Jeannie T. Lee

During early development, female mammals inactivate one X chromosome to balance their X-linked gene dosage with males. While allelic choice is random in inbred mouse populations, choice can be significantly skewed in interstrain hybrids. The genetic basis of skewing has long been attributed to the mysterious “X chromosome controlling element(s)” ( Xce ) with different strengths among species, subspecies, and strains. When two X-chromosomes with different Xce strengths are inherited by offspring, the X chromosome with the stronger Xce will have a higher probability of remaining active. Here, we provide evidence that L1Tf repeats—a subfamily of long interspersed nuclear elements 1—plays a role in determining Xce strength. L1Tf elements form a condensed core within the inactive X (Xi) territory. Mouse strains with varying Xce strengths differ in the L1Tf copy number on the X chromosome, with the strength of the Xce allele being inversely related to L1Tf copy number. L1Tf expression mediates the Xce effect. However, in contrast to a prior report, L1Tf RNA does not coat the Xi. Rather, L1Tf promotes condensation of the Xi core. Intriguingly, L1Tfs recruit and sequester YY1 from active genes, accelerating XCI in cis. Thus, L1Tf copy number, expression, and binding of YY1 are key defining features of the Xce . We propose a model in which the Xce influences the choice of Xist alleles by promoting YY1 binding to the nucleation site for the initiation of Xist spreading.

Sirtuins are deacetylases implicated in stress responses and longevity in mammals^1,2. Although their differential impact on disease for the two sexes has been noted^3-7, the underlying reasons are unclear. Here, using Sirt7 as a model in mice, we examine the mechanisms leading to sex differences and find that Sirt7^-/- female mice have decreased fitness throughout their lifespan. Notably, SIRT7 preferentially localizes to the sex chromosomes. In female individuals, SIRT7 loss affects X-chromosome inactivation, the first arm of dosage compensation that equalizes X-linked gene expression between males and females^8-10. Xist is overexpressed and gene silencing becomes more efficient. However, SIRT7 loss has greatest impact on the active X (X_a) chromosome. The X_a chromosome becomes hyperacetylated at Lys36 of histone H3, structurally disorganized, prone to DNA damage and overexpressed. Increased X_a-chromosome expression leads to genome imbalance and augmented X-chromosome upregulation-the second arm of dosage compensation that balances X-chromosome versus autosomal gene expression. These data reveal an essential crosstalk between sirtuins and the sex chromosomes, with SIRT7 safeguarding X-chromosome integrity and dosage balance with autosomes. We propose that the sex bias in SIRT7 biology can be explained in part by unequal effects on the sex chromosomes.

The SINE-encoded B2 retrotransposon is an RNA Polymerase III (POL-III)-derived transcript whose expression is substantially upregulated during various cellular stress responses. Beyond retrotransposition, the B2 non-coding RNA can directly bind and repress the activity of RNA Polymerase II (POL-II), leading to a significant downregulation of transcripts during stress. Notably, our recent findings have shown that B2 is a self-cleaving ribozyme whose activity can be induced by interactions with chromatin-modifying factors through non-canonical epigenetic mechanisms that co-regulate its function across distinct chromatin-binding target loci. Here, by integrating RNA chemical probing, small-angle X-ray scattering, and 3D motif modeling, we determine structural ensemble-to-function relations for the B2 SINE ribozyme RNA. Genetic perturbations of the RNA suggest that the B2 SINE ribozyme has a well-defined secondary and dynamic tertiary structure that depends on the integrity of the critical region, which confers ribozymatic activity and repressive extent by POL-II. Using an RNA engineering approach, we examine the effects of point mutations, deletions of the main cleavage site, and deletions of the cleavage domain on the structural ensemble of the RNA. Combining this approach with in vitro and in vivo functional perturbation methods highlights the relationships between structural ensembles and various biologically relevant functional outcomes.

In mammals, X-linked dosage compensation involves X-chromosome inactivation to balance X chromosome dosage between males and females, and hyperactivation of the remaining X-chromosome (Xa-hyperactivation) to achieve X-autosome balance in both sexes. Studies of both processes have largely focused on coding genes and have not accounted for transposable elements which comprise 50% of the X-chromosome with numerous epigenetic functions. Here we develop a new bioinformatic pipeline tailored to repetitive elements with capability for allelic discrimination. We then apply the pipeline to our recent So-Smart-Seq analysis of single embryos to comprehensively interrogate whether X-linked transposable elements are subject to either X-chromosome inactivation or Xa-hyperactivation. We observe significant differences in repeat silencing in parentally driven “imprinted” versus zygotically driven “random” X-chromosome inactivation. Chromosomal positioning, genetic background and evolutionary age impact their silencing. In contrast, transposable elements do not undergo Xa-hyperactivation. Evolutionary and functional implications are discussed.

RNA G-quadruplexes (rG4s) are unusual RNA secondary structures formed by stacking arrays of guanine tetrads. Although thousands of potential rG4-forming motifs occur throughout the mammalian transcriptome, many single-stranded RNA (ssRNA) viruses are thought to be depleted of rG4-forming sequences. Using in silico methods, we examine rG4-forming potential in single-stranded RNA (ssRNA) viruses and observe that, while canonical rG4 motifs are depleted, non-canonical rG4 motifs occur at comparable or higher frequencies relative to the mammalian transcriptome. We ask if the non-canonical rG4’s can be leveraged to block viral replication and control infection using OC43, the coronavirus believed to be responsible for the 1889 “Russian Flu” pandemic. Profiling with “d-rG4-seq” confirms a dearth of folded rG4 in the OC43 RNA genome during natural infection. Intriguingly, rG4 ligands induce synthetic rG4 structures of a non-canonical nature. Significantly, induced rG4 inhibits viral replication and reduces infectivity. We show that the rG4 ligands act by disrupting the unique pattern of OC43 “discontinuous transcription”. Thus, rG4-targeting compounds present a potential therapeutic approach for targeting ssRNA viruses.

Xist, a pivotal player in X chromosome inactivation (XCI), has long been perceived as a cis-acting long noncoding RNA that binds exclusively to the inactive X chromosome (Xi). However, Xist’s ability to diffuse under select circumstances has also been documented, leading us to suspect that Xist RNA may have targets and functions beyond the Xi. Here, using female mouse embryonic stem cells (ES) and mouse embryonic fibroblasts (MEF) as models, we demonstrate that Xist RNA indeed can localize beyond the Xi. However, its binding is limited to ~100 genes in cells undergoing XCI (ES cells) and in post-XCI cells (MEFs). The target genes are diverse in function but are unified by their active chromatin status. Xist binds discretely to promoters of target genes in neighborhoods relatively depleted for Polycomb marks, contrasting with the broad, Polycomb-enriched domains reported for human XIST RNA. We find that Xist binding is associated with down-modulation of autosomal gene expression. However, unlike on the Xi, Xist binding does not lead to full silencing and also does not spread beyond the target gene. Over-expressing Xist in transgenic ES cells similarly leads to autosomal gene suppression, while deleting Xist’s Repeat B motif reduces autosomal binding and perturbs autosomal down-regulation. Furthermore, treating female ES cells with the Xist inhibitor, X1, leads to loss of autosomal suppression. Altogether, our findings reveal that Xist targets ~100 genes beyond the Xi, identify Repeat B as a crucial domain for its in-trans function in mice, and indicate that autosomal targeting can be disrupted by a small molecule inhibitor.

A major unsolved problem in epigenetics is how RNA regulates Polycomb repressive complex 2 (PRC2), a complex that trimethylates histone H3 Lys27 (H3K27me3) to form repressive chromatin. Key questions include how PRC2 binds RNA in vivo and what the functional consequences of binding are. In this Perspective, we expound on the viewpoint that RNA is integral to the stepwise regulation of PRC2 activity. Using the long non-coding RNA XIST and X chromosome inactivation as a model, we discuss evidence indicating that RNA is involved in PRC2 recruitment onto chromatin, in induction of its catalytic activity and in its eviction from chromatin. Studies have also implicated RNA in controlling promoter-proximal pausing of RNA polymerase II. The cumulative data argue that the functional consequences of PRC2–RNA interactions crucially depend on RNA conformation. We recognize that alternative hypotheses exist and therefore we attempt to integrate contrary data. Thus, although an RNA-rich landscape is emerging for Polycomb complexes, additional work is required to resolve a broad range of data interpretations.

Non-coding 7SL RNA is an ancestor to mammalian Alu and B1 SINE RNAs and is thought to function exclusively within the Signal Recognition Particle (SRP), aiding in the translocation of secretory proteins into the endoplasmic reticulum for export. Here, we discover a function of 7SL/SRP unrelated to protein secretion. Under acute heat shock, 7SL and SRP together selectively arrest cellular transcription and translation machineries during early response to stress. Under thermal stress, 7SL is upregulated, accumulates in the nucleus, and binds to target genes repressed by heat shock. Concurrently, in the cytosol, SRP binds to ribosomes and inhibits new protein synthesis. Translational suppression occurs independently of the signal peptide and is abrogated by depleting SRP. Translation inhibition extends to the mitochondria, as nuclear-encoded genes with mitochondrial functions are enriched among SRP targets. Thus, apart from its role in protein export, 7SL/SRP orchestrates a global response to acute stress that encompasses the nucleus, cytosol, and mitochondria across transcription and translation.