ATR phosphorylates DHX9 at serine 321 to suppress R-loop accumulation upon genotoxic stress.

Aberrant DNA/RNA hybrids (R-loops) formed during transcription and replication disturbances pose threats to genome stability. DHX9 is an RNA helicase involved in R-loop resolution, but how DHX9 is regulated in response to genotoxic stress remains unclear. Here we report that DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation primarily induced by ATR after DNA damage. Phosphorylation of DHX9 at S321 promotes its interaction with γH2AX, BRCA1 and RPA, and is required for its association with R-loops under genotoxic stress. Inhibition of ATR or expression of the non-phosphorylatable DHX9S321A prevents DHX9 from interacting with RPA and R-loops, leading to the accumulation of stress-induced R-loops. Furthermore, depletion of RPA reduces the association between DHX9 and γH2AX, and in vitro binding analysis confirms a direct interaction between DHX9 and RPA. Notably, cells with the non-phosphorylatable DHX9S321A variant exhibit hypersensitivity to genotoxic stress, while those expressing the phosphomimetic DHX9S321D variant prevent R-loop accumulation and display resistance to DNA damage agents. In summary, we uncover a new mechanism by which ATR directly regulates DHX9 through phosphorylation to eliminate stress-induced R-loops.

TERRA regulates DNA G-quadruplex formation and ATRX recruitment to chromatin.

The genome consists of non-B-DNA structures such as G-quadruplexes (G4) that are involved in the regulation of genome stability and transcription. Telomeric-repeat containing RNA (TERRA) is capable of folding into G-quadruplex and interacting with chromatin remodeler ATRX. Here we show that TERRA modulates ATRX occupancy on repetitive sequences and over genes, and maintains DNA G-quadruplex structures at TERRA target and non-target sites in mouse embryonic stem cells. TERRA prevents ATRX from binding to subtelomeric regions and represses H3K9me3 formation. G4 ChIP-seq reveals that G4 abundance decreases at accessible chromatin regions, particularly at transcription start sites (TSS) after TERRA depletion; such G4 reduction at TSS is associated with elevated ATRX occupancy and differentially expressed genes. Loss of ATRX alleviates the effect of gene repression caused by TERRA depletion. Immunostaining analyses demonstrate that knockdown of TERRA diminishes DNA G4 signals, whereas silencing ATRX elevates G4 formation. Our results uncover an epigenetic regulation by TERRA that sequesters ATRX and preserves DNA G4 structures.

XPF activates break-induced telomere synthesis.

Alternative Lengthening of Telomeres (ALT) utilizes a recombination mechanism and break-induced DNA synthesis to maintain telomere length without telomerase, but it is unclear how cells initiate ALT. TERRA, telomeric repeat-containing RNA, forms RNA:DNA hybrids (R-loops) at ALT telomeres. We show that depleting TERRA using an RNA-targeting Cas9 system reduces ALT-associated PML bodies, telomere clustering, and telomere lengthening. TERRA interactome reveals that TERRA interacts with an extensive subset of DNA repair proteins in ALT cells. One of TERRA interacting proteins, the endonuclease XPF, is highly enriched at ALT telomeres and recruited by telomeric R-loops to induce DNA damage response (DDR) independent of CSB and SLX4, and thus triggers break-induced telomere synthesis and lengthening. The attraction of BRCA1 and RAD51 at telomeres requires XPF in FANCM-deficient cells that accumulate telomeric R-loops. Our results suggest that telomeric R-loops activate DDR via XPF to promote homologous recombination and telomere replication to drive ALT.

Jpx RNA regulates CTCF anchor site selection and formation of chromosome loops.

Chromosome loops shift dynamically during development, homeostasis, and disease. CCCTC-binding factor (CTCF) is known to anchor loops and construct 3D genomes, but how anchor sites are selected is not yet understood. Here, we unveil Jpx RNA as a determinant of anchor selectivity. Jpx RNA targets thousands of genomic sites, preferentially binding promoters of active genes. Depleting Jpx RNA causes ectopic CTCF binding, massive shifts in chromosome looping, and downregulation of >700 Jpx target genes. Without Jpx, thousands of lost loops are replaced by de novo loops anchored by ectopic CTCF sites. Although Jpx controls CTCF binding on a genome-wide basis, it acts selectively at the subset of developmentally sensitive CTCF sites. Specifically, Jpx targets low-affinity CTCF motifs and displaces CTCF protein through competitive inhibition. We conclude that Jpx acts as a CTCF release factor and shapes the 3D genome by regulating anchor site usage.

Embryonic Steroids Control Developmental Programming of Energy Balance.

