Epigenetic Manipulation of Transposable and Repetitive Elements.

This protocol describes methods to design, assemble, and validate tools for targeted activation or repression of single-copy and multi-copy genes, including repetitive and transposable elements. It uses transcription activator-like effector (TALE) technology combined with VP64 activator or Kruppel-associated box (KRAB) repressor, both of which are potent transcriptional regulators that modify the epigenetic state of endogenous DNA loci. This protocol has been successfully used to simultaneously modify expression patterns of thousands of LINE-1 transposable elements and satellite repeats, both in cell culture model systems and in preimplantation mouse embryos.

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome.

Although thousands of long non-coding RNAs (lncRNAs) are encoded in mammalian genomes, their mechanisms of action are poorly understood, in part because they are often expressed at lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes (X) to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a much larger number of target genes while maintaining specificity exclusively to genes on the X within each cell is not well understood. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP (also known as SPEN) to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. Expression of Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: chromosome-wide gene silencing and specificity to the X. This suggests a more general mechanism by which other low-abundance lncRNAs could balance specificity to, and robust control of, their regulatory targets.

SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding.

A fundamental question in gene regulation is how cell-type-specific gene expression is influenced by the packaging of DNA within the nucleus of each cell. We recently developed Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables mapping of higher-order interactions within the nucleus. SPRITE works by cross-linking interacting DNA, RNA and protein molecules and then mapping DNA-DNA spatial arrangements through an iterative split-and-pool barcoding method. All DNA molecules within a cross-linked complex are barcoded by repeatedly splitting complexes across a 96-well plate, ligating molecules with a unique tag sequence, and pooling all complexes into a single well before repeating the tagging. Because all molecules in a cross-linked complex are covalently attached, they will sort together throughout each round of split-and-pool and will obtain the same series of SPRITE tags, which we refer to as a barcode. The DNA fragments and their associated barcodes are sequenced, and all reads sharing identical barcodes are matched to reconstruct interactions. SPRITE accurately maps pairwise DNA interactions within the nucleus and measures higher-order spatial contacts occurring among up to thousands of simultaneously interacting molecules. Here, we provide a detailed protocol for the experimental steps of SPRITE, including a video ( https://youtu.be/6SdWkBxQGlg ). Furthermore, we provide an automated computational pipeline available on GitHub that allows experimenters to seamlessly generate SPRITE interaction matrices starting with raw fastq files. The protocol takes ~5 d from cell cross-linking to high-throughput sequencing for the experimental steps and 1 d for data processing.

Single-cell measurement of higher-order 3D genome organization with scSPRITE.

Although three-dimensional (3D) genome organization is central to many aspects of nuclear function, it has been difficult to measure at the single-cell level. To address this, we developed 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE). scSPRITE uses split-and-pool barcoding to tag DNA fragments in the same nucleus and their 3D spatial arrangement. Because scSPRITE measures multiway DNA contacts, it generates higher-resolution maps within an individual cell than can be achieved by proximity ligation. We applied scSPRITE to thousands of mouse embryonic stem cells and detected known genome structures, including chromosome territories, active and inactive compartments, and topologically associating domains (TADs) as well as long-range inter-chromosomal structures organized around various nuclear bodies. We observe that these structures exhibit different levels of heterogeneity across the population, with TADs representing dynamic units of genome organization across cells. We expect that scSPRITE will be a critical tool for studying genome structure within heterogeneous populations.

Xist nucleates local protein gradients to propagate silencing across the X chromosome.

The lncRNA Xist forms ∼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain ∼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.

RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

Deletion of LBR N-terminal domains recapitulates Pelger-Huet anomaly phenotypes in mouse without disrupting X chromosome inactivation.

Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia (GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes, hindering efforts to understand the molecular etiology of this disorder. Here we show, using CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr gene, generating a protein missing the N-terminal domains of LBR, presents a superior model of human PHA. Further, we address recent reports of a link between Lbr and defects in X chromosome inactivation (XCI) and show that our mouse mutant displays minor X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We suggest that our N-terminal deletion model provides a valuable pre-clinical tool to the research community and will aid in further understanding the etiology of PHA and the diverse functions of LBR.

LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo.

After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.

LINEs in mice: features, families, and potential roles in early development.

Approximately half of the mammalian genome is composed of repetitive elements, including LINE-1 (L1) elements. Because of their potential ability to transpose and integrate into other regions of the genome, their activation represents a threat to genome stability. Molecular pathways have emerged to tightly regulate and repress their transcriptional activity, including DNA methylation, histone modifications, and RNA pathways. It has become evident that Line-L1 elements are evolutionary diverse and dedicated repression pathways have been recently uncovered that discriminate between evolutionary old and young elements, with RNA-directed silencing mechanisms playing a prominent role. During periods of epigenetic reprogramming in development, specific classes of repetitive elements are upregulated, presumably due to the loss of most heterochromatic marks in this process. While we have learnt a lot on the molecular mechanisms that regulate Line-L1 expression over the last years, it is still unclear whether reactivation of Line-L1 after fertilization serves a functional purpose or it is a simple side effect of reprogramming.

Heterochromatin establishment at pericentromeres depends on nuclear position.

Mammalian development begins with fertilization of an oocyte by the sperm followed by genome-wide epigenetic reprogramming. This involves de novo establishment of chromatin domains, including the formation of pericentric heterochromatin. We dissected the spatiotemporal kinetics of the first acquisition of heterochromatic signatures of pericentromeric chromatin and found that the heterochromatic marks follow a temporal order that depends on a specific nuclear localization. We addressed whether nuclear localization of pericentric chromatin is required for silencing by tethering it to the nuclear periphery and show that this results in defective silencing and impaired development. Our results indicate that reprogramming of pericentromeric heterochromatin is functionally linked to its nuclear localization.