Local Chromatin Environment Research Articles

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.

DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures.

Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; “DIP-SC-seq”) on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.

Local chromatin environment of a Polycomb target gene instructs its own epigenetic inheritance.

Inheritance of gene expression states is fundamental for cells to 'remember' past events, such as environmental or developmental cues. The conserved Polycomb Repressive Complex 2 (PRC2) maintains epigenetic repression of many genes in animals and plants and modifies chromatin at its targets. Histones modified by PRC2 can be inherited through cell division. However, it remains unclear whether this inheritance can direct long-term memory of individual gene expression states (cis memory) or instead if local chromatin states are dictated by the concentrations of diffusible factors (trans memory). By monitoring the expression of two copies of the Arabidopsis Polycomb target gene FLOWERING LOCUS C (FLC) in the same plants, we show that one copy can be repressed while the other is active. Furthermore, this 'mixed' expression state is inherited through many cell divisions as plants develop. These data demonstrate that epigenetic memory of FLC expression is stored not in trans but in cis.

Tuning noise in gene expression.

The relative contribution of promoter architecture and the associated chromatin environment in regulating gene expression noise has remained elusive. In their recent work, Arkin, Schaffer and colleagues (Dey et al, 2015) show that mean expression and noise for a given promoter at different genomic loci are uncorrelated and influenced by the local chromatin environment.

The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly.

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).

Transcription Kinetics Heterogeneity of Highly Mobile Identical Genes Revealed by Simultaneous Measurement at the Single Cell Level

2560-Plat Transcription Kinetics Heterogeneity of Highly Mobile Identical Genes Revealed by Simultaneous Measurement at the Single Cell Level Enrico Gratton, Paolo Annibale. Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. Fluorescence microscopy was used in combination with molecular constructs to measure in real time and in living cells the transcriptional activity of individ- ual genes and their displacement within the nucleus. The question whether local mobility of individual chromatin regions is correlated to their transcriptional output is still the subject of active research. There is a consensus that the local chromatin environment and physical accessibility may play a role in modu- lating transcription, possibly determining the large heterogeneity observed in RNA Polymerase II (PolII) elongation rates measured from fluorescence as- says. Employing high-speed fluorescence nanoimaging we measure here at ms-temporal resolution PolII elongation and we are able to resolve the minute displacements of identical active genes surrounding a labeled transgene array. We observe a correlation between the transcriptional activity and sub- micrometer movements of the active genes that is evidence of an active molec- ular mechanism determining displacements of the active genes following transcriptional bursts. Furthermore, we detect a significant heterogeneity in the kinetics of identical genes measured simultaneously and at sub-second tem- poral resolution within the same cell. Together these observations rule out cell to cell variability as the underlying cause for the observed kinetic heterogeneity and point to the local chromatin environment and physical accessibility as the source of PolII elongation variability. Work supported in part by NIH P41- GM103540 and NIH P50-GM076516

Repression of flowering under a noninductive photoperiod by the HDA9-AGL19-FT module in Arabidopsis.

Posttranslational acetylation of histones is reversibly regulated by histone deacetylases (HDACs). Despite the evident significance of HDACs in Arabidopsis development, the biological roles and underlying molecular mechanisms of many HDACs are yet to be elucidated. By a reverse-genetic approach, we isolated an hda9 mutant and performed phenotypic analyses on it. In order to address the role of HDA9 in flowering, genetic, molecular, and biochemical approaches were employed. hda9 flowered early under noninductive short-day (SD) conditions and had increased expression of the floral integrator FLOWERING LOCUS T (FT) and the floral activator AGAMOUS-LIKE 19 (AGL19) compared with the wild-type. The hda9 mutation increased histone acetylation and RNA polymerase II occupancy at AGL19 but not at FT during active transcription, and the HDA9 protein directly targeted AGL19. AGL19 expression was higher under SD than under inductive long-day (LD) conditions, and an AGL19 overexpression caused a strong up-regulation of FT. A genetic analysis showed that an agl19 mutation is epistatic to the hda9 mutation, masking both the early flowering and the increased FT expression of hda9. Taken together, our data indicate that HDA9 prevents precocious flowering under SD conditions by curbing the hyperactivation of AGL19, an upstream activator of FT, through resetting the local chromatin environment.

TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity.

Despite overwhelming evidence that transcriptional activation by TP53 is critical for its tumor suppressive activity, the mechanisms by which TP53 engages the genome in the context of chromatin to activate transcription are not well understood. Using a compendium of novel and existing genome-wide data sets, we examined the relationship between TP53 binding and the dynamics of the local chromatin environment. Our analysis revealed three distinct categories of TP53 binding events that differ based on the dynamics of the local chromatin environment. The first class of TP53 binding events occurs near transcriptional start sites (TSS) and is defined by previously characterized promoter-associated chromatin modifications. The second class comprises a large cohort of preestablished, promoter-distal enhancer elements that demonstrates dynamic histone acetylation and transcription upon TP53 binding. The third class of TP53 binding sites is devoid of classic chromatin modifications and, remarkably, fall within regions of inaccessible chromatin, suggesting that TP53 has intrinsic pioneer factor activity and binds within structurally inaccessible regions of chromatin. Intriguingly, these inaccessible TP53 binding sites feature several enhancer-like properties in cell types within the epithelial lineage, indicating that TP53 binding events include a group of “proto-enhancers” that become active enhancers given the appropriate cellular context. These data indicate that TP53, along with TP63, may act as pioneer factors to specify epithelial enhancers. Further, these findings suggest that rather than following a global cell-type invariant stress response program, TP53 may tune its response based on the lineage-specific epigenomic landscape.

Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter

Genomic engineering by the guide RNA (gRNA)-directed CRISPR/CAS9 is rapidly becoming a method of choice for various biological systems. However, pressing concerns remain regarding its off-target activities and wide variations in efficacies. While next generation sequencing (NGS) has been primarily used to evaluate the efficacies and off-target activities of gRNAs, it only detects the imperfectly repaired double strand DNA breaks (DSB) by the error-prone non-homologous end joining (NHEJ) mechanism and may not faithfully represent the DSB activities because the efficiency of NHEJ-mediated repair varies depending on the local chromatin environment. Here we describe a reporter system for unbiased detection and comparison of DSB activities that promises to improve the chance of success in genomic engineering and to facilitate large-scale screening of CAS9 activities and gRNA libraries. Additionally, we demonstrated that the tolerances to mismatches between a gRNA and the corresponding target DNA can occur at any position of the gRNA, and depend on both specific gRNA sequences and CAS9 constructs used.

Transcription Factors Modulate c-Fos Transcriptional Bursts

SUMMARYTranscription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)—especially in rapidly inducible systems—is largely unknown. Using fluorescence in situ hybridization and computational models, we quantified the transcriptional activity of the proto-oncogene c-Fos with single mRNA accuracy at individual endogenous alleles. We showed that, during MAPK induction, the TF concentration modulates the burst frequency of c-Fos, whereas other bursting parameters remain mostly unchanged. By using synthetic TFs with TALE DNA-binding domains, we systematically altered different aspects of these bursts. Specifically, we linked the polymerase initiation frequency to the strength of the transactivation domain and the burst duration to the TF lifetime on the promoter. Our results show how TFs and promoter binding domains collectively act to regulate different bursting parameters, offering a vast, evolutionarily tunable regulatory range for individual genes.

GPS2/KDM4A Pioneering Activity Regulates Promoter-Specific Recruitment of PPARγ

Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however, the regulatory strategies underlying each factor's effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of peroxisome proliferator-activator receptor γ (PPARγ) in adipocytes requires G protein suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARγ-mediated regulation of a specific transcriptional program, including the lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.

PARP1 orchestrates variant histone exchange in signal-mediated transcriptional activation.

Transcriptional activation is accompanied by multiple molecular events that remodel the local chromatin environment in promoter regions. These molecular events are often orchestrated in response to the activation of signalling pathways, as exemplified by the response of immediate early genes such as FOS to ERK MAP kinase signalling. Here, we demonstrate that inducible NFI recruitment permits PARP1 binding to the FOS promoter by a mutually reinforcing loop. PARP1 and its poly(ADP-ribosyl)ation activity are required for maintaining FOS activation kinetics. We also show that the histone variant H2A.Z associates with the FOS promoter and acts in a transcription-suppressive manner. However, in response to ERK pathway signalling, H2A.Z is replaced by H2A; PARP1 activity is required to promote this exchange. Thus, our work has revealed an additional facet of PARP1 function in promoting dynamic remodelling of promoter-associated nucleosomes to allow transcriptional activation in response to cellular signalling.

Change point analysis of histone modifications reveals epigenetic blocks linking to physical domains

Histone modification is a vital epigenetic mechanism for transcriptional control in eukaryotes. High-throughput techniques have enabled whole-genome analysis of histone modifications in recent years. However, most studies assume one combination of histone modification invariantly translates to one transcriptional output regardless of local chromatin environment. In this study we hypothesize that, the genome is organized into local domains that manifest similar enrichment pattern of histone modification, which leads to orchestrated regulation of expression of genes with relevant bio- logical functions. We propose a multivariate Bayesian Change Point (BCP) model to segment the Drosophila melanogaster genome into consecutive blocks on the basis of combinatorial patterns of histone marks. By modeling the sparse distribution of histone marks across the chromosome with a zero-inflated Gaussian mixture, our partitions capture local BLOCKs that manifest relatively homogeneous enrichment pattern of histone modifications. We further characterized BLOCKs by their transcription levels, distribution of genes, degree of co-regulation and GO enrichment. Our results demonstrate that these BLOCKs, although inferred merely from histone modifications, reveal strong relevance with physical domains, which suggest their important roles in chromatin organization and coordinated gene regulation.

Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes

Protein complexes that bind to ‘GAGA’ DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p<1×10−8). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution.

E2F-7 couples DNA damage-dependent transcription with the DNA repair process

The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.

Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins.

We have devised a method for isolating virtually pure and comprehensive libraries of restriction fragments that contained replication initiation sites (bubbles) in vivo. We have now sequenced and mapped the bubble-containing fragments from GM06990, a near-normal EBV-transformed lymphoblastoid cell line, and have compared origin distributions with a comprehensive replication timing study recently published for this cell line. We find that early-firing origins, which represent ∼32% of all origins, overwhelmingly represent zones, associate only marginally with active transcription units, are localized within large domains of open chromatin, and are significantly associated with DNase I hypersensitivity. Origin “density” falls from early- to mid-S-phase, but rises again in late S-phase to levels only 17% lower than in early S-phase. Unexpectedly, late origin density calculated on the 1-Mb scale increases as a function of increasing chromatin compaction. Furthermore, the median efficiency of origins in late-replicating, heterochromatic domains is only 25% lower than in early-replicating euchromatic loci. Thus, the activation of early- and late-firing origins must be regulated by quintessentially different mechanisms. The aggregate data can be unified into a model in which initiation site selection is driven almost entirely by epigenetic factors that fashion both the long-range and local chromatin environments, with underlying DNA sequence and local transcriptional activity playing only minor roles. Importantly, the comprehensive origin map we have prepared for GM06990 overlaps moderately well with origin maps recently reported for the genomes of four different human cell lines based on the distributions of small nascent strands.

A Prototypic Lysine Methyltransferase 4 from Archaea with Degenerate Sequence Specificity Methylates Chromatin Proteins Sul7d and Cren7 in Different Patterns

The origin of eukaryotic histone modification enzymes still remains obscure. Prototypic KMT4/Dot1 from Archaea targets chromatin proteins (Sul7d and Cren7) and shows increased activity on Sul7d, but not Cren7, in the presence of DNA. Promiscuous aKMT4 could be regulated by chromatin environment. This study supports the prokaryotic origin model of eukaryotic histone methyltransferases and sheds light on chromatin dynamics in Archaea. Histone methylation is one of the major epigenetic modifications even in early diverging unicellular eukaryotes. We show that a widespread lysine methyltransferase from Archaea (aKMT4), bears striking structural and functional resemblance to the core of distantly related eukaryotic KMT4/Dot1. aKMT4 methylates a set of various proteins, including the chromatin proteins Sul7d and Cren7, and RNA exosome components. Csl4- and Rrp4-exosome complexes are methylated in different patterns. aKMT4 can self-methylate intramolecularly and compete with other proteins for the methyl group. Automethylation is inhibited by suitable substrates or DNA in a concentration-dependent manner. The automethylated enzyme shows relatively compromised activity. aKMT4-8A mutant with abrogated automethylation shows a more than 150% increase in methylation of substrates, suggesting a possible mechanism to regulate methyltransferase activity. More interestingly, methylation of Sul7d, but not Cren7, by aKMT4 is significantly enhanced by DNA. MS/MS and kinetic analysis further suggest that aKMT4 methylates Sul7d in the chromatin context. These data provide a clue to the possible regulation of aKMT4 activity by the local chromatin environment, albeit as a promiscuous enzyme required for extensive and variegated lysine methylation in Sulfolobus. This study supports the prokaryotic origin model of eukaryotic histone modification enzymes and sheds light on regulation of archaeal chromatin.

HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster

Heterochromatin protein 1a (HP1a) is a chromatin-associated protein important for the formation and maintenance of heterochromatin. In Drosophila, the two histone methyltransferases SETDB1 and Su(var)3-9 mediate H3K9 methylation marks that initiates the establishment and spreading of HP1a-enriched chromatin. Although HP1a is generally regarded as a factor that represses gene transcription, several reports have linked HP1a binding to active genes, and in some cases, it has been shown to stimulate transcriptional activity. To clarify the function of HP1a in transcription regulation and its association with Su(var)3-9, SETDB1 and the chromosome 4-specific protein POF, we conducted genome-wide expression studies and combined the results with available binding data in Drosophila melanogaster. The results suggest that HP1a, SETDB1 and Su(var)3-9 repress genes on chromosome 4, where non-ubiquitously expressed genes are preferentially targeted, and stimulate genes in pericentromeric regions. Further, we showed that on chromosome 4, Su(var)3-9, SETDB1 and HP1a target the same genes. In addition, we found that transposons are repressed by HP1a and Su(var)3-9 and that the binding level and expression effects of HP1a are affected by gene length. Our results indicate that genes have adapted to be properly expressed in their local chromatin environment.

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

A persistent question in biology is how cis-acting sequence elements influence trans-acting factors and the local chromatin environment to modulate gene expression. We reported previously that the DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster. We have now generated a collection of variegating phiC31 landing-pad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360-sensitive silencing. Many 1360-sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360-dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360-sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360-sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis-acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue.