Enhancer switching in cell lineage priming is linked to eRNA, Brg1's AT-hook, and SWI/SNF recruitment.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.

Improved functions for non-linear sequence comparison using SEEKR.

SEquence Evaluation through k -mer Representation (SEEKR) is a method of sequence comparison that utilizes sequence substrings called k -mers to quantify non-linear similarity between nucleic acid species. We describe the development of new functions within SEEKR that enable end-users to estimate p-values that ascribe statistical significance to SEEKR-derived similarities as well as visualize different aspects of k -mer similarity. We apply the new functions to identify chromatin-enriched long noncoding RNAs (lncRNAs) that harbor XIST -like sequence fragments and show that several of these fragments are bound by XIST -associated proteins. We also highlight the best practice of using RNA-Seq data to evaluate support for lncRNA annotations prior to their in-depth study in cell types of interest.

SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells.

Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1, which also contains a purine-rich region in its 5' end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA.

Proximity-dependent recruitment of Polycomb repressive complexes by the lncRNA Airn.

During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between the Airn lncRNA and chromatin correlated with underlying intensity of PRC recruitment and PRC-directed modifications. Deletion of CpG islands that contact the Airn locus altered long-distance repression and PRC activity in a manner that correlated with changes in chromatin architecture. Our data imply that the extent to which Airn expression recruits PRCs to chromatin is controlled by DNA regulatory elements that modulate proximity of the Airn lncRNA product to its target DNA.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Ectopically expressed Airn lncRNA deposits Polycomb with a potency that rivals Xist.

We report that when expressed at similar levels from an isogenic locus, the Airn lncRNA induces Polycomb deposition with a potency that rivals Xist . However, when subject to the same degree of promoter activation, Xist is more abundant and more potent than Airn . Our data definitively demonstrate that the Airn lncRNA is functional and suggest that Xist achieved extreme potency in part by evolving mechanisms to promote its own abundance.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Oncogenic transcription factors and neogenes: New opportunities for cancer immunotherapy?

Vibert et al. (2022) demonstrate that oncogenic transcription factor fusion proteins bind otherwise silent genomic regions, producing RNAs that can be spliced, exported, and translated. These "neogenes" represent possible targets for immunotherapy and may even be universal byproducts of altered transcription in cancer.

Movement ecology of vulnerable lowland tapirs between areas of varying human disturbance.

BACKGROUND: Animal movement is a key ecological process that is tightly coupled to local environmental conditions. While agriculture, urbanisation, and transportation infrastructure are critical to human socio-economic improvement, these have spurred substantial changes in animal movement across the globe with potential impacts on fitness and survival. Notably, however, human disturbance can have differential effects across species, and responses to human activities are thus largely taxa and context specific. As human disturbance is only expected to worsen over the next decade it is critical to better understand how species respond to human disturbance in order to develop effective, case-specific conservation strategies. METHODS: Here, we use an extensive telemetry dataset collected over 22 years to fill a critical knowledge gap in the movement ecology of lowland tapirs (Tapirus terrestris) across areas of varying human disturbance within three biomes in southern Brazil: the Pantanal, Cerrado, and Atlantic Forest. RESULTS: From these data we found that the mean home range size across all monitored tapirs was 8.31 km2 (95% CI 6.53-10.42), with no evidence that home range sizes differed between sexes nor age groups. Interestingly, although the Atlantic Forest, Cerrado, and Pantanal vary substantially in habitat composition, levels of human disturbance, and tapir population densities, we found that lowland tapir movement behaviour and space use were consistent across all three biomes. Human disturbance also had no detectable effect on lowland tapir movement. Lowland tapirs living in the most altered habitats we monitored exhibited movement behaviour that was comparable to that of tapirs living in a near pristine environment. CONCLUSIONS: Contrary to our expectations, although we observed individual variability in lowland tapir space use and movement, human impacts on the landscape also had no measurable effect on their movement. Lowland tapir movement behaviour thus appears to exhibit very little phenotypic plasticity in response to human disturbance. Crucially, the lack of any detectable response to anthropogenic disturbance suggests that human modified habitats risk being ecological traps for tapirs and this information should be factored into conservation actions and species management aimed towards protecting lowland tapir populations.

The control of polycomb repressive complexes by long noncoding RNAs.

