Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin. Here, discontinuous iodixanol gradients are used as a gentle preparation of the nuclei from Xenopus embryos. Short DNase I digestion times are followed by size selection of digested genomic DNA, yielding DHS fragments. These DNA fragments are subjected to real-time quantitative polymerase chain reaction (qPCR) and sequencing library construction. A library generation method and pipeline for analyzing DNase-seq data are also described.

For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.

Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.

The addition of dietary proteases (PRO) to weaner diets hydrolyzes soybean-based anti-nutritive factors and improves weaned pig's dietary digestibility and growth performance. Therefore, this study explores the effects of PRO in a lower crude protein (CP) level diet than that in a commercial diet on the growth performance, nutrient digestibility, and intestinal morphology of weaned pigs. A total of 90 weaned pigs were randomly assigned to 3 dietary treatments with 6 pigs per pen and 5 replicated pens per treatment using a randomized complete block design (block = body weight [BW]): 1) a commercial weaner diet as a positive control (PC; phase1 CP = 23.71%; phase2 CP: 22.36%), 2) lower CP diet than PC as a negative control (NC; 0.61% less CP than PC), and 3) an NC diet with 0.02% PRO. Pigs fed PC and PRO had higher (p < 0.05) final BW, average daily gain, and/or gain to feed ratio for the first three weeks and the overall experimental period than NC. The PC and PRO groups had greater (p < 0.05) apparent ileal digestibility of dry matter, CP, and energy than the NC group. Moreover, pigs fed PC and PRO increased (p < 0.05) apparent total tract digestibility of CP compared with those fed NC. In addition, the PRO group had a higher number of goblet cells than the PC and NC groups. However, pig fed PC and PRO increased (p < 0.05) villus height and height to crypt depth ratio in the ileum compared with those fed NC. In conclusion, PRO supplementation in a commercial weaner diet with low CP levels improves growth rate and nutrient digestibility by modulating the intestinal morphology of weaned pigs.

This research was performed to investigate the hypothesis that dietary mono-component protease (PRO) might improve growth performance, nutrient digestibility, and carcass characteristics of growing-finishing pigs. A total of eighty-four pigs [Duroc × (Landrace × Yorkshire), 25.3 ± 2.16 kg initial body weight] were randomly assigned to three dietary treatments (7 replicates/treatment; 2 barrows and 2 gilts/replicate) in a randomized complete block design (block = sex). The dietary treatments were prepared as follows; (1) a positive control (PC) as a typical growing-finishing diet based on corn and soybean meal, (2) PC added with 0.015% of PRO (PCPRO), and (3) a negative control (NC) added with 0.015% of PRO (NCPRO). The NC had a lower concentration of crude protein (CP) compared with PC. The PRO was a commercial product that contained 75,000 protease units/g and derived from Nocardiopsis prasina produced in Bacillus licheniformis. Dietary treatments were offered to pigs during growing and finishing periods. Measurements were growth performance, apparent total tract digestibility (ATTD) of nutrients, and carcass characteristics. The PCPRO and/or NCPRO increased average daily gain (ADG) and gain to feed ratio (G:F) during growing (p < 0.10), finishing (p < 0.05), and growing-finishing periods (p < 0.10) compared with PC. Furthermore, pigs fed PCPRO and NCPRO had higher (p < 0.05) ATTD of CP and energy during growing and/or finishing periods than those fed PC. In conclusion, the supplementation of PRO in diets improved growth performance and protein digestibility of growing-finishing pigs.

The purpose of the current study was to assess the effects of substituting corn with ground brown rice on growth performance, immune status, and gut microbiota in weanling pigs. Seventy-two weanling pigs (28 d old with 6.78 ± 0.94 kg body weight [BW]) were randomly allotted to two dietary treatments with six pens and six pigs (three barrows and gilts) per pen within a randomized complete block design. The control pigs were fed a typical diet for weanling pigs based on corn and soybean meal diet (control diet: CON), and the other pigs were fed a formulated diet with 100% replacement of corn with ground brown rice for 35d (treatment diet: GBR). Growth performance, immune status, and gut microbiota of weanling pigs were measured. The substitution of corn with GBR did not affect growth performance or diarrhea frequency. Additionally, there were no differences in white blood cell number, hematocrit, cortisol, C-reactive protein, and serum tumor necrosis factor-alpha levels between pigs fed CON or GBR for the first 2 wk after weaning. However, weanling pigs fed GBR had lower (P < 0.05) serum transforming growth factor-beta 1 level than those fed CON. Furthermore, weanling pigs fed GBR had increased (P < 0.05) relative abundance of phylum Firmicutes and genus Lactobacillus and Streptococcus and decreased (P < 0.05) relative abundance of phylum Bacteroidetes and genus Clostridium and Prevotella in the gut microbiota compared with those fed CON. In conclusion, there was no significant difference in growth performance when corn was replaced with ground brown rice in diets for weanling pigs. Furthermore, the substitution of corn with ground brown rice in weaning diet modulated immune status and gut microbiota of pigs by increasing beneficial microbial communities and reducing harmful microbial communities. Overall, ground brown rice-based diet is a potential alternative to corn-based diet without negative effects on growth performance, immune status, and gut microbiota changes of weanling pigs.

Dietary glutamic acid (GLU) is used as a feed additive because of its functional characteristics that may affect the growth performance and health of pigs. This study was carried out to determine the effects of dietary GLU on growth performance, nutrient digestibility, immune responses, and intestinal health of weaned pigs. A total of ninety-six weaned pigs (8.07 ± 1.17 kg of body weight; 28 days of age) were assigned to two dietary treatments (8 pigs/pen; 6 replicates/treatment) in a randomized complete block design (block: body weight): (1) a typical weaner diet (CON) and (2) CON supplemented with 0.5% GLU. The experimental period was for 4 weeks. All data and sample collections were performed at the specific time points during the experimental period. Pigs fed GLU had higher average daily gain and average daily feed intake for the first two weeks and nutrient digestibility than pigs fed CON. In addition, dietary GLU increased villus height to crypt depth ratio, number of goblet cells, and ileal gene expression of claudin family and occludin compared with CON, but decreased serum TNF-α and IL-6 and ileal gene expression of TNF-α. Moreover, pigs fed GLU had increased relative composition of bacterial communities of genus Prevotella and Anaerovibrio and decreased genus Clostridium and Terrisporobacter compared with those fed CON. This study suggests that dietary GLU influences growth performance and health of weaned pigs by modulating nutrient digestibility, intestinal morphology, ileal gene expression of tight junction proteins and cytokines, immune responses, and microbial community in the gut.

The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-β signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied by Foxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.

Dietary yeast cell wall products (YCW) are recognized as a feed additive due to multifunctional benefits by the biological response modulators. Thus, this study was conducted to verify a potential advantage of YCW for improving growth performance, nutrient digestibility, immune responses, and intestinal health and microbiota of weaned pigs. A total of 112 weaned pigs (7.99 ± 1.10 kg of body weight; 28 days old) were arbitrarily allocated to two experimental treatments with eight pigs (four barrows and four gilts) per pen and seven replicate pens per treatment in a completely randomized block design (block = BW and sex): (1) a basal diet based on corn and soybean meal (CON) and (2) CON + 0.05% YCW. The experimental period was for 4 weeks. There were no differences in final body weight, average daily feed intake, and gain-to-feed ratio between dietary treatments. In contrast, pigs fed YCW had higher average daily gain (p = 0.088) and apparent ileal digestibility of DM (p < 0.05) and energy (p = 0.052) and lower diarrhea frequency (p = 0.083) than those fed control diet (CON). Pigs fed YCW also had a higher (p < 0.05) ratio between villus height and crypt depth, villus width and area, and goblet cell counts in the duodenum and/or jejunum than those fed CON. Dietary YCW decreased (p < 0.05) serum TNF-α and IL-1β of weaned pigs on day 7 and 14, respectively, compared with CON. Furthermore, pigs fed YCW had higher (p < 0.05) ileal gene expression of claudin family, occludin, MUC1, INF-γ, and IL-6 and lower (p < 0.05) that of TNF-α than those fed CON. Lastly, there were no differences in the relative abundance of bacteria at the phylum level between CON and YCW. However, dietary YCW increased (p < 0.05) the relative abundance of genera Prevotella and Roseburia compared with CON. This study provided that dietary YCW improved growth rate, nutritional digestibility, and intestinal health and modified immune responses and intestinal microbiota of weaned pigs.