The effect of corn silage hybrid and inclusion on performance of finishing steers and silage hybrid effects on digestibility and performance of growing steers.

Three experiments evaluated the effects of three corn silage hybrids, inclusion, and nutrient digestibility in growing and finishing diets. The three hybrids tested included a control (CON), a hybrid containing a brown midrib (bm3) trait (BM3), and an experimental bm3 hybrid with the soft endosperm trait (BM3-SOFT). Experiment 1 utilized 360 crossbred steers (body weight [BW] = 334; SD = 25 kg) to evaluate inclusion of silage in a finishing diet at (15% or 45% of diet dry matter [DM]) and silage hybrid (CON, BM3, or BM3-SOFT). Experiment 2 and 3 utilized 216 crossbred steers (BW = 324; SD = 10 kg) and six ruminally fistulated steers (BW = 274; SD = 27 kg), respectively, to evaluate effects of either CON, BM3, or BM3-SOFT silage hybrids on performance and nutrient digestibility in growing diets. In Exp. 1, there was a silage inclusion × hybrid interaction for average daily gain (ADG) and gain-to-feed ratio (G:F). All treatments with 15% silage had greater (P ≤ 0.04) ADG and G:F compared with 45% silage. Cattle fed BM3-SOFT had greater ADG and G:F than cattle fed CON or BM3 when silage was included at 15% of the diet. When silage was fed at 45% of the diet DM, ADG did not differ between cattle fed either bm3 hybrid. Cattle fed BM3 had the greatest G:F (P < 0.01), with no difference between BM3-SOFT and CON. At 15% silage inclusion, hot carcass weight (HCW) was greater (P < 0.01) for cattle fed BM3-SOFT compared with cattle fed CON and BM3 but did not differ between cattle fed BM3 and CON. At 45% silage inclusion, steers fed either bm3 hybrid did not differ in HCW but were both heavier (P < 0.01) compared with cattle fed CON. In Exp. 2, ending BW, dry matter intake (DMI), and ADG were greater (P < 0.01) for steers fed either bm3 hybrid compared to steers fed the CON, but not different between steers fed the bm3 hybrids. There were no differences (P = 0.26) in G:F between the silage hybrids. In Exp. 3, steers fed either bm3 had greater (P < 0.01) neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility than steers fed the CON. Ruminal pH was lower (P < 0.01), and total volatile fatty acid (VFA) concentration was greater (P < 0.01) for steers fed bm3 hybrids compared to steers fed CON. Feeding silage with the bm3 trait improved fiber digestibility, which increased DMI and subsequent ADG in high-forage growing diets. Feeding corn silage with the bm3 trait improved performance compared to non-bm3 corn silage when included above typical roughage concentration.

HIV chromatin is a preferred target for drugs that bind in the DNA minor groove.

The HIV genome is rich in A but not G or U and deficient in C. This nucleotide bias controls HIV phenotype by determining the highly unusual composition of all major HIV proteins. The bias is also responsible for the high frequency of narrow DNA minor groove sites in the double-stranded HIV genome as compared to cellular protein coding sequences and the bulk of the human genome. Since drugs that bind in the DNA minor groove disrupt nucleosomes on sequences that contain closely spaced oligo-A tracts which are prevalent in HIV DNA because of its bias, it was of interest to determine if these drugs exert this selective inhibitory effect on HIV chromatin. To test this possibility, nucleosomes were reconstituted onto five double-stranded DNA fragments from the HIV-1 pol gene in the presence and in the absence of several minor groove binding drugs (MGBDs). The results demonstrated that the MGBDs inhibited the assembly of nucleosomes onto all of the HIV-1 segments in a manner that was proportional to the A-bias, but had no detectable effect on the formation of nucleosomes on control cloned fragments or genomic DNA from chicken and human. Nucleosomes preassembled onto HIV DNA were also preferentially destabilized by the drugs as evidenced by enhanced nuclease accessibility in physiological ionic strength and by the preferential loss of the histone octamer in hyper-physiological salt solutions. The drugs also selectively disrupted HIV-containing nucleosomes in yeast as revealed by enhanced nuclease accessibility of the in vivo assembled HIV chromatin and reductions in superhelical densities of plasmid chromatin containing HIV sequences. A comparison of these results to the density of A-tracts in the HIV genome indicates that a large fraction of the nucleosomes that make up HIV chromatin should be preferred in vitro targets for the MGBDs. These results show that the MGBDs preferentially disrupt HIV-1 chromatin in vitro and in vivo and raise the possibility that non-toxic derivatives of certain MGBDs might serve as a novel class of anti-HIV agents.

