and 106 genes related to the activation of protein kinase, which is up-regulated by butyrate. The possibility exists that the modification of p53 is affected by butyrate, directly or indirectly. Clearly, more studies are still required to understand the exact roles that TP53 plays in butyrate-induced biological effects.
In conclusion, the acetylation of histone tails is essential for diverse cellular processes, such as DNA replication and cell cycle progression. Butyrate-induced histone hyper-acetylation, however, has some divergent activities, including the induction of cell cycle arrest, gene expression, and apoptosis 
. The transcriptome characterization of bovine cells using RNAseq identified transcriptional control mechanisms via butyrate. Our results extended our knowledge of the regulatory effects of butyrate on gene expression and will undoubtedly provide insight into the molecular mechanisms of in vivo
butyrate-induced epigenomic regulation.