The histone variants H3.3 and H2A.Z have recently emerged as two of the most important features in transcriptional regulation, the molecular mechanism of which still remains poorly understood. In this study, we investigated the regulation of H3.3 and H2A.Z on chromatin dynamics during transcriptional activation. Our in vitro biophysical and biochemical investigation showed that H2A.Z promoted chromatin compaction and repressed transcriptional activity. Surprisingly, with only four to five amino acid differences from the canonical H3, H3.3 greatly impaired higher-ordered chromatin folding and promoted gene activation, although it has no significant effect on the stability of mononucleosomes. We further demonstrated that H3.3 actively marks enhancers and determines the transcriptional potential of retinoid acid (RA)-regulated genes via creating an open chromatin signature that enables the binding of RAR/RXR. Additionally, the H3.3-dependent recruitment of H2A.Z on promoter regions resulted in compaction of chromatin to poise transcription, while RA induction results in the incorporation of H3.3 on promoter regions to activate transcription via counteracting H2A.Zmediated chromatin compaction. Our results provide key insights into the mechanism of how histone variants H3.3 and H2A.Z function together to regulate gene transcription via the modulation of chromatin dynamics over the enhancer and promoter regions.

gThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ϳ1% and ϳ0.1% of the total non-rRNAs, respectively. The majority (ϳ98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (ϳ82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. In nature, the cow rumen represents a highly specialized bioreactor wherein plant cell wall polysaccharides (PCWPs) are efficiently deconstructed. The extraordinary efficiency results from the concerted action of various enzymes produced by rumen-resident bacteria, archaea, fungi, and protozoa. Three rumen bacteria, i.e., Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes, which can be isolated and cultivated in the laboratory, have been thought to serve a predominant role in the degradation of cellulosic PCWPs in this niche (1, 2). However, metagenomic quantitations based on 16S rRNA gene analysis indicate that these three species of bacteria account for only less than 5% of the total rumen microorganisms (3). In addition, Koike et al. estimated that ϳ77% of the rumen microorganisms attached to solid fibers are uncultured, as their 16S rRNA gene sequences share less than 97% similarity with those of known isolates (4).To bypass the cultivation step, metagenomic approaches involving the direct analysis of total DNA sequences have been extensively used to investigate the PCWP-degrading gastrointestinal microbes in a variety of herbivores, such as termite hindguts (5); cow (6-8), yak (9), and Svalbard reindeer (10) ...