Chengzu Long

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus–9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA

Genome editing corrects a muscle disease Patients with Duchenne muscular dystrophy find their muscles growing progressively weaker. Studies identified dystrophin as the culprit gene, which galvanized research into gene-targeted therapies. Long et al. apply genome editing to “correct” the disease-causing mutation in mice genetically destined to develop the disease. This germline editing strategy kept muscles from degenerating, even in mice harboring only a small percentage of corrected cells. Although not feasible for humans, this proof of concept sets the stage for applying genome editing to specific cell types involved in the disease. Science, this issue p. 1184 A mutation that causes muscular dystrophy in mice can be corrected by genome editing, which prevents the disease from developing. Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened life span, and there is no effective treatment. We used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)–mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germ line of mdx mice, a model for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle. With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategy may one day allow correction of disease-causing mutations in the muscle tissue of patients with DMD.

The Phosphothreonine Lyase Activity of a Bacterial Type III Effector Family

Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal–regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the α proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.

Partitioning of Histone H3-H4 Tetramers During DNA Replication-Dependent Chromatin Assembly

Histone Inheritance Chromatin, the packaging material for eukaryotic genomes, is a potential repository for epigenetic information. The core structure of chromatin is the nucleosome, which consists of an octamer of histone proteins, two dimers each of histones H2A and H2B, and histones 3 and 4. Histones 3 and 4, in particular, carry a series of covalent modifications presumed to be passed on through cell division. Using mass spectrometry of tagged and isotope labeled histones, Xu et al. (p. 94; see the Perspective byRay-Gallet and Almouzni) followed the inheritance of the histones themselves through mitosis. The H2A-H2B dimers were inherited randomly through cell division, correlating with their lack of major covalent marks. In comparison, replication-deposited H3.1-H4 dimers did not separate through cell division, implying that H3 and H4 histone modifications might be maintained by copying from neighboring preexisting histones. Intriguingly, up to one-quarter of the nonreplication-deposited H3.3-H4 dimers, which mark active chromatin, did split during cell division. Inheritance of histones H3 and H4 implies that epigenetic marks are copied between nucleosomes. Semiconservative DNA replication ensures the faithful duplication of genetic information during cell divisions. However, how epigenetic information carried by histone modifications propagates through mitotic divisions remains elusive. To address this question, the DNA replication–dependent nucleosome partition pattern must be clarified. Here, we report significant amounts of H3.3-H4 tetramers split in vivo, whereas most H3.1-H4 tetramers remained intact. Inhibiting DNA replication–dependent deposition greatly reduced the level of splitting events, which suggests that (i) the replication-independent H3.3 deposition pathway proceeds largely by cooperatively incorporating two new H3.3-H4 dimers and (ii) the majority of splitting events occurred during replication-dependent deposition. Our results support the idea that “silent” histone modifications within large heterochromatic regions are maintained by copying modifications from neighboring preexisting histones without the need for H3-H4 splitting events.

Two Prp19-Like U-Box Proteins in the MOS4-Associated Complex Play Redundant Roles in Plant Innate Immunity

Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein–mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity.

Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.

Heterogeneous Nuclear Ribonucleoprotein L Is a Subunit of Human KMT3a/Set2 Complex Required for H3 Lys-36 Trimethylation Activity in Vivo

The presence of histone H3 lysine 36 methylation (H3K36me) correlates with actively transcribed genes. In yeast, histone H3K36me mediated by KMT3 (also known as Set2) recruits a histone deacetylase complex, Rpd3s, to ensure the fidelity of transcription initiation. We report the purification of human KMT3a (also known as HYPB or hSet2) complex and the identification of a novel, higher eukaryotic specific subunit, heterogeneous nuclear ribonucleoprotein L (HnRNP-L). Interestingly, although KMT3a has intrinsic activity in vitro, HnRNP-L is essential in vivo. Moreover, KMT3a generates mono-, di-, and trimethylated products in vitro, but RNA interference against KMT3a or HnRNP-L down-regulates exclusively the H3K36me3 mark in vivo.

A bacterial type III effector family uses the papain-like hydrolytic activity to arrest the host cell cycle

Pathogenic bacteria deliver effector proteins into host cells through the type III secretion apparatus to modulate the host function. We identify a family of proteins, homologous to the type III effector Cif from enteropathogenic Escherichia coli, in pathogens including Yersinia, Photorhabdus, and Burkholderia that contain functional type III secretion systems. Like Cif, this family of proteins is capable of arresting the host cell cycle at G2/M. Structure of one of the family members, Cif homolog in Burkholderia pseudomallei (CHBP), reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad despite the lack of primary sequence identity. For CHBP and Cif, only the putative catalytic Cys is susceptible to covalent modification by E-64, a specific inhibitor of papain-like cysteine proteases. Unlike papain-like enzymes where the S2 site is the major determinant of cleavage-site specificity, CHBP has a characteristic negatively charged pocket occupying surface areas corresponding to the S1/S1′ site in papain-like proteases. The negative charge is provided by a conserved aspartate, and the pocket best fits an arginine, as revealed by molecular docking analysis. Mutation analysis establishes the essential role of the catalytic triad and the negatively charged pocket in inducing cell cycle arrest in host cells. Our results demonstrate that bacterial pathogens have evolved a unique papain-like hydrolytic activity to block the normal host