Covalent histone modifications are highly conserved and play multiple roles in eukaryotic transcription regulation. Here, we mapped 26 histone modifications genome-wide in exponentially growing yeast ...and during a dramatic transcriptional reprogramming—the response to diamide stress. We extend prior studies showing that steady-state histone modification patterns reflect genomic processes, especially transcription, and display limited combinatorial complexity. Interestingly, during the stress response we document a modest increase in the combinatorial complexity of histone modification space, resulting from roughly 3% of all nucleosomes transiently populating rare histone modification states. Most of these rare histone states result from differences in the kinetics of histone modification that transiently uncouple highly correlated marks, with slow histone methylation changes often lagging behind the more rapid acetylation changes. Explicit analysis of modification dynamics uncovers ordered sequences of events in gene activation and repression. Together, our results provide a comprehensive view of chromatin dynamics during a massive transcriptional upheaval.
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•Comprehensive map of 26 histone marks during a transcriptional response in yeast•Stress does not alter global relationships between marks and transcription•Limited combinatorial complexity with transient modest increase during response•Ordered waves of histone modifications during transcriptional reprogramming
Weiner et al. carry out a thorough analysis of histone modification localization in budding yeast responding to stress. Histone modifications are highly correlated throughout the stress response, but a small number of nucleosomes enter unusual regions of 26-dimensional modification space during stress.
Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis ...and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients.
Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of ...only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression.
The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was ...considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.
Replicating chromatin involves disruption of histone-DNA contacts and subsequent reassembly of maternal histones on the new daughter genomes. In bulk, maternal histones are randomly segregated to the ...two daughters, but little is known about the fine details of this process: do maternal histones re-assemble at preferred locations or close to their original loci? Here, we use a recently developed method for swapping epitope tags to measure the disposition of ancestral histone H3 across the yeast genome over six generations. We find that ancestral H3 is preferentially retained at the 5' ends of most genes, with strongest retention at long, poorly transcribed genes. We recapitulate these observations with a quantitative model in which the majority of maternal histones are reincorporated within 400 bp of their pre-replication locus during replication, with replication-independent replacement and transcription-related retrograde nucleosome movement shaping the resulting distributions of ancestral histones. We find a key role for Topoisomerase I in retrograde histone movement during transcription, and we find that loss of Chromatin Assembly Factor-1 affects replication-independent turnover. Together, these results show that specific loci are enriched for histone proteins first synthesized several generations beforehand, and that maternal histones re-associate close to their original locations on daughter genomes after replication. Our findings further suggest that accumulation of ancestral histones could play a role in shaping histone modification patterns.
Chromatin domains are believed to spread via a polymerization‐like mechanism in which modification of a given nucleosome recruits a modifying complex, which can then modify the next nucleosome in the ...polymer. In this study, we carry out genome‐wide mapping of the Sir3 component of the Sir silencing complex in budding yeast during a time course of establishment of heterochromatin. Sir3 localization patterns do not support a straightforward model for nucleation and polymerization, instead showing strong but spatially delimited binding to silencers, and weaker and more variable Ume6‐dependent binding to novel secondary recruitment sites at the seripauperin (PAU) genes. Genome‐wide nucleosome mapping revealed that Sir binding to subtelomeric regions was associated with overpackaging of subtelomeric promoters. Sir3 also bound to a surprising number of euchromatic sites, largely at genes expressed at high levels, and was dynamically recruited to GAL genes upon galactose induction. Together, our results indicate that heterochromatin complex localization cannot simply be explained by nucleation and linear polymerization, and show that heterochromatin complexes associate with highly expressed euchromatic genes in many different organisms.
This paper reports the genome‐wide binding pattern and temporal spreading of the key SIR complex component Sir3. These results lend further support to the notion that heterochromatin complexes associate with highly expressed euchromatic genes in many organisms and reveal that nucleation/linear polymerization is not sufficient to explain heterochromatin complex localization.
The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that ...promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures.
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•H3-K56Ac and H2A.Z stimulate expression of promoter-associated, divergent ncRNAs•Global transcriptional roles for H3-K56Ac are masked by the nuclear RNA exosome•H2A.Z functions with the RNA exosome to repress expression of a subset of ncRNAs•H3-K56ac and H2A.Z facilitate formation of chromosome interaction domains
The histone modification H3-K56Ac and variant H2A.Z are known to enhance chromatin dynamics. Here, Rege et al. find that these features have global impacts on coding and noncoding RNA expression only after RNA exosome inactivation. This may be due, in part, to their roles in the formation of chromosome interaction domains.
In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a ...major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.
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•A new method for profiling the perturbation and transcriptome in the same cell•An integrated approach for CRISPR-pooled screens with single-cell transcriptomics•Immune development and signaling-dependent circuits revealed by CRISP-seq•CRISP-seq uncovers gene modules regulated by Stat1 and Rela in myeloid cells
Integrating massively parallel single-cell RNA-seq and CRISPR-pooled screens allows reconstruction of the regulatory networks controlling myeloid cell differentiation and their response to pathogens.
Alzheimer’s disease (AD) is a detrimental neurodegenerative disease with no effective treatments. Due to cellular heterogeneity, defining the roles of immune cell subsets in AD onset and progression ...has been challenging. Using transcriptional single-cell sorting, we comprehensively map all immune populations in wild-type and AD-transgenic (Tg-AD) mouse brains. We describe a novel microglia type associated with neurodegenerative diseases (DAM) and identify markers, spatial localization, and pathways associated with these cells. Immunohistochemical staining of mice and human brain slices shows DAM with intracellular/phagocytic Aβ particles. Single-cell analysis of DAM in Tg-AD and triggering receptor expressed on myeloid cells 2 (Trem2)−/− Tg-AD reveals that the DAM program is activated in a two-step process. Activation is initiated in a Trem2-independent manner that involves downregulation of microglia checkpoints, followed by activation of a Trem2-dependent program. This unique microglia-type has the potential to restrict neurodegeneration, which may have important implications for future treatment of AD and other neurodegenerative diseases.
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•Single-cell RNA-seq in Alzheimer’s model identified a novel microglia type (DAM)•DAM are Alzheimer’s disease-associated phagocytic cells conserved in mice and human•DAM are activated sequentially by Trem2-independent and -dependent pathways•DAM activation requires downregulation of microglia inhibitory-checkpoint pathways
A new type of microglia associated with restricting neurodegeneration may have important implications for treatment of Alzheimer’s and related diseases.
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