3D organization of enhancers in MuSCs.

Skeletal muscle stem cells (MuSCs), also known as satellite cells, are essential for muscle growth and injury induced regeneration. In healthy adult muscle, MuSCs remain in a quiescent state located in a specialized niche beneath the basal lamina. Upon injury, these dormant MuSCs can quickly activate to re-enter the cell cycle and differentiate into new myofibers, while a subset undergoes self-renewal and returns to quiescence to restore the stem cell pool. The myogenic lineage progression is intricately controlled by complex intrinsic and extrinsic cues and coupled with dynamic transcriptional programs. In transcriptional regulation, enhancers are key regulatory elements controlling spatiotemporal gene expression through physical contacting promoters of target genes. The three-dimensional (3D) chromatin architecture is known to orchestrate the establishment of proper enhancer-promoter interactions throughout development and aging. However, studies dissecting the 3D organization of enhancers in MuSCs are just emerging. Here, we provide an overview of the general properties of enhancers and newly developed methods for assessing their activity. In particular, we summarize recent discoveries regarding the 3D rewiring of enhancers during MuSC specification, lineage progression as well as aging.

ATF3 induction prevents precocious activation of skeletal muscle stem cell by regulating H2B expression.

Skeletal muscle stem cells (also called satellite cells, SCs) are important for maintaining muscle tissue homeostasis and damage-induced regeneration. However, it remains poorly understood how SCs enter cell cycle to become activated upon injury. Here we report that AP-1 family member ATF3 (Activating Transcription Factor 3) prevents SC premature activation. Atf3 is rapidly and transiently induced in SCs upon activation. Short-term deletion of Atf3 in SCs accelerates acute injury-induced regeneration, however, its long-term deletion exhausts the SC pool and thus impairs muscle regeneration. The Atf3 loss also provokes SC activation during voluntary exercise and enhances the activation during endurance exercise. Mechanistically, ATF3 directly activates the transcription of Histone 2B genes, whose reduction accelerates nucleosome displacement and gene transcription required for SC activation. Finally, the ATF3-dependent H2B expression also prevents genome instability and replicative senescence in SCs. Therefore, this study has revealed a previously unknown mechanism for preserving the SC population by actively suppressing precocious activation, in which ATF3 is a key regulator.

In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing.

Skeletal muscle satellite cells (SCs) are adult stem cells responsible for muscle development and injury-induced muscle regeneration. Functional elucidation of intrinsic regulatory factors governing SC activity is constrained partially by the technological limitations in editing SCs in vivo. Although the power of CRISPR/Cas9 in genome manipulation has been widely documented, its application in endogenous SCs remains largely untested. Our recent study generates a muscle-specific genome editing system leveraging the Cre-dependent Cas9 knockin mice and AAV9-mediated sgRNAs delivery, which allows gene disruption in SCs in vivo. Here, we illustrate the step-by-step procedure for achieving efficient editing using the above system.

Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export.

Skeletal muscle stem cells (also known as satellite cells [SCs]) are essential for muscle regeneration and the regenerative activities of SCs are intrinsically governed by gene regulatory mechanisms, but the post-transcriptional regulation in SCs remains largely unknown. N(6)-methyladenosine (m6A) modification of RNAs is the most pervasive and highly conserved RNA modification in eukaryotic cells; it exerts powerful impact on almost all aspects of mRNA processing that is mainly endowed by its binding with m6A reader proteins. In this study, we investigate the previously uncharacterized regulatory roles of YTHDC1, an m6A reader in mouse SCs. Our results demonstrate that YTHDC1 is an essential regulator of SC activation and proliferation upon acute injury-induced muscle regeneration. The induction of YTHDC1 is indispensable for SC activation and proliferation; thus, inducible YTHDC1 depletion almost abolishes SC regenerative capacity. Mechanistically, transcriptome-wide profiling using LACE-seq in both SCs and mouse C2C12 myoblasts identifies m6A-mediated binding targets of YTHDC1. Next, splicing analysis defines splicing mRNA targets of m6A-YTHDC1. Furthermore, nuclear export analysis also leads to the identification of potential mRNA export targets of m6A-YTHDC1 in SCs and C2C12 myoblasts;interestingly, some mRNAs can be regulated at both splicing and export levels. Lastly, we map YTHDC1 interacting protein partners in myoblasts and unveil a myriad of factors governing mRNA splicing, nuclear export, and transcription, among which hnRNPG appears to be a bona fide interacting partner of YTHDC1. Altogether, our findings uncover YTHDC1 as an essential factor controlling SC regenerative ability through multifaceted gene regulatory mechanisms in mouse myoblast cells.

Multiscale 3D genome reorganization during skeletal muscle stem cell lineage progression and aging.

Little is known about three-dimensional (3D) genome organization in skeletal muscle stem cells [also called satellite cells (SCs)]. Here, we comprehensively map the 3D genome topology reorganization during mouse SC lineage progression. Specifically, rewiring at the compartment level is most pronounced when SCs become activated. Marked loss in topologically associating domain (TAD) border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can form TAD clusters and super-enhancer-containing TAD clusters orchestrate stage-specific gene expression. Furthermore, we uncover that transcription factor PAX7 is pivotal in enhancer-promoter (E-P) loop formation. We also identify cis-regulatory elements that are crucial for local chromatin organization at Pax7 locus and Pax7 expression. Lastly, we unveil that geriatric SC displays a prominent gain in long-range contacts and loss of TAD border insulation. Together, our results uncover that 3D chromatin extensively reorganizes at multiple architectural levels and underpins the transcriptome remodeling during SC lineage development and SC aging.

seRNA PAM controls skeletal muscle satellite cell proliferation and aging through trans regulation of Timp2 expression synergistically with Ddx5.

Muscle satellite cells (SCs) are responsible for muscle homeostasis and regeneration and lncRNAs play important roles in regulating SC activities. Here, in this study, we identify PAM (Pax7 Associated Muscle lncRNA) that is induced in activated/proliferating SCs upon injury to promote SC proliferation as myoblast cells. PAM is generated from a myoblast-specific super-enhancer (SE); as a seRNA it binds with a number of target genomic loci predominantly in trans. Further studies demonstrate that it interacts with Ddx5 to tether PAM SE to its inter-chromosomal targets Timp2 and Vim to activate the gene expression. Lastly, we show that PAM expression is increased in aging SCs, which leads to enhanced inter-chromosomal interaction and target genes upregulation. Altogether, our findings identify PAM as a previously unknown lncRNA that regulates both SC proliferation and aging through its trans gene regulatory activity.

Lockd promotes myoblast proliferation and muscle regeneration via binding with DHX36 to facilitate 5' UTR rG4 unwinding and Anp32e translation.

Adult muscle stem cells, also known as satellite cells (SCs), play pivotal roles in muscle regeneration, and long non-coding RNA (lncRNA) functions in SCs remain largely unknown. Here, we identify a lncRNA, Lockd, which is induced in activated SCs upon acute muscle injury. We demonstrate that Lockd promotes SC proliferation; deletion of Lockd leads to cell-cycle arrest, and in vivo repression of Lockd in mouse muscles hinders regeneration process. Mechanistically, we show that Lockd directly interacts with RNA helicase DHX36 and the 5'end of Lockd possesses the strongest binding with DHX36. Furthermore, we demonstrate that Lockd stabilizes the interaction between DHX36 and EIF3B proteins; synergistically, this complex unwinds the RNA G-quadruplex (rG4) structure formed at Anp32e mRNA 5' UTR and promotes the translation of ANP32E protein, which is required for myoblast proliferation. Altogether, our findings identify a regulatory Lockd/DHX36/Anp32e axis that promotes myoblast proliferation and acute-injury-induced muscle regeneration.

