Deciphering eukaryotic gene-regulatory logic with 100 million random promotersThe MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters.

Blood contains cfDNA fragments derived from dying cells 1 . cfDNA has a half-life of ~15 min 2 and, therefore, represents events that occurred close to sampling time. cfDNA analysis is used for assessment of fetus chromosomal aberrations, graft rejection, monitoring tumor dynamics and targeted treatment [3][4][5][6][7] . These applications rely on genetic differences between the host and the tissue of interest. Analysis of CpG methylation in cfDNA is emerging as an alternative independent of genetic alteration 5,[8][9][10][11] . CpG methylation profiles are determined during differentiation and are stable afterwards and, thus, are highly informative about cell identity (for example, liver or lung). However, genetic and methylation-based approaches do not report on recent transcriptional events, as mutations and methylation changes occur over developmental time scales.The basic repeating unit of chromatin is the nucleosome, which is a histone-DNA complex encompassing ~150 base pairs (bp) of DNA 12 . Histone proteins are subject to multiple covalent modifications, which are involved in nearly all aspects of messenger RNA (mRNA) biogenesis [13][14][15][16] . Histone modification patterns reflect recent events related to chromatin regulation and activity of RNA polymerase 13,15 , and different combinations of such modifications mark the location and activity of non-coding regions, enhancers, promoters and gene bodies [17][18][19][20][21][22] . Chromatin immunoprecipitation and sequencing (ChIP-seq) enables genome-wide mapping of histone modifications and provides detailed understanding of the regulatory activity within cells [17][18][19][23][24][25][26][27] .Upon cell death, the genome is fragmented, and chromatin, mostly in the form of nucleosomes, is released into the circulation as cell-free nucleosomes (cf-nucleosomes) 28-30 that retain some histone modifications [31][32][33] . We reasoned that capturing and DNA sequencing of modified nucleosomes from plasma might inform on DNA-related activities, including transcription, within the cells of origin (Fig. 1a). This currently inaccessible epigenetic information extends beyond cfDNA modalities examined to date [4][5][6][7][8][9][10][11][34][35][36][37][38][39][40][41][42][43] .In this study, we performed chromatin immunoprecipitation and sequencing of cell-free nucleosomes directly from human plasma (cfChIP-seq). We show that cfChIP-seq recapitulates the original genomic distribution of modifications associated with transcriptionally active promoters, enhancers and gene bodies, demonstrating that plasma nucleosomes retain the epigenetic information of their

The ubiquitin-proteasome system (UPS) for protein degradation has been under intensive study, and yet, we have only partial understanding of mechanisms by which proteins are selected to be targeted for proteolysis. One of the obstacles in studying these recognition pathways is the limited repertoire of known degradation signals (degrons). To better understand what determines the susceptibility of intracellular proteins to degradation by the UPS, we developed an unbiased method for large-scale identification of eukaryotic degrons. Using a reporter-based high-throughput competition assay, followed by deep sequencing, we measured a degradation potency index for thousands of native polypeptides in a single experiment. We further used this method to identify protein quality control (PQC)-specific and compartment-specific degrons. Our method provides an unprecedented insight into the yeast degronome, and it can readily be modified to study protein degradation signals and pathways in other organisms and in various settings.

The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K 2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4a) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R 2 correlation of 0.94 between TC50 values obtained in stem cellderived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. Conclusion: Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology. (HEPATOLOGY 2015;62:265-278) T he liver is the largest internal organ in the human body, responsible for protein synthesis and metabolic homeostasis. Metabolic transformation of lipids is primarily carried out by the cytochrome P450 (CYP450) family of monooxygenases, also responsible for xenobiotic transformation.

Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, while a few ‘locked’ nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.

SummaryTranscription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP (short-fragment-enriched, low-input, indexed MNase ChIP), which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal −1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcription. SLIM-ChIP is an attractive solution for mapping DNA binding proteins and charting the surrounding chromatin occupancy landscape at a single-cell level.

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SummaryChromatin immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our current view of chromatin structure and function. It allows genome-wide mapping of histone marks, which demarcate biologically relevant domains. However, ChIP-seq is an ensemble measurement reporting the average occupancy of individual marks in a cell population. Consequently, our understanding of the combinatorial nature of chromatin states relies almost exclusively on correlation between the genomic distributions of individual marks. Here, we report the development of combinatorial-iChIP to determine the genome-wide co-occurrence of histone marks at single-nucleosome resolution. By comparing to a null model, we show that certain combinations of overlapping marks (H3K36me3 and H3K79me3) co-occur more frequently than would be expected by chance, while others (H3K4me3 and H3K36me3) do not, reflecting differences in the underlying chromatin pathways. We further use combinatorial-iChIP to illuminate aspects of the Set2-RPD3S pathway. This approach promises to improve our understanding of the combinatorial complexity of chromatin.