Maintenance of CTCF- and Transcription Factor-Mediated Interactions from the Gametes to the Early Mouse Embryo.

The epigenetic information present in mammalian gametes and whether it is transmitted to the progeny are relatively unknown. We find that many promoters in mouse sperm are occupied by RNA polymerase II (Pol II) and Mediator. The same promoters are accessible in GV and MII oocytes and preimplantation embryos. Sperm distal ATAC-seq sites containing motifs for various transcription factors are conserved in monkeys and humans. ChIP-seq analyses confirm that Foxa1, ERα, and AR occupy distal enhancers in sperm. Accessible sperm enhancers containing H3.3 and H2A.Z are also accessible in oocytes and preimplantation embryos. Furthermore, their interactions with promoters in the gametes persist during early development. Sperm- or oocyte-specific interactions mediated by CTCF and cohesin are only present in the paternal or maternal chromosomes, respectively, in the zygote and 2-cell stages. These interactions converge in both chromosomes by the 8-cell stage. Thus, mammalian gametes contain complex patterns of 3D interactions that can be transmitted to the zygote after fertilization.

Architectural Proteins and Pluripotency Factors Cooperate to Orchestrate the Transcriptional Response of hESCs to Temperature Stress.

Cells respond to temperature stress via up- and downregulation of hundreds of genes. This process is thought to be regulated by the heat shock factor HSF1, which controls the release of RNAPII from promoter-proximal pausing. Here, we analyze the events taking place in hESCs upstream of RNAPII release. We find that temperature stress results in the activation or decommissioning of thousands of enhancers. This process involves alterations in the occupancy of transcription factors HSF1, AP-1, NANOG, KLF4, and OCT4 accompanied by nucleosome remodeling by BRG1 and changes in H3K27ac. Furthermore, redistribution of RAD21 and CTCF results in the formation and disassembly of interactions mediated by these two proteins. These alterations tether and untether enhancers to their cognate promoters or refashion insulated neighborhoods, thus transforming the landscape of enhancer-promoter interactions. Details of the 3D interactome remodeling process support loop extrusion initiating at random sites as a mechanism for the establishment of CTCF/cohesin loops.

The zinc-finger protein Z4 cooperates with condensin II to regulate somatic chromosome pairing and 3D chromatin organization.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

Evolutionarily Conserved Principles Predict 3D Chromatin Organization.

Topologically associating domains (TADs), CTCF loop domains, and A/B compartments have been identified as important structural and functional components of 3D chromatin organization, yet the relationship between these features is not well understood. Using high-resolution Hi-C and HiChIP, we show that Drosophila chromatin is organized into domains we term compartmental domains that correspond precisely with A/B compartments at high resolution. We find that transcriptional state is a major predictor of Hi-C contact maps in several eukaryotes tested, including C. elegans and A. thaliana. Architectural proteins insulate compartmental domains by reducing interaction frequencies between neighboring regions in Drosophila, but CTCF loops do not play a distinct role in this organism. In mammals, compartmental domains exist alongside CTCF loop domains to form topological domains. The results suggest that compartmental domains are responsible for domain structure in all eukaryotes, with CTCF playing an important role in domain formation in mammals.

Organizational principles of 3D genome architecture.

Studies of 3D chromatin organization have suggested that chromosomes are hierarchically organized into large compartments composed of smaller domains called topologically associating domains (TADs). Recent evidence suggests that compartments are smaller than previously thought and that the transcriptional or chromatin state is responsible for interactions leading to the formation of small compartmental domains in all organisms. In vertebrates, CTCF forms loop domains, probably via an extrusion process involving cohesin. CTCF loops cooperate with compartmental domains to establish the 3D organization of the genome. The continuous extrusion of the chromatin fibre by cohesin may also be responsible for the establishment of enhancer-promoter interactions and stochastic aspects of the transcription process. These observations suggest that the 3D organization of the genome is an emergent property of chromatin and its components, and thus may not be only a determinant but also a consequence of its function.

Minute-Made Data Analysis: Tools for Rapid Interrogation of Hi-C Contacts.

Juicer and Juicebox, described by Durand et al. (2016a, 2016b), are two new tools for fast and reliable processing of Hi-C data, providing approaches for read processing, multiple normalization schemes, feature annotation, and dynamic browsing of chromatin contacts, thus reducing arduous Hi-C analysis into an easy yet flexible pipeline.

A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis.

DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. 24-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ∼ 20-60 nt in length, often having the same 5' ends but differing in 3' ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3'-5' exonucleases. Our findings suggest an alternative route for siRNA biogenesis. Precursor transcripts are bound by AGO4 and subsequently subjected to 3'-5' exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals.

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome-wide condensation in C. elegans hermaphrodites.

The inhibition of LSD1 via sequestration contributes to tau-mediated neurodegeneration.

