Distinct Modes of Regulation by Chromatin Encoded through Nucleosome Positioning Signals

The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by the genomic DNA sequence. However, less is known about the functional consequences of this encoding. Here, we address this question using a genome-wide map of ∼380,000 yeast nucleosomes that we sequenced in their entirety. Utilizing the high resolution of our map, we refine our understanding of how nucleosome organizations are encoded by the DNA sequence and demonstrate that the genomic sequence is highly predictive of the in vivo nucleosome organization, even across new nucleosome-bound sequences that we isolated from fly and human. We find that Poly(dA:dT) tracts are an important component of these nucleosome positioning signals and that their nucleosome-disfavoring action results in large nucleosome depletion over them and over their flanking regions and enhances the accessibility of transcription factors to their cognate sites. Our results suggest that the yeast genome may utilize these nucleosome positioning signals to regulate gene expression with different transcriptional noise and activation kinetics and DNA replication with different origin efficiency. These distinct functions may be achieved by encoding both relatively closed (nucleosome-covered) chromatin organizations over some factor binding sites, where factors must compete with nucleosomes for DNA access, and relatively open (nucleosome-depleted) organizations over other factor sites, where factors bind without competition

Gene expression divergence in yeast is coupled to evolution of DNA-encoded nucleosome organization

Eukaryotic transcription occurs within a chromatin environment, whose organization plays an important regulatory role and is partly encoded in cis by the DNA sequence itself1-6. Here, we examine whether evolutionary changes in gene expression are linked to changes in the DNA-encoded nucleosome organization of promoters. We find that in aerobic yeast species, where cellular respiration genes are active under typical growth conditions, the promoter sequences of these genes encode a relatively open (nucleosome-depleted) chromatin organization. This nucleosome-depleted organization requires only DNA sequence information, is independent of any co-factors and of transcription, and is a general property of growth-related genes. In contrast, in anaerobic yeast species, where cellular respiration genes are inactive under typical growth conditions, respiration gene promoters encode relatively closed (nucleosome-occupied) chromatin organizations. Thus, our results suggest a previously unidentified genetic mechanism underlying phenotypic diversity, consisting of DNA sequence changes that directly alter the DNA-encoded nucleosome organization of promoters

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns

High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans

We mapped nucleosome occupancy by paired-end Illumina sequencing in C. elegans embryonic cells, adult somatic cells, and a mix of adult somatic and germ cells. In all three samples, the nucleosome occupancy of gene promoters on the X chromosome differed from autosomal promoters. While both X and autosomal promoters exhibit a typical nucleosome-depleted region upstream of transcript start sites and a well-positioned +1 nucleosome, X-linked gene promoters on average exhibit higher nucleosome occupancy relative to autosomal promoters. We show that the difference between X and autosomes does not depend on the somatic dosage compensation machinery. Instead, the chromatin difference at promoters is partly encoded by DNA sequence, because a model trained on nucleosome sequence preferences from S. cerevisiae in vitro data recapitulate nearly completely the experimentally observed difference between X and autosomal promoters. The model predictions also correlate very well with experimentally determined occupancy values genome-wide. The nucleosome occupancy differences observed on X promoters may bear on mechanisms of X chromosome dosage compensation in the soma, and chromosome-wide repression of X in the germline

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns

p53 binds preferentially to genomic regions with high DNA-encoded nucleosome occupancy

The human transcription factor TP53 is a pivotal roadblock against cancer. A key unresolved question is how the p53 protein selects its genomic binding sites in vivo out of a large pool of potential consensus sites. We hypothesized that chromatin may play a significant role in this site-selection process. To test this, we used a custom DNA microarray to measure p53 binding at approximately 2000 sites predicted to possess high-sequence specificity, and identified both strongly bound and weakly bound sites. When placed within a plasmid, weakly bound sites become p53 responsive and regain p53 binding when stably integrated into random genomic locations. Notably, strongly bound sites reside preferentially within genomic regions whose DNA sequence is predicted to encode relatively high intrinsic nucleosome occupancy. Using in vivo nucleosome occupancy measurements under conditions where p53 is inactive, we experimentally confirmed this prediction. Furthermore, upon p53 activation, nucleosomes are partially displaced from a relatively broad region surrounding the bound p53 sites, and this displacement is rapidly reversed upon inactivation of p53. Thus, in contrast to the general assumption that transcription-factor binding is preferred in sites that have low nucleosome occupancy prior to factor activation, we find that p53 binding occurs preferentially within a chromatin context of high intrinsic nucleosome occupancy

Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein

Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function

Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER+ Breast Cancer

Inhibició de PI3K-alfa; Càncer de mama; Resposta al tractamentInhibición de PI3K-alfa; Cáncer de mama; Respuesta al tratamientoPI3K alpha inhibition; Breast cancer; Treatment responsePIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER+ breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[18F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance.This work was supported by a CRUK Programme grant (C197/A28289) and by the CRUK-EPSRC Imaging Centre (C197/A16465), and by NIH grants P30 CA008748 and R01CA190642-01, the Breast Cancer Research Foundation, and by grants from Stand Up To Cancer (Cancer Drug Combination Convergence Team), the V Foundation, and the National Science Foundation

Single cell Hi-C identifies plastic chromosome conformations underlying the gastrulation enhancer landscape.

Embryonic development involves massive proliferation and differentiation of cell lineages. This must be supported by chromosome replication and epigenetic reprogramming, but how proliferation and cell fate acquisition are balanced in this process is not well understood. Here we use single cell Hi-C to map chromosomal conformations in post-gastrulation mouse embryo cells and study their distributions and correlations with matching embryonic transcriptional atlases. We find that embryonic chromosomes show a remarkably strong cell cycle signature. Despite that, replication timing, chromosome compartment structure, topological associated domains (TADs) and promoter-enhancer contacts are shown to be variable between distinct epigenetic states. About 10% of the nuclei are identified as primitive erythrocytes, showing exceptionally compact and organized compartment structure. The remaining cells are broadly associated with ectoderm and mesoderm identities, showing only mild differentiation of TADs and compartment structures, but more specific localized contacts in hundreds of ectoderm and mesoderm promoter-enhancer pairs. The data suggest that while fully committed embryonic lineages can rapidly acquire specific chromosomal conformations, most embryonic cells are showing plastic signatures driven by complex and intermixed enhancer landscapes

Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein

Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function