Glucose is a major energy source for growth. At birth, neonates must change their energy source from maternal supply to its own glucose production. The mechanism of this transition has not been clearly elucidated. To evaluate the possible roles of steroids in this transition, here we examine the defects associated with energy production of a mouse line that cannot synthesize steroids de novo due to the disruption of its Cyp11a1 (cytochrome P450 family 11 subfamily A member 1) gene. The Cyp11a1 null embryos had insufficient blood insulin and failed to store glycogen in the liver since embryonic day 16.5. Their blood glucose dropped soon after maternal deprivation, and the expression of hepatic gluconeogenic and glycogenic genes were reduced. Insulin was synthesized in the mutant fetal pancreas but failed to be secreted. Maternal glucocorticoid supply rescued the amounts of blood glucose, insulin, and liver glycogen in the fetus but did not restore expression of genes for glycogen synthesis, indicating the requirement of de novo glucocorticoid synthesis for glycogen storage. Thus, our investigation of Cyp11a1 null embryos reveals that the energy homeostasis is established before birth, and fetal steroids are required for the regulation of glycogen synthesis, hepatic gluconeogenesis, and insulin secretion at the fetal stage.

iDRiP for the systematic discovery of proteins bound directly to noncoding RNA.

More than 90% of the human genome is transcribed into noncoding RNAs, but their functional characterization has lagged behind. A major bottleneck in the understanding of their functions and mechanisms has been a dearth of systematic methods for identifying interacting protein partners. There now exist several methods, including identification of direct RNA interacting proteins (iDRiP), chromatin isolation by RNA purification (ChIRP), and RNA antisense purification, each previously applied towards identifying a proteome for the prototype noncoding RNA, Xist. iDRiP has recently been modified to successfully identify proteomes for two additional noncoding RNAs of interest, TERRA and U1 RNA. Here we describe the modified protocol in detail, highlighting technical differences that facilitate capture of various noncoding RNAs. The protocol can be applied to short and long RNAs in both cultured cells and tissues, and requires ~1 week from start to finish. Here we also perform a comparative analysis between iDRiP and ChIRP. We obtain partially overlapping profiles, but find that iDRiP yields a greater number of specific proteins and fewer mitochondrial contaminants. With an increasing number of essential long noncoding RNAs being described, robust RNA-centric protein capture methods are critical for the probing of noncoding RNA function and mechanism.

Expression of Telomeric Repeat-Containing RNA Decreases in Sarcopenia and Increases after Exercise and Nutrition Intervention.

Sarcopenia is defined as aging-related loss of muscle mass and function. Telomere length in chromosomes shortens with age and is modulated by telomeric repeat-containing RNA (TERRA). This study aimed to explore the impact of aging and sarcopenia on telomere length and TERRA expression, and changes following strengthening exercise and nutrition intervention (supplement of branched-chain amino acids, calcium and vitamin D3) for 12 weeks in the sarcopenic population. Older adults (≥65 years old) were divided into non-sarcopenic controls (n = 36) and sarcopenic individuals (n = 36) after measurement of grip strength and body composition. The relative telomere length of leukocytes in all research participants was evaluated using the T/S ratio (telomere/single copy gene), and relative TERRA expression of leukocytes was determined by reverse-transcription qPCR (RT-qPCR). Generalized estimating equation (GEE) was used to analyze the influence of sarcopenia and intervention on the outcomes. There was no significant difference in telomere length between control subjects and participants with sarcopenia. TERRA expression was lower in sarcopenic participants compared to that in non-sarcopenic controls (5.18 ± 2.98 vs. 2.51 ± 1.89; p < 0.001). In the sarcopenic group, intervention significantly increased TERRA expression, but not telomere length. The GEE analysis demonstrated that TERRA expression was negatively associated with sarcopenia (ß coefficient = -2.705, p < 0.001) but positively associated with intervention (ß coefficient = 1.599, p = 0.023). Sarcopenia is associated with a decrease in TERRA expression in leukocytes. Rebound TERRA expression (returning to the level similar to the non-sarcopenic controls) was observed in the sarcopenic group after exercise and nutrition intervention. Future studies are warranted to examine the potential of TERRA as a biomarker for sarcopenia and its subsequent responses to intervention.

Functions and properties of nuclear lncRNAs-from systematically mapping the interactomes of lncRNAs.

Protein and DNA have been considered as the major components of chromatin. But beyond that, an increasing number of studies show that RNA occupies a large amount of chromatin and acts as a regulator of nuclear architecture. A significant fraction of long non-coding RNAs (lncRNAs) prefers to stay in the nucleus and cooperate with protein complexes to modulate epigenetic regulation, phase separation, compartment formation, and nuclear organization. An RNA strand also can invade into double-stranded DNA to form RNA:DNA hybrids (R-loops) in living cells, contributing to the regulation of gene expression and genomic instability. In this review, we discuss how nuclear lncRNAs orchestrate cellular processes through their interactions with proteins and DNA and summarize the recent genome-wide techniques to study the functions of lncRNAs by revealing their interactomes in vivo.

