SIRT6 Suppresses Pancreatic Cancer through Control of Lin28b.

Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD(+)-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at the Lin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%-40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset. PAPERCLIP.

The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.

A novel quantitative trait locus implicates Msh3 in the propensity for genome-wide short tandem repeat expansions in mice.

Short tandem repeats (STRs) are a class of rapidly mutating genetic elements typically characterized by repeated units of 1-6 bp. We leveraged whole-genome sequencing data for 152 recombinant inbred (RI) strains from the BXD family of mice to map loci that modulate genome-wide patterns of new mutations arising during parent-to-offspring transmission at STRs. We defined quantitative phenotypes describing the numbers and types of germline STR mutations in each strain and performed quantitative trait locus (QTL) analyses for each of these phenotypes. We identified a locus on Chromosome 13 at which strains inheriting the C57BL/6J (B) haplotype have a higher rate of STR expansions than those inheriting the DBA/2J (D) haplotype. The strongest candidate gene in this locus is Msh3, a known modifier of STR stability in cancer and at pathogenic repeat expansions in mice and humans, as well as a current drug target against Huntington's disease. The D haplotype at this locus harbors a cluster of variants near the 5' end of Msh3, including multiple missense variants near the DNA mismatch recognition domain. In contrast, the B haplotype contains a unique retrotransposon insertion. The rate of expansion covaries positively with Msh3 expression-with higher expression from the B haplotype. Finally, detailed analysis of mutation patterns showed that strains carrying the B allele have higher expansion rates, but slightly lower overall total mutation rates, compared with those with the D allele, particularly at tetranucleotide repeats. Our results suggest an important role for inherited variants in Msh3 in modulating genome-wide patterns of germline mutations at STRs.

Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.

Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.

H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration.

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.

A flexible ChIP-sequencing simulation toolkit.

BACKGROUND: A major challenge in evaluating quantitative ChIP-seq analyses, such as peak calling and differential binding, is a lack of reliable ground truth data. Accurate simulation of ChIP-seq data can mitigate this challenge, but existing frameworks are either too cumbersome to apply genome-wide or unable to model a number of important experimental conditions in ChIP-seq. RESULTS: We present ChIPs, a toolkit for rapidly simulating ChIP-seq data using statistical models of key experimental steps. We demonstrate how ChIPs can be used for a range of applications, including benchmarking analysis tools and evaluating the impact of various experimental parameters. ChIPs is implemented as a standalone command-line program written in C++ and is available from https://github.com/gymreklab/chips . CONCLUSIONS: ChIPs is an efficient ChIP-seq simulation framework that generates realistic datasets over a flexible range of experimental conditions. It can serve as an important component in various ChIP-seq analyses where ground truth data are needed.

Study of mitotic chromatin supports a model of bookmarking by histone modifications and reveals nucleosome deposition patterns.

Mitosis encompasses key molecular changes including chromatin condensation, nuclear envelope breakdown, and reduced transcription levels. Immediately after mitosis, the interphase chromatin structure is reestablished and transcription resumes. The reestablishment of the interphase chromatin is probably achieved by "bookmarking," i.e., the retention of at least partial information during mitosis. To gain a deeper understanding of the contribution of histone modifications to the mitotic bookmarking process, we merged proteomics, immunofluorescence, and ChIP-seq approaches. We focused on key histone modifications and employed HeLa-S3 cells as a model system. Generally, in spite of the general hypoacetylation observed during mitosis, we observed a global concordance between the genomic organization of histone modifications in interphase and mitosis, suggesting that the epigenomic landscape may serve as a component of the mitotic bookmarking process. Next, we investigated the nucleosome that enters nucleosome depleted regions (NDRs) during mitosis. We observed that in ∼60% of the NDRs, the entering nucleosome is distinct from the surrounding highly acetylated nucleosomes and appears to have either low levels of acetylation or high levels of phosphorylation in adjacent residues (since adjacent phosphorylation may interfere with the ability to detect acetylation). Inhibition of histone deacetylases (HDACs) by the small molecule TSA reverts this pattern, suggesting that these nucleosomes are specifically deacetylated during mitosis. Altogether, by merging multiple approaches, our study provides evidence to support a model where histone modifications may play a role in mitotic bookmarking and uncovers new insights into the deposition of nucleosomes during mitosis.

Dissecting neural differentiation regulatory networks through epigenetic footprinting.

Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

Role of transcription complexes in the formation of the basal methylation pattern in early development.

Following erasure in the blastocyst, the entire genome undergoes de novo methylation at the time of implantation, with CpG islands being protected from this process. This bimodal pattern is then preserved throughout development and the lifetime of the organism. Using mouse embryonic stem cells as a model system, we demonstrate that the binding of an RNA polymerase complex on DNA before de novo methylation is predictive of it being protected from this modification, and tethering experiments demonstrate that the presence of this complex is, in fact, sufficient to prevent methylation at these sites. This protection is most likely mediated by the recruitment of enzyme complexes that methylate histone H3K4 over a local region and, in this way, prevent access to the de novo methylation complex. The topological pattern of H3K4me3 that is formed while the DNA is as yet unmethylated provides a strikingly accurate template for modeling the genome-wide basal methylation pattern of the organism. These results have far-reaching consequences for understanding the relationship between RNA transcription and DNA methylation.

A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals.

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.

Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.

Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output, with important functional consequences. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs or proteins simultaneously, because genomic profiling methods could not be applied to single cells until very recently. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.

Charting histone modifications and the functional organization of mammalian genomes.

A succession of technological advances over the past decade have enabled researchers to chart maps of histone modifications and related chromatin structures with increasing accuracy, comprehensiveness and throughput. The resulting data sets highlight the interplay between chromatin and genome function, dynamic variations in chromatin structure across cellular conditions, and emerging roles for large-scale domains and higher-ordered chromatin organization. Here we review a selection of recent studies that have probed histone modifications and successive layers of chromatin structure in mammalian genomes, the patterns that have been identified and future directions for research.

Optimization of transcription factor binding map accuracy utilizing knockout-mouse models.

Genome-wide assessment of protein-DNA interaction by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) is a key technology for studying transcription factor (TF) localization and regulation of gene expression. Signal-to-noise-ratio and signal specificity in ChIP-seq studies depend on many variables, including antibody affinity and specificity. Thus far, efforts to improve antibody reagents for ChIP-seq experiments have focused mainly on generating higher quality antibodies. Here we introduce KOIN (knockout implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout mice as a critical control for ChIP-seq data experiments. Additionally, KOIN can identify 'hyper ChIPable regions' as another source of false-positive signals. As the use of the KOIN algorithm reduces false-positive results and thereby prevents misinterpretation of ChIP-seq data, it should be considered as the gold standard for future ChIP-seq analyses, particularly when developing ChIP-assays with novel antibody reagents.

Digital transcriptome profiling from attomole-level RNA samples.

Accurate profiling of minute quantities of RNA in a global manner can enable key advances in many scientific and clinical disciplines. Here, we present low-quantity RNA sequencing (LQ-RNAseq), a high-throughput sequencing-based technique allowing whole transcriptome surveys from subnanogram RNA quantities in an amplification/ligation-free manner. LQ-RNAseq involves first-strand cDNA synthesis from RNA templates, followed by 3' polyA tailing of the single-stranded cDNA products and direct single molecule sequencing. We applied LQ-RNAseq to profile S. cerevisiae polyA+ transcripts, demonstrate the reproducibility of the approach across different sample preparations and independent instrument runs, and establish the absolute quantitative power of this method through comparisons with other reported transcript profiling techniques and through utilization of RNA spike-in experiments. We demonstrate the practical application of this approach to define the transcriptional landscape of mouse embryonic and induced pluripotent stem cells, observing transcriptional differences, including over 100 genes exhibiting differential expression between these otherwise very similar stem cell populations. This amplification-independent technology, which utilizes small quantities of nucleic acid and provides quantitative measurements of cellular transcripts, enables global gene expression measurements from minute amounts of materials and offers broad utility in both basic research and translational biology for characterization of rare cells.

Chromatin profiling by directly sequencing small quantities of immunoprecipitated DNA.

Chromatin structure and transcription factor localization can be assayed genome-wide by sequencing genomic DNA fractionated by protein occupancy or other properties, but current technologies involve multiple steps that introduce bias and inefficiency. Here we apply a single-molecule approach to directly sequence chromatin immunoprecipitated DNA with minimal sample manipulation. This method is compatible with just 50 pg of DNA and should thus facilitate charting chromatin maps from limited cell populations.

Polymorphic short tandem repeats make widespread contributions to blood and serum traits.

Short tandem repeats (STRs) are genomic regions consisting of repeated sequences of 1-6 bp in succession. Single-nucleotide polymorphism (SNP)-based genome-wide association studies (GWASs) do not fully capture STR effects. To study these effects, we imputed 445,720 STRs into genotype arrays from 408,153 White British UK Biobank participants and tested for association with 44 blood phenotypes. Using two fine-mapping methods, we identify 119 candidate causal STR-trait associations and estimate that STRs account for 5.2%-7.6% of causal variants identifiable from GWASs for these traits. These are among the strongest associations for multiple phenotypes, including a coding CTG repeat associated with apolipoprotein B levels, a promoter CGG repeat with platelet traits, and an intronic poly(A) repeat with mean platelet volume. Our study suggests that STRs make widespread contributions to complex traits, provides stringently selected candidate causal STRs, and demonstrates the need to consider a more complete view of genetic variation in GWASs.

The use of base editing technology to characterize single nucleotide variants.

Single nucleotide variants (SNVs) represent the most common type of polymorphism in the human genome. However, in many cases the phenotypic impacts of such variants are not well understood. Intriguingly, while some SNVs cause debilitating diseases, other variants in the same gene may have no, or limited, impact. The mechanisms underlying these complex patterns are difficult to study at scale. Additionally, current data and research is mainly focused on European populations, and the mechanisms underlying genetic traits in other populations are poorly studied. Novel technologies may be able to mitigate this disparity and improve the applicability of personalized healthcare to underserved populations. In this review we discuss base editing technologies and their potential to accelerate progress in this field, particularly in combination with single-cell RNA sequencing. We further explore how base editing screens can help link SNVs to distinct disease phenotypes. We then highlight several studies that take advantage of single-cell RNA sequencing and CRISPR screens to emphasize the current limitations and future potential of this technique. Lastly, we consider the use of such approaches to potentially accelerate the study of genetic mechanisms in non-European populations.

Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3, and SIRT1.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

Missing heritability may be hiding in repeats.

Unexplored variable number tandem repeats make a large contribution to complex traits.