Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the expression of sexspecific lincRNAs and their potential for regulation of sex differences in liver physiology and disease. High-throughput sequencing of mammalian transcriptomes has revealed nearly ubiquitous transcription of the genome and the generation of large numbers of noncoding transcripts. Noncoding RNAs (ncRNAs) have drawn much attention as potential chromatin regulators, exemplified by classical ncRNAs, such as Xist (1). Several thousand ncRNAs have been discovered in human (2, 3), mouse (4-7), zebrafish (8,9), and fruit fly (10,11). Individual ncRNAs were shown to play diverse regulatory roles in gene expression (9,(12)(13)(14)(15); however, the vast majority of ncRNAs are poorly characterized, both computationally and experimentally. Many ncRNAs share salient features of protein-coding genes, including transcription by RNA polymerase II, 5= capping, splicing, polyadenylation, and deposition of histone marks associated with transcription, specifically H3K4me3 at the promoter and H3K36me3 across the gene body (4). These ncRNAs are typically Ͼ200 nucleo...

Sex-specific temporal patterns of pituitary growth hormone (GH) secretion determine the sex-biased transcription of hundreds of genes in the liver and impart important sex differences in liver physiology, metabolism, and disease. Sex differences in hepatic gene expression vary widely, ranging from less than twofold to >1000-fold in the mouse. Here, we use small RNA sequencing to discover 24 sex-biased mouse liver microRNAs (miRNAs), and then investigate the roles of two of these miRNAs in GH-regulated liver sex differences. Studies in prepubertal and young adult mice, and in mice in which pituitary hormones are ablated or where sex-specific hepatic GH signaling is dysregulated, demonstrated that the male-biased miR-1948 and the female-biased miR-802 are both regulated by sex-specific pituitary GH secretory patterns, acquire sex specificity at puberty, and are dependent on the GH-activated transcription factor STAT5 for their sex-specific expression. Both miRNAs are within genomic regions characterized by sex-biased chromatin accessibility. miR-1948, an uncharacterized miRNA, has essential features for correct Drosha/Dicer processing, generates a bona fide mature miRNA with strong strand bias for the 5p arm, and is bound by Argonaute in liver tissue, as is miR-802. In vivo studies using inhibitory locked nucleic acid sequences revealed that miR-1948-5p preferentially represses female-biased messenger RNAs (mRNAs) and induces male-biased mRNAs in male liver; conversely, miR-802-5p preferentially represses male-biased mRNAs and increases levels of female-biased mRNAs in female liver. Cytochrome P450 mRNAs were strongly enriched as targets of both miRNAs. Thus, miR-1948-5p and miR-802-5p are functional components of the GH regulatory network that shapes sex-differential gene expression in mouse liver.

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies.

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2–6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research.

Quantitation of rare somatic mutations is essential for basic research and translational clinical applications including minimal residual disease (MRD) detection. Though unique molecular identifier (UMI) has suppressed errors for rare mutation detection, the sequencing depth requirement is high. Here, we present Quantitative Blocker Displacement Amplification (QBDA) which integrates sequence-selective variant enrichment into UMI quantitation for accurate quantitation of mutations below 0.01% VAF at only 23,000X depth. Using a panel of 20 genes recurrently altered in acute myeloid leukemia, we demonstrate quantitation of various mutations including single base substitutions and indels down to 0.001% VAF at a single locus with less than 4 million sequencing reads, allowing sensitive MRD detection in patients during complete remission. In a pan-cancer panel and a melanoma hotspot panel, we detect mutations down to 0.1% VAF using only 1 million reads. QBDA provides a convenient and versatile method for sensitive mutation quantitation using low-depth sequencing.