Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

Nucleosome density shapes kilobase-scale regulation by a mammalian chromatin remodeler.

Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.

The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis.

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs).

Massively multiplex single-molecule oligonucleosome footprinting.

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.

Non-B-Form DNA Is Enriched at Centromeres.

Animal and plant centromeres are embedded in repetitive "satellite" DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10-bp dyad symmetries predicted to adopt non-B-form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence-specific DNA-binding proteins with DNA-bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non-B-form DNA and are associated with binding sites for the conserved DNA-binding protein CENP-B, which is required for artificial centromere function but is paradoxically nonessential. We also detected dyad symmetries and predicted non-B-form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non-B-form DNA because of dyad symmetries or are strengthened by sequence-specific DNA binding proteins. This may resolve the CENP-B paradox and provide a general basis for centromere specification.

Simple and Complex Centromeric Satellites in Drosophila Sibling Species.

Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats-often 100-400 bp in length-found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5-12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. Here we use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5- and 10-bp repeats; but in closely related D. simulans, the centromeres comprise more complex repeats. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster.

Remarkable Evolutionary Plasticity of Centromeric Chromatin.

Centromeres were familiar to cell biologists in the late 19th century, but for most eukaryotes the basis for centromere specification has remained enigmatic. Much attention has been focused on the cenH3 (CENP-A) histone variant, which forms the foundation of the centromere. To investigate the DNA sequence requirements for centromere specification, we applied a variety of epigenomic approaches, which have revealed surprising diversity in centromeric chromatin properties. Whereas each point centromere of budding yeast is occupied by a single precisely positioned tetrameric nucleosome with one cenH3 molecule, the "regional" centromeres of fission yeast contain unphased presumably octameric nucleosomes with two cenH3s. In Caenorhabditis elegans, kinetochores assemble all along the chromosome at sites of cenH3 nucleosomes that resemble budding yeast point centromeres, whereas holocentric insects lack cenH3 entirely. The "satellite" centromeres of most animals and plants consist of cenH3-containing particles that are precisely positioned over homogeneous tandem repeats, but in humans, different α-satellite subfamilies are occupied by CENP-A nucleosomes with very different conformations. We suggest that this extraordinary evolutionary diversity of centromeric chromatin architectures can be understood in terms of the simplicity of the task of equal chromosome segregation that is continually subverted by selfish DNA sequences.

Corrigendum: ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.

This corrects the article DOI: 10.1038/ncomms15643.

ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.

Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.

A unique chromatin complex occupies young α-satellite arrays of human centromeres.

The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100-base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C-containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes.

Mapping regulatory factors by immunoprecipitation from native chromatin.

Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors.

Acceleration of genetic gain in cattle by reduction of generation interval.

Genomic selection (GS) approaches, in combination with reproductive technologies, are revolutionizing the design and implementation of breeding programs in livestock species, particularly in cattle. GS leverages genomic readouts to provide estimates of breeding value early in the life of animals. However, the capacity of these approaches for improving genetic gain in breeding programs is limited by generation interval, the average age of an animal when replacement progeny are born. Here, we present a cost-effective approach that combines GS with reproductive technologies to reduce generation interval by rapidly producing high genetic merit calves.

5-Aza-CdR delivers a gene body blow.

In this issue of Cancer Cell, Yang et al. describe a causal relationship between gene body methylation and gene expression and a role for genic methylation in response to clinical DNA methylation inhibitors, which suggests that the mechanism of action of these inhibitors includes gene body hypomethylation-induced downregulation of cancer-associated genes.

High-resolution mapping defines the cooperative architecture of Polycomb response elements.

Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb response elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of nonhistone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor ADF1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished by enhanced Polycomb recruitment both to PREs and to target promoters of repressed genes. These results suggest that the stability of multifactor complexes at promoters and regulatory elements is a crucial aspect of developmentally regulated gene expression.

High-resolution mapping of transcription factor binding sites on native chromatin.

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling.

An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.

Nuclear alpha1-adrenergic receptors signal activated ERK localization to caveolae in adult cardiac myocytes.

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.