Development in mammals is accompanied by specific de novo and demethylation events that are thought to stabilize differentiated cell phenotypes. We demonstrate that a large percentage of the tissue-specific methylation pattern is generated postnatally. Demethylation in the liver is observed in thousands of enhancer-like sequences associated with genes that undergo activation during the first few weeks of life. Using. conditional gene ablation strategy we show that the removal of these methyl groups is stable and necessary for assuring proper hepatocyte gene expression and function through its effect on chromatin accessibility. These postnatal changes in methylation come about through exposure to hormone signaling. These results define the molecular rules of 5-methyl-cytosine regulation as an epigenetic mechanism underlying cellular responses to. changing environment.

Short tandem repeats (STRs) have been implicated in a variety of complex traits in humans. However, genome-wide studies of the effects of STRs on gene expression thus far have had limited power to detect associations and provide insights into putative mechanisms. Here, we leverage whole-genome sequencing and expression data for 17 tissues from the Genotype-Tissue Expression Project to identify more than 28,000 STRs for which repeat number is associated with expression of nearby genes (eSTRs). We use fine-mapping to quantify the probability that each eSTR is causal and characterize the top 1,400 fine-mapped eSTRs. We identify hundreds of eSTRs linked with published genome-wide association study signals and implicate specific eSTRs in complex traits, including height, schizophrenia, inflammatory bowel disease and intelligence. Overall, our results support the hypothesis that eSTRs contribute to a range of human phenotypes, and our data should serve as a valuable resource for future studies of complex traits.

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.

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.

Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4(+) memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4(+) T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4(+) T cell memory.

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.

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.

The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion.

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.

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.

Alzheimer's disease (AD) is a progressive amnestic dementia that involves post-translational hyperphosphorylation, enzymatic cleavage, and conformational alterations of the microtubule-associated protein tau. The truncation state of tau influences many of its pathologic characteristics, including its ability to assume AD-related conformations and to assemble into filaments. Cleavage also appears to be an important marker in AD progression. Although C-terminal truncation of tau at D421 has recently been attributed to the apoptotic enzyme caspase-3, N-terminal processing of the protein remains mostly uncharacterized. Here, we report immunohistochemical staining in a cohort of 35 cases ranging from noncognitively impaired to early AD with a panel of three N-terminal anti-tau antibodies: Tau-12, 5A6, and 9G3-pY18. Of these three, the phosphorylation-independent epitope of 5A6 was the earliest to emerge in the pathological lesions of tau, followed by the appearance of the Tau-12 epitope. The unmasking of the Tau-12 epitope in more mature 5A6-positive tangles was not correlated with tau phosphorylation at tyrosine 18 (9G3-pY18). Still, later in the course of tangle evolution, the extreme N terminus of tau was lost, correlating temporally with the appearance of a C-terminal caspase-truncated epitope lacking residues 422-441. In addition, caspase-6 cleaved the N terminus of tau in vitro, preventing immunoreactivity with both Tau-12 and 5A6. Mass spectrometry confirmed that the in vitro caspase-6 truncation site is D13, a semicanonical and hitherto undescribed caspase cleavage site in tau. Collectively, these results suggest a role for caspase-6 and N-terminal truncation of tau during neurofibrillary tangle evolution and the progression of Alzheimer's disease.

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.

The robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of all meningiomas, but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas had simple genomes, with fewer mutations, rearrangements and copy-number alterations than reported in other tumors in adults. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements, including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers in an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (p.Glu17Lys) and SMO (p.Trp535Leu) and exhibited immunohistochemical evidence of activation of these pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets.

Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5' end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation.

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.

Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.

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.