Tauopathies are a class of neurodegenerative diseases associated with pathological tau. Despite many advances in our understanding of these diseases, the direct mechanism through which tau contributes to neurodegeneration remains poorly understood. Previously, our laboratory implicated the histone demethylase LSD1 in tau-induced neurodegeneration by showing that LSD1 localizes to pathological tau aggregates in Alzheimer's disease cases, and that it is continuously required for the survival of hippocampal and cortical neurons in mice. Here, we utilize the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. In addition, we show that reducing LSD1 in these mice is sufficient to highly exacerbate tau-mediated neurodegeneration and tau-induced gene expression changes. Finally, we find that overexpressing LSD1 in the hippocampus of tauopathy mice, even after pathology has formed, is sufficient to significantly delay neurodegeneration and counteract tau-induced expression changes. These results suggest that inhibiting LSD1 via sequestration contributes to tau-mediated neurodegeneration. Thus, LSD1 is a promising therapeutic target for tauopathies such as Alzheimer's disease.

A SWI/SNF chromatin-remodeling complex acts in noncoding RNA-mediated transcriptional silencing.

RNA-mediated transcriptional silencing prevents deleterious effects of transposon activity and controls the expression of protein-coding genes. It involves long noncoding RNAs (lncRNAs). In Arabidopsis thaliana, some of those lncRNAs are produced by a specialized RNA Polymerase V (Pol V). The mechanism by which lncRNAs affect chromatin structure and mRNA production remains mostly unknown. Here we identify the SWI/SNF ATP-dependent nucleosome-remodeling complex as a component of the RNA-mediated transcriptional silencing pathway. We found that SWI3B, an essential subunit of the SWI/SNF complex, physically interacts with a lncRNA-binding protein, IDN2. SWI/SNF subunits contribute to lncRNA-mediated transcriptional silencing. Moreover, Pol V mediates stabilization of nucleosomes on silenced regions. We propose that Pol V-produced lncRNAs mediate transcriptional silencing by guiding the SWI/SNF complex and establishing positioned nucleosomes on specific genomic loci. We further propose that guiding ATP-dependent chromatin-remodeling complexes may be a more general function of lncRNAs.

Evolutionary History and Activity of RNase H1-Like Proteins in Arabidopsis thaliana.

RNase H1 is an endonuclease specific toward the RNA strand of RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. Arabidopsis thaliana has been previously shown to contain three genes encoding RNase H1 proteins that localize to three distinct cellular compartments. We show that these genes originate from two gene duplication events. One occurred in the common ancestor of dicots and produced nuclear and organellar RNase H1 paralogs. Second duplication occurred in the common ancestor of Brassicaceae and produced mitochondrial- and plastid-localized proteins. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for endonucleolytic digestion. Analysis of mutants in the RNase H1 genes revealed that the nuclear RNH1A and mitochondrial RNH1B are dispensable for development under normal growth conditions. However, the presence of at least one organellar RNase H1 (RNH1B or RNH1C) is required for embryonic development. The plastid-localized RNH1C affects plastid DNA copy number and sensitivity to replicative stress. Our results present the evolutionary history of RNH1 proteins in A. thaliana, demonstrate their canonical RNase H1 activity and indicate their role in early embryonic development.

Long-range control of gene expression via RNA-directed DNA methylation.

RNA-mediated transcriptional silencing, in plants known as RNA-directed DNA methylation (RdDM), is a conserved process where small interfering RNA (siRNA) and long non-coding RNA (lncRNA) help establish repressive chromatin modifications. This process represses transposons and affects the expression of protein-coding genes. We found that in Arabidopsis thaliana AGO4 binding sites are often located distant from genes differentially expressed in ago4. Using Hi-C to compare interactions between genotypes, we show that RdDM-targeted loci have the potential to engage in chromosomal interactions, but these interactions are inhibited in wild-type conditions. In mutants defective in RdDM, the frequency of chromosomal interactions at RdDM targets is increased. This includes increased frequency of interactions between Pol V methylated sites and distal genes that are repressed by RdDM. We propose a model, where RdDM prevents the formation of chromosomal interactions between genes and their distant regulatory elements.

Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome.

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture.

Eukaryotic gene expression is regulated by enhancer-promoter interactions but the molecular mechanisms that govern specificity have remained elusive. Genome-wide studies utilizing STARR-seq identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). We hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. By evaluating ChIP-seq occupancy of architectural proteins, typical enhancer-associated proteins, and histone modifications, we determine that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, we map the interactions of each enhancer class utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity.

RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA.

Ribonucleic acid-mediated transcriptional gene silencing (known as RNA-directed DNA methylation, or RdDM, in Arabidopsis thaliana) is important for influencing gene expression and the inhibition of transposons by the deposition of repressive chromatin marks such as histone modifications and DNA methylation. A key event in de novo methylation of DNA by RdDM is the production of long non-coding RNA (lncRNA) by RNA polymerase V (Pol V). Little is known about the events that connect Pol V transcription to the establishment of repressive chromatin modifications. Using RNA immunoprecipitation, we elucidated the order of events downstream of lncRNA production and discovered interdependency between lncRNA-associated proteins. We found that the effector protein ARGONAUTE4 (AGO4) binds lncRNA independent of the RNA-binding protein INVOLVED IN DE NOVO2 (IDN2). In contrast, IDN2 binds lncRNA in an AGO4-dependent manner. We further found that the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) also associates with lncRNA produced by Pol V and that this event depends on AGO4 and IDN2. We propose a model where the silencing proteins AGO4, IDN2 and DRM2 bind to lncRNA in a stepwise manner, resulting in DNA methylation of RdDM target loci.

RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes.

Transcriptional gene silencing controls transposons and other repetitive elements through RNA-directed DNA methylation (RdDM) and heterochromatin formation. A key component of the Arabidopsis RdDM pathway is ARGONAUTE4 (AGO4), which associates with siRNAs to mediate DNA methylation. Here, we show that AGO4 preferentially targets transposable elements embedded within promoters of protein-coding genes. This pattern of AGO4 binding cannot be simply explained by the sequences of AGO4-bound siRNAs; instead, AGO4 binding to specific gene promoters is also mediated by long non-coding RNAs (lncRNAs) produced by RNA polymerase V. lncRNA-mediated AGO4 binding to gene promoters directs asymmetric DNA methylation to these genomic regions and is involved in regulating the expression of targeted genes. Finally, AGO4 binding overlaps sites of DNA methylation affected by the biotic stress response. Based on these findings, we propose that the targets of AGO4-directed RdDM are regulatory units responsible for controlling gene expression under specific environmental conditions.

Analysis of long non-coding RNAs produced by a specialized RNA polymerase in Arabidopsis thaliana.

Long non-coding RNAs (lncRNAs) play important roles in several processes including control of gene expression. In Arabidopsis thaliana, a class of lncRNAs is produced by a specialized RNA Polymerase V (Pol V), which is involved in controlling genome activity by transcriptional gene silencing. lncRNAs produced by Pol V have been proposed to serve as scaffolds for binding of several silencing factors which further mediate the establishment of repressive chromatin modifications. We present methods for discovery and characterization of lncRNAs produced by Pol V. Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-seq) allows discovery of genomic regions bound by proteins in a manner dependent on either Pol V or transcripts produced by Pol V. RNA Immunoprecipitation (RIP) allows testing lncRNA-protein interactions at identified loci. Finally, real-time RT-PCR allows detection of low abundance Pol V transcripts from total RNA. These methods may be more broadly applied to discovery and characterization of RNAs produced by distinct RNA Polymerases.

Independent chromatin binding of ARGONAUTE4 and SPT5L/KTF1 mediates transcriptional gene silencing.

Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism. Expression of these elements is suppressed by establishment of repressive chromatin modifications. In Arabidopsis thaliana, they are silenced by the siRNA-mediated transcriptional gene silencing pathway where long non-coding RNAs (lncRNAs) produced by RNA Polymerase V (Pol V) guide ARGONAUTE4 (AGO4) to chromatin and attract enzymes that establish repressive chromatin modifications. It is unknown how chromatin modifying enzymes are recruited to chromatin. We show through chromatin immunoprecipitation (ChIP) that SPT5L/KTF1, a silencing factor and a homolog of SPT5 elongation factors, binds chromatin at loci subject to transcriptional silencing. Chromatin binding of SPT5L/KTF1 occurs downstream of RNA Polymerase V, but independently from the presence of 24-nt siRNA. We also show that SPT5L/KTF1 and AGO4 are recruited to chromatin in parallel and independently of each other. As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.

Mechanistic drivers of chromatin organization into compartments.

The human genome is not just a simple string of DNA, it is a complex and dynamic entity intricately folded within the cell's nucleus. This three-dimensional organization of chromatin, the combination of DNA and proteins in the nucleus, is crucial for many biological processes and has been prominently studied for its intricate relationship to gene expression. Indeed, the transcriptional machinery does not operate in isolation but interacts intimately with the folded chromatin structure. Techniques for chromatin conformation capture, including genome-wide sequencing approaches, have revealed key organizational features of chromatin, such as the formation of loops by CCCTC-binding factor (CTCF) and the division of loci into chromatin compartments. While much of the recent research and reviews have focused on CTCF loops, we discuss several new revelations that have emerged concerning chromatin compartments, with a particular focus on what is known about mechanistic drivers of compartmentalization. These insights challenge the traditional views of chromatin organization and reveal the complexity behind the formation and maintenance of chromatin compartments.

HiCrayon reveals distinct layers of multi-state 3D chromatin organization.

The co-visualization of chromatin conformation with 1D 'omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayon's visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization. Local version: https://github.com/JRowleyLab/HiCrayon Web version: https://jrowleylab.com/HiCrayon.