The polycomb repressive complexes 1 and 2 (PRCs; PRC1 and PRC2) are conserved histone-modifying enzymes that often function cooperatively to repress gene expression. The PRCs are regulated by long noncoding RNAs (lncRNAs) in complex ways. On the one hand, specific lncRNAs cause the PRCs to engage with chromatin and repress gene expression over genomic regions that can span megabases. On the other hand, the PRCs bind RNA with seemingly little sequence specificity, and at least in the case of PRC2, direct RNA-binding has the effect of inhibiting the enzyme. Thus, some RNAs appear to promote PRC activity, while others may inhibit it. The reasons behind this apparent dichotomy are unclear. The most potent PRC-activating lncRNAs associate with chromatin and are predominantly unspliced or harbor unusually long exons. Emerging data imply that these lncRNAs promote PRC activity through internal RNA sequence elements that arise and disappear rapidly in evolutionary time. These sequence elements may function by interacting with common subsets of RNA-binding proteins that recruit or stabilize PRCs on chromatin. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Classification of Long Noncoding RNAs by k-mer Content.

K-mer based comparisons have emerged as powerful complements to BLAST-like alignment algorithms, particularly when the sequences being compared lack direct evolutionary relationships. In this chapter, we describe methods to compare k-mer content between groups of long noncoding RNAs (lncRNAs), to identify communities of lncRNAs with related k-mer contents, to identify the enrichment of protein-binding motifs in lncRNAs, and to scan for domains of related k-mer contents in lncRNAs. Our step-by-step instructions are complemented by Python code deposited in Github. Though our chapter focuses on lncRNAs, the methods we describe could be applied to any set of nucleic acid sequences.

Analysis of RNA-protein networks with RNP-MaP defines functional hubs on RNA.

RNA-protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells.

Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs.

Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA's intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of interest. At least two approaches have been used to approximate lncRNA intracellular abundance: single-molecule sensitivity RNA fluorescence in situ hybridization (smFISH) and single-gene, calibrated reverse-transcription followed by quantitative PCR (RT-qPCR). However, like all experimental approaches, these methods have their limitations. smFISH, when analyzed using diffraction-limited microscopy, can underestimate intracellular abundance, especially for lncRNAs that accumulate in focused subcellular regions. Calibrated RT-qPCR may return inaccurate estimates of abundance because individual PCR amplicons spaced across the length of a transcript can vary in their efficiency of reverse transcription. Here, we describe a sequencing-based approach that is straightforward, orthogonal to smFISH and RT-qPCR, and can be used to approximate the intracellular abundance for most expressed long RNAs (lncRNAs and mRNAs) in a cell type of interest. Firstly, the average weight of total RNA per cell for the cell type of interest is estimated by replicate rounds of RNA purification from a known number of cells. Secondly, an rRNA-depletion RNA-Seq protocol is performed after adding spike-in control RNAs to a known quantity of total cellular RNA. Lastly, by comparing read counts per transcript to a standard curve derived from the spiked-in RNAs, the intracellular abundance for each transcript is estimated. The sequencing-based approach provides a powerful complement to existing methods, particularly in situations where it is desirable to quantify the abundance of multiple lncRNAs and/or mRNAs simultaneously.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity.

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

Multimodal Long Noncoding RNA Interaction Networks: Control Panels for Cell Fate Specification.

Lineage specification in early development is the basis for the exquisitely precise body plan of multicellular organisms. It is therefore critical to understand cell fate decisions in early development. Moreover, for regenerative medicine, the accurate specification of cell types to replace damaged/diseased tissue is strongly dependent on identifying determinants of cell identity. Long noncoding RNAs (lncRNAs) have been shown to regulate cellular plasticity, including pluripotency establishment and maintenance, differentiation and development, yet broad phenotypic analysis and the mechanistic basis of their function remains lacking. As components of molecular condensates, lncRNAs interact with almost all classes of cellular biomolecules, including proteins, DNA, mRNAs, and microRNAs. With functions ranging from controlling alternative splicing of mRNAs, to providing scaffolding upon which chromatin modifiers are assembled, it is clear that at least a subset of lncRNAs are far from the transcriptional noise they were once deemed. This review highlights the diversity of lncRNA interactions in the context of cell fate specification, and provides examples of each type of interaction in relevant developmental contexts. Also highlighted are experimental and computational approaches to study lncRNAs.

lncRNA-Induced Spread of Polycomb Controlled by Genome Architecture, RNA Abundance, and CpG Island DNA.

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.