CRISPR/Cas9/AAV9-mediated in vivo editing identifies MYC regulation of 3D genome in skeletal muscle stem cell.

Skeletal muscle satellite cells (SCs) are stem cells responsible for muscle development and regeneration. Although CRISPR/Cas9 has been widely used, its application in endogenous SCs remains elusive. Here, we generate mice expressing Cas9 in SCs and achieve robust editing in juvenile SCs at the postnatal stage through AAV9-mediated short guide RNA (sgRNA) delivery. Additionally, we reveal that quiescent SCs are resistant to CRISPR/Cas9-mediated editing. As a proof of concept, we demonstrate efficient editing of master transcription factor (TF) Myod1 locus using the CRISPR/Cas9/AAV9-sgRNA system in juvenile SCs. Application on two key TFs, MYC and BCL6, unveils distinct functions in SC activation and muscle regeneration. Particularly, we reveal that MYC orchestrates SC activation through regulating 3D genome architecture. Its depletion results in strengthening of the topologically associating domain boundaries thus may affect gene expression. Altogether, our study establishes a platform for editing endogenous SCs that can be harnessed to elucidate the functionality of key regulators governing SC activities.

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions.

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.

MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation.

Emerging evidence supports roles of enhancer RNAs (eRNAs) in regulating target gene. Here, we study eRNA regulation and function during skeletal myoblast differentiation. We provide a panoramic view of enhancer transcription and categorization of eRNAs. Master transcription factor MyoD is crucial in activating eRNA production. Super enhancer (se) generated seRNA-1 and -2 promote myogenic differentiation in vitro and in vivo. seRNA-1 regulates expression levels of two nearby genes, myoglobin (Mb) and apolipoprotein L6 (Apol6), by binding to heterogeneous nuclear ribonucleoprotein L (hnRNPL). A CAAA tract on seRNA-1 is essential in mediating seRNA-1/hnRNPL binding and function. Disruption of seRNA-1-hnRNPL interaction attenuates Pol II and H3K36me3 deposition at the Mb locus, in coincidence with the reduction of its transcription. Furthermore, analyses of hnRNPL binding transcriptome-wide reveal its association with eRNAs is a general phenomenon in multiple cells. Collectively, we propose that eRNA-hnRNPL interaction represents a mechanism contributing to target mRNA activation.

MyoD- and FoxO3-mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation.

Super-enhancers (SEs) are cis-regulatory elements enriching lineage specific key transcription factors (TFs) to form hotspots. A paucity of identification and functional dissection promoted us to investigate SEs during myoblast differentiation. ChIP-seq analysis of histone marks leads to the uncovering of SEs which remodel progressively during the course of differentiation. Further analyses of TF ChIP-seq enable the definition of SE hotspots co-bound by the master TF, MyoD and other TFs, among which we perform in-depth dissection for MyoD/FoxO3 interaction in driving the hotspots formation and SE activation. Furthermore, using Myogenin as a model locus, we elucidate the hierarchical and complex interactions among hotspots during the differentiation, demonstrating SE function is propelled by the physical and functional cooperation among hotspots. Finally, we show MyoD and FoxO3 are key in orchestrating the Myogenin hotspots interaction and activation. Altogether our results identify muscle-specific SEs and provide mechanistic insights into the functionality of SE.

The role of FADD in pancreatic cancer cell proliferation and drug resistance.