FANCM suppresses DNA replication stress at ALT telomeres by disrupting TERRA R-loops.

Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. Among the many prominent features of ALT cells, C-circles (CC) formation is considered to be the most specific and quantifiable biomarker of ALT. However, the molecular mechanism behind the initiation and maintenance of CC formation in ALT cells is still largely unknown. We reported previously that depletion of the FANCM complex (FANCM-FAAP24-MHF1&2) in ALT cells induced pronounced replication stress, which primarily takes place at their telomeres. Here, we characterized the changes in ALT associated phenotypes in cells deficient of the FANCM complex. We found that depletion of FAAP24 or FANCM, but not MHF1&2, induces a dramatic increase of CC formation. Most importantly, we identified multiple DNA damage response (DDR) and DNA repair pathways that stimulate the dramatic increase of CC formation in FANCM deficient cells, including the dissolvase complex (BLM-TOP3A-RMI1/2, or BTR), DNA damage checkpoint kinases (ATR and Chk1), HR proteins (BRCA2, PALB2, and Rad51), as well as proteins involved in Break-Induced Replication (BIR) (POLD1 and POLD3). In addition, FANCD2, another Fanconi Anemia (FA) protein, is also required for CC formation, likely through promoting the recruitment of BLM to the replication stressed ALT telomeres. Finally, we demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. Taken together, our data suggest that FANCM prevents replisomes from stalling/collapsing at ALT telomeres by disrupting TERRA R-loops.

SMCHD1 Merges Chromosome Compartments and Assists Formation of Super-Structures on the Inactive X.

Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.

TERRA RNA Antagonizes ATRX and Protects Telomeres.

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.

PAR-TERRA directs homologous sex chromosome pairing.

In mammals, homologous chromosomes rarely pair outside meiosis. One exception is the X chromosome, which transiently pairs during X-chromosome inactivation (XCI). How two chromosomes find each other in 3D space is not known. Here, we reveal a required interaction between the X-inactivation center (Xic) and the telomere in mouse embryonic stem (ES) cells. The subtelomeric, pseudoautosomal regions (PARs) of the two sex chromosomes (X and Y) also undergo pairing in both female and male cells. PARs transcribe a class of telomeric RNA, dubbed PAR-TERRA, which accounts for a vast majority of all TERRA transcripts. PAR-TERRA binds throughout the genome, including to the PAR and Xic. During X-chromosome pairing, PAR-TERRA anchors the Xic to the PAR, creating a 'tetrad' of pairwise homologous interactions (Xic-Xic, PAR-PAR, and Xic-PAR). Xic pairing occurs within the tetrad. Depleting PAR-TERRA abrogates pairing and blocks initiation of XCI, whereas autosomal PAR-TERRA induces ectopic pairing. We propose a 'constrained diffusion model' in which PAR-TERRA creates an interaction hub to guide Xic homology searching during XCI.

Destabilization of B2 RNA by EZH2 Activates the Stress Response.

More than 98% of the mammalian genome is noncoding, and interspersed transposable elements account for ∼50% of noncoding space. Here, we demonstrate that a specific interaction between the polycomb protein EZH2 and RNA made from B2 SINE retrotransposons controls stress-responsive genes in mouse cells. In the heat-shock model, B2 RNA binds stress genes and suppresses their transcription. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as a "speed bump" against advancement of RNA polymerase II, and temperature stress releases the brakes on transcriptional elongation. These data attribute a new function to EZH2 that is independent of its histone methyltransferase activity and reconcile how EZH2 can be associated with both gene repression and activation. Our study reveals that EZH2 and B2 together control activation of a large network of genes involved in thermal stress.

Paradoxical Roles of Elongation Factor-2 Kinase in Stem Cell Survival.

Protein synthesis inhibition is an immediate response during stress to switch the composition of protein pool in order to adapt to the new environment. It was reported that this response could be either protective or deleterious. However, how cells choose to live or die upon protein synthesis inhibition is largely unknown. Previously, we have shown that elongation factor-2 kinase (eEF2K), a protein kinase that suppresses protein synthesis during elongation phase, is a positive regulator of apoptosis both in vivo and in vitro Consistently, here we report that knock-out of eEF2K protects mice from a lethal dose of whole-body ionizing radiation at 8 Gy by reducing apoptosis levels in both bone marrow and gastrointestinal tracts. Surprisingly, similar to the loss of p53, eEF2K deficiency results in more severe damage to the gastrointestinal tract at 20 Gy with the increased mitotic cell death in small intestinal stem cells. Furthermore, using epithelial cell lines, we showed that eEF2K is required for G2/M arrest induced by radiation to prevent mitotic catastrophe in a p53-independent manner. Specifically, we observed the elevation of Akt/ERK activity as well as the reduction of p21 expression in Eef2k(-/-) cells. Therefore, eEF2K also provides a protective strategy to maintain genomic integrity by arresting cell cycle in response to stress. Our results suggest that protective versus pro-apoptotic roles of eEF2K depend on the type of cells: eEF2K is protective in highly proliferative cells, such as small intestinal stem cells and cancer cells, which are more susceptible to mitotic catastrophe.

Germline quality control: eEF2K stands guard to eliminate defective oocytes.

The control of germline quality is critical to reproductive success and survival of a species; however, the mechanisms underlying this process remain unknown. Here, we demonstrate that elongation factor 2 kinase (eEF2K), an evolutionarily conserved regulator of protein synthesis, functions to maintain germline quality and eliminate defective oocytes. We show that disruption of eEF2K in mice reduces ovarian apoptosis and results in the accumulation of aberrant follicles and defective oocytes at advanced reproductive age. Furthermore, the loss of eEF2K in Caenorhabditis elegans results in a reduction of germ cell death and significant decline in oocyte quality and embryonic viability. Examination of the mechanisms by which eEF2K regulates apoptosis shows that eEF2K senses oxidative stress and quickly downregulates short-lived antiapoptotic proteins, XIAP and c-FLIPL by inhibiting global protein synthesis. These results suggest that eEF2K-mediated inhibition of protein synthesis renders cells susceptible to apoptosis and functions to eliminate suboptimal germ cells.

Human CYP11A1 promoter drives Cre recombinase expression in the brain in addition to adrenals and gonads.

The first step of steroid biosynthesis is catalyzed by cytochrome P450scc, encoded by CYP11A1. To achieve steroidogenic tissue-specific inactivation of genes in vivo by the Cre-loxP approach, we used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the tissues that produce steroids. The resulting SCC-Cre mice express high levels of Cre in the adrenal cortex and gonads at the same sites as that for the endogenous CYP11A1 expression. In addition, Cre activity was found in the diencephalon and midbrain. In the developing brain, the Cre activity was first detected in the embryonic day 10.5. Our study is the first to show that the 4.4-kb CYP11A1 promoter is transcriptionally active in the brain in vivo.

Alpha-kinase 1, a new component in apical protein transport.

A key aspect in the structure of epithelial cells is the maintenance of a polarized organization based on a highly specific sorting machinery for cargo destined for the apical or the basolateral membrane domain at the exit site of the trans-Golgi network. We could recently identify two distinct post-trans-Golgi network vesicle populations that travel along separate routes to the plasma membrane, a lipid raft-dependent and a lipid raft-independent pathway. A new component of raft-carrying apical vesicles is alpha-kinase 1 (ALPK1), which was identified in immunoisolated vesicles carrying raft-associated sucrase-isomaltase (SI). This kinase was absent from vesicles carrying raft-non-associated lactase-phlorizin hydrolase. The expression of ALPK1 increases by the time of epithelial cell differentiation, whereas the intracellular localization of ALPK1 on apical transport vesicles was confirmed by confocal analysis. A phosphorylation assay on isolated SI-carrying vesicles revealed the phosphorylation of a protein band of about 105 kDa, which could be identified as the motor protein myosin I. Finally, a specific reduction of ALPK1-expression by RNA interference results in a significant decrease in the apical delivery of SI. Taken together, our data suggest that the phosphorylation of myosin I by ALPK1 is an essential process in the apical trafficking of raft-associated SI.

Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1.

Steroid deficiencies are diseases affecting salt levels, sugar levels, and sexual differentiation. To study steroid deficiency in more detail, we used a gene-targeting technique to insert a neo gene into the first exon to disrupt Cyp11a1, the first gene in steroid biosynthetic pathways. Cyp11a1 null mice do not synthesize steroids. They die shortly after birth, but can be rescued by steroid injection. Due to the lack of feedback inhibition by glucocorticoid, their circulating ACTH levels are exceedingly high; this results in ectopic Cyp21 gene expression in the testis. Male Cyp11a1 null mice are feminized with female external genitalia and underdeveloped male accessory sex organs. Their testis, epididymis, and vas deferens are present, but undersized. In addition, their adrenals and gonads accumulate excessive amounts of lipid. The lack of steroid production, abnormal gene expression, and aberrant reproductive organ development resemble various steroid deficiency syndromes, making these mice good models for studies of steroid function and regulation.