Links between DNA methylation and nucleosome occupancy in the human genome.

BACKGROUND: DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. RESULTS: The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. CONCLUSIONS: Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

Effects of DNA methylation on nucleosome stability.

Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

Oligonucleotide sequence motifs as nucleosome positioning signals.

To gain a better understanding of the sequence patterns that characterize positioned nucleosomes, we first performed an analysis of the periodicities of the 256 tetranucleotides in a yeast genome-wide library of nucleosomal DNA sequences that was prepared by in vitro reconstitution. The approach entailed the identification and analysis of 24 unique tetranucleotides that were defined by 8 consensus sequences. These consensus sequences were shown to be responsible for most if not all of the tetranucleotide and dinucleotide periodicities displayed by the entire library, demonstrating that the periodicities of dinucleotides that characterize the yeast genome are, in actuality, due primarily to the 8 consensus sequences. A novel combination of experimental and bioinformatic approaches was then used to show that these tetranucleotides are important for preferred formation of nucleosomes at specific sites along DNA in vitro. These results were then compared to tetranucleotide patterns in genome-wide in vivo libraries from yeast and C. elegans in order to assess the contributions of DNA sequence in the control of nucleosome residency in the cell. These comparisons revealed striking similarities in the tetranucleotide occurrence profiles that are likely to be involved in nucleosome positioning in both in vitro and in vivo libraries, suggesting that DNA sequence is an important factor in the control of nucleosome placement in vivo. However, the strengths of the tetranucleotide periodicities were 3-4 fold higher in the in vitro as compared to the in vivo libraries, which implies that DNA sequence plays less of a role in dictating nucleosome positions in vivo. The results of this study have important implications for models of sequence-dependent positioning since they suggest that a defined subset of tetranucleotides is involved in preferred nucleosome occupancy and that these tetranucleotides are the major source of the dinucleotide periodicities that are characteristic of positioned nucleosomes.

Nucleosome positioning determinants.

A previous report demonstrated that one site in a nucleosome assembled onto a synthetic positioning sequence known as Fragment 67 is hypersensitive to permanganate. The site is required for positioning activity and is located 1.5 turns from the dyad, which is a region of high DNA curvature in the nucleosome. Here, the permanganate sensitivity of the nucleosome positioning Fragment 601 was examined in order to expand the dataset of nucleosome sequences containing KMnO(4) hypersensitive sites. The hyperreactive T residue in the six sites detected as well as the one in Fragment 67 and three in the 5 S rDNA positioning sequence were contained within a TA step. Seven of the ten sequences were of the form CTAGPuG or the related sequence TTAAPu. These motifs were also found in the binding sites of several transcriptional regulatory proteins that kink DNA. In order to assess the significance of these sites, the 10 bp positioning determinant in Fragment 67 was removed and replaced by the nine sequences from the 5 S rDNA and Fragment 601. The results demonstrated that these derivative fragments promoted high nucleosome stability and positioning as compared to a control sequence that contained an AT step in place of the TA step. The importance of the TA step was further tested by making single base-pair substitutions in Fragment 67 and the results revealed that stability and positioning activity followed the order: TA>TG>TT>/=TC approximately GG approximately GA approximately AT. Sequences flanking the TA step were also shown to be critical for nucleosome stability and positioning. Nucleosome positioning was restored to near wild-type levels with (CTG)(3), which can form slipped stranded structures and with one base bulges that kink DNA. The results of this study suggest that local DNA structures are important for positioning and that single base-pair changes at these sites could have profound effects on those genomic functions that depend on ordered nucleosomes.