Pancreatic cancer has one of the poorest patient outcomes and is highly resistant to chemotherapy. Identifying the molecular mechanisms involved in drug resistance is critical in the development of novel strategies to treat pancreatic cancer. The results of the present study demonstrate that Fas-associated death domain protein (FADD), a classical adaptor protein mediating apoptotic stimuli-induced cell death, protects pancreatic cancer cells from drug-induced apoptosis. In contrast to its classical apoptotic roles, it was observed that FADD is required for pancreatic cancer cell proliferation and that it is overexpressed to varying degrees in various types of pancreatic cancer cell. This leads to differing levels of drug resistance in pancreatic cancer cells, where drug resistance is positively correlated with FADD expression. Notably, the results of the present study demonstrate that FADD protects pancreatic cancer cells from drug-induced apoptosis, while RNA interference of FADD sensitizes drug-resistant cells to Adriamycin®-mediated apoptosis. The results of the present study reveal unexpected roles for FADD in pancreatic cancer cell proliferation and drug resistance.

Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity.

Malat1 is one of the most abundant long non-coding RNAs in various cell types; its exact cellular function is still a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both in vitro and in vivo assays, we demonstrate that Malat1 has a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knockdown of Malat1 accelerates the myogenic differentiation in cultured cells. Consistently, Malat1 knockout mice display enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic mdx mice also improves the muscle regeneration. Mechanistically, in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD-binding loci, causing trimethylation of histone 3 lysine 9 (H3K9me3), which suppresses the target gene expression. Upon differentiation, the pro-myogenic miR-181a is increased and targets the nuclear Malat1 transcripts for degradation through Ago2-dependent nuclear RNA-induced silencing complex machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1ß/HDAC1-repressive complex and displacement by a Set7-containing activating complex, which allows MyoD trans-activation to occur. Together, our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation.

Fas-associated protein with death domain (FADD) regulates autophagy through promoting the expression of Ras homolog enriched in brain (Rheb) in human breast adenocarcinoma cells.

FADD (Fas-associated protein with death domain) is a classical adaptor protein in apoptosis. Increasing evidences have shown that FADD is also implicated in cell cycle progression, proliferation and tumorigenesis. The role of FADD in cancer remains largely unexplored. In this study, In Silico Analysis using Oncomine and Kaplan Meier plotter revealed that FADD is significantly up-regulated in breast cancer tissues and closely associated with a poor prognosis in patients with breast cancer. To better understanding the FADD functions in breast cancer, we performed proteomics analysis by LC-MS/MS detection and found that Rheb-mTORC1 pathway was dysregulated in MCF-7 cells when FADD knockdown. The mTORC1 pathway is a key regulator in many processes, including cell growth, metabolism and autophagy. Here, FADD interference down-regulated Rheb expression and repressed mTORC1 activity in breast cancer cell lines. The autophagy was induced by FADD deficiency in MCF7 or MDA-231 cells but rescued by recovering Rheb expression. Similarly, growth defect in FADD-knockdown cells was also restored by Rheb overexpression. These findings implied a novel role of FADD in tumor progression via Rheb-mTORC1 pathway in breast cancer.

Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces.

Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, "Candidatus Saccharibacteria," Verrucomicrobia, Tenericutes, and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

Quantitative analysis of annexin V-membrane interaction by flow cytometry.

We constructed a green fluorescent phosphatidylserine (PS)-binding probe, which was generated by fusing enhanced green fluorescent protein (EGFP) to the C terminus of human annexin V (anxV). With this probe, we investigated anxV-membrane interaction under different calcium and anxV-EGFP concentrations through flow cytometry (FCM). A mathematical description of the binding characteristics is proposed and validated to quantify the relationship concerning the relative concentration of membrane-bound anxV (B), calcium concentration ([C]), and protein concentration ([P]). Further analyses reveal that [Formula: see text] is linear with [Formula: see text] or [Formula: see text] when [P] and [C] are fixed, respectively, which indicates that the anxV-membrane binding reaction may involve sequential multiple steps. Our study provides a reference for application of anxV in apoptosis detection. The mathematical expression facilitates exploration of the possible interactions between calcium, anxV, and membrane. The corresponding mathematical analysis strengthens the interpretation of the interaction data.

Regulation of T lymphocyte activation by microRNA-21.

MicroRNAs are small noncoding RNAs that act as posttranscriptional regulators of gene expression. To identify microRNAs involved in T cell activation, we performed a microRNA array profiling with Jurkat cells. We found that microRNA-21 (miR-21), which is upregulated in many tumors by targeting a series of tumor suppressor genes to promote tumor growth, was significantly increased in activated Jurkat cells and primary CD4(+) T lymphocytes compared with that in quiescent counterparts. By using a signaling network building tool, miR-21 was predicted regulates ERK and JNK signaling in activated Jurkat cells. Indeed, miR-21 promotes ERK and JNK signaling in activated T cells. Sprouty1, a direct target of miR-21 that has been shown an inhibitor of ERK and JNK, was also inhibited by forced miR-21 expression in activated T cells. Reciprocally, miR-21 levels were induced by MEK or JNK signaling response to T cell receptor (TCR) engagement. Furthermore, transfection with miR-21 mimic promotes activator protein 1 (AP-1) activity and interleukin-2 (IL-2) expression. These results provide a missing function of miR-21 in TCR-mediated signaling transduction in T lymphocytes, suggesting that miR-21 may augment T cell immune response by a positive feedback mechanism.

Self-renewal and differentiation of muscle satellite cells are regulated by the Fas-associated death domain.

Making the decision between self-renewal and differentiation of adult stem cells is critical for tissue repair and homeostasis. Here we show that the apoptotic adaptor Fas-associated death domain (FADD) regulates the fate decisions of muscle satellite cells (SCs). FADD phosphorylation was specifically induced in cycling SCs, which was high in metaphase and declined in later anaphase. Furthermore, phosphorylated FADD at Ser-191 accumulated in the uncommitted cycling SCs and was asymmetrically localized in the self-renewing daughter SCs. SCs containing a phosphoryl-mimicking mutation at Ser-191 of FADD (FADD-D) expressed higher levels of stem-like markers and reduced commitment-associated markers. Moreover, a phosphoryl-mimicking mutation at Ser-191 of FADD suppressed SC activation and differentiation, which promoted the cycling SCs into a reversible quiescent state. Therefore, these data indicate that FADD regulates the fate determination of cycling SCs.

A critical role of Fas-associated protein with death domain phosphorylation in intracellular reactive oxygen species homeostasis and aging.

AIM: Reactive oxygen species (ROS) plays important roles in aging. However, the specific mechanisms for intracellular ROS accumulation, especially during aging, remain elusive. RESULTS: We have reported that Fas-associated protein with death domain (FADD) phosphorylation abolishes the recruitment of phosphatase type 2A C subunit (PP2Ac) to protein kinase C (PKC)ßII, which specifically regulates mitochondrial ROS generation by p66shc. Here, we have studied the role of FADD phosphorylation in an FADD constitutive-phosphorylation mutation (FADD-D) mouse model. In FADD-D mice, the constitutive FADD phosphorylation led to ROS accumulation (hydrogen peroxide [H2O2]), in a process that was dependent on PKCß and accompanied by increased PKCß and p66shc phosphorylation, impaired mitochondrial integrity, and enhanced sensitivity to oxidative stress-mediated apoptosis. Moreover, FADD-D mice exhibited premature aging-like phenotypes, including DNA damage, cellular senescence, and shortened lifespan. In addition, we demonstrate that FADD phosphorylation and the recruitment of PP2A and FADD to PKCß are induced responses to oxidative stress, and that the extent of FADD phosphorylation in wild-type mice was augmented during aging, accompanied by impairment of the interaction between PKCß and PP2A. INNOVATION: The present study first addresses the role of FADD phosphorylation in aging through controlling mitochondrial ROS specifically generated by PKCß. CONCLUSION: These data identify that FADD phosphorylation is critical for the PKCß-p66shc signaling route to generate H2O2 and to implicate enhanced FADD phosphorylation as a primary cause of ROS accumulation during aging.