Butyrate is one of important short-chain fatty acids (SCFAs). Produced in the rumen and hindgut by gut microorganisms, butyrate is rapidly absorbed and utilized by the rumen and colon epithelium, contributing to 75% of the total energy requirement in ruminants and ~10% for humans 
. Additionally, butyrate is shown to reinforce intestinal barriers and modulate motility and visceral sensitivity of the intestine 
. As a signaling molecule, butyrate induces apoptosis and inhibits cell proliferation, and therefore, it has antitumorigenic properties. Butyrate is an inhibitor of histone deacetylases (HDACs), one of three classes of enzymes epigenetically modifying chromatin histones. Mounting evidence suggests histone acetylation plays a major role in controlling transcriptional activities of genes 
. HDAC inhibitors such as butyrate induce hyperacetylation of histones, and therefore increase transcriptional activities 
. This was supported by our observation that the total number of genes transcribed in butyrate-treated cells (mean ± sd =19,322±155) was significantly higher than in untreated control cells (17,626±125) (P
<0.00001). Transcriptional effects of butyrate have been extensively investigated in various cell types, in both in vitro
and in vivo
. Indeed, previous studies have demonstrated that butyrate down-regulates genes controlling cell proliferation in vitro
, resulting in the inhibition of the proliferation of epithelial cells 
. Microarray results show that butyrate has a profound effect in global gene expression, including up-regulation of genes related to apoptosis and differentiation 
. Our recent transcriptome studies using RNA-seq technology demonstrate that a 24-h butyrate treatment significantly affects the transcript abundance of 11,408 of the 17,625 genes detected in the bovine epithelial cell, representing ~65% of the entire transcriptome 
. However, these studies focus on the transcription at a gene level. The effect of butyrate on individual transcript isoforms and alternative splicing has been systematically studied only recently in human cells 
. In this study, we examined the regulation of alternative splicing by butyrate in bovine epithelial cells. Our results should facilitate a better understanding of alternative splicing in the development of epithelial cells-derived diseases.
Of four classes of histone deacetylases, butyrate inhibits enzymatic activities of most HDACs in Class I, II, and IV, which are zinc-dependent, except HDAC6 and HDAC10 
. Class III HDACs (also called sirtuins or SIRTs) depend on nicotinamide adenine dinucleotide for their catalytic activity 
. SIRTs are associated with chromtain regulation and affect genome stability in yeast and may represent pivotal regulators of lifespan and aging 
. SIRTs catalyze two major biochemical reactions: deacetylation on lysine residues of target proteins by altering cellular [NAD+
]/[NADH] ratios (SIRT1, SIRT2, SIRT3, SIRT5, and SIRT7) and ADP-ribosylation (SIRT4 and SIRT6) 
. In neuronal cells, SIRT1, SIRT5, and SIRT6 are down-regulated, whereas SIRT2, SIRT4, and SIRT7 up-regulated by butyrate 
. Our RNA-seq data suggest that butyrate regulated the transcript abundance or gene expression of the majority of HDACs (). Butyrate significantly increased the expression of HDAC3, HDAC5, and HDAC11. On the other hand, the expression level of HDACs7-10 was significantly down-regulated. The mRNA levels of SIRT4 and SIRT6 were strongly up-regulated while SIRT1 was significantly down-regulated by butyrate (). However, the relative abundance of HDAC1, HDAC2, HDAC4, and HDAC6 remained unchanged by butyrate. In addition to its effect on the expression at the whole gene level, butyrate selectively regulated the transcript abundance of different mRNA isoforms. While the abundance of both short and long isoforms of HDAC5 was significantly enhanced by butyrate, only the long and dominant isoform of HDAC10 was significantly down-regulated. Intriguingly, butyrate is unable to inhibit the catalytic activity of HDAC10 
. Nevertheless, butyrate may still exert its control on the deacetylase activity of HDAC10 via transcriptional regulation at the mRNA level. Our future work will focus on the biological relevance of various HDAC isoforms induced by butyrate, especially various SIRTs and their roles in cell senescence and aging.
The expression of histone deacetylases affected by butyrate in the bovine epithelial cells.
Distant gene fusion events are well known in tumors, often resulting from genomic abnormalities such as chromosomal translocation. These events, such as BCL-ABL, lead to the formation of a novel chimeric protein with different functions and are one of the common mechanisms for oncogene activation 
. Recently, a new type of fusion involving two adjacent genes in the same orientation on the same chromosome has been described 
. Adjacent genes are normally transcripted independently. However, a single transcript can be occasionally formed to include at least part of one exon from each of two or more distinct genes 
. This phenomenon, Transcription Induced Chimeras (TICs) 
or Conjoined Genes (CGs) 
, is widespread in mammalian genomes. It is estimated that at least 4%-5% of the tandem gene pairs in the human genome can be transcripted into TICs. Moreover, these TICs may possess novel functions because >70% of them are conserved in other vertebrate genomes 
. In this study, we detected 13 TICs that were supported by multiple junction reads. Intriguingly, these TICs were also differentially regulated by butyrate in bovine epithelial cells. Approximately 46% of these fusion events were involved in the members of gene families, which is much higher than 11% as previously reported 
. For example, TICs were formed between 2 homeobox genes, HOXA6 and HOXA5 and between 2 keratin genes, KRT8 and KRT4. In addition, the fusion between zinc finger proteins ZNF865 and ZNF524 was also supported by multiple junction reads, and a significantly higher number of reads was detected in untreated control cells than in butyrate-treated cells. A similar fusion event between ZNF649 and ZNF577 was identified in prostate tumors 
. A relatively higher percentage of TICs between genes with related functions identified in this study should be further examined. Most importantly, the functional significance of these fusion events, especially their possible role in transcription regulation, should be experimentally determined.
One advantage of GSNAP algorithm is its potential to identify novel splice sites and therefore possibly novel transcript isoforms. The algorithm relies on a maximum entropy model and uses frequencies of nucleotides neighboring a donor and acceptor splice site to discriminate between true and false splicing sites 
. The power of this approach was exemplified by a case study involving the prohibitin gene (PHB). The prohibitin protein complex, located in mitochondrial inner membrane, is formed by heteromeric binding of both PHB and PHB2 
and is involved in transcription regulation and cell cycle progression by blocking the G1/S transition of the cell cycle 
. Prohibitin induces apoptosis by interacting with the retinoblastoma protein as well as being involved in the p53
pathway. Its 3′ untranslated region (UTR) acts as a novel class of non-coding regulatory RNAs. Additionally, PHB expression is up-regulated in the retina in aging and diabetic models and may serve as an oxidative marker 
. A recent study using thyroid tumor cell lines demonstrates that butyrate increases PHB mRNA expression. Furthermore, butyrate as well as other HDAC inhibitors, such as trichostatin A, affects PHB splicing 
, leading to the over-expression of the longer isoform with 3′ UTR. Both inhibitors decrease the mRNA levels of the shorter isoform but increase those of the longer isoform, which exerts a growth-suppressive action. Our results showed that butyrate significantly down-regulated the mRNA expression of both PHB and PHB2 in the bovine epithelial cell (FDR <0.0001). No known isoforms in both genes have been annotated in cattle so far. Annotated PHB and PHB2 genes have 7 and 10 exons, respectively. GSNAP correctly identified all normally splicing exon-exon junctions. Moreover, GSNAP detected novel splice sites. For example, several junction reads detected in the untreated control cells suggest a possible exon skipping event that may result in skipping of Exon#2 in PHB2. Such reads were not detectable in the butyrate-treated cells. In the PHB gene, significantly higher numbers of junction reads in the control group than the butyrate-treated group indicated that multiple alternative splicing events involved Exon#1 and Exon#2 and the intron between them. These events occurred in the 5′ UTR and did not seem to alter its primary protein structure. In humans, the 3′ UTR of PHB is attributed to its anti-tumorigenic and anti-proliferative properties 
. The biological implication of various splicing events in the 5′ UTR of PHB genes in cattle is worthy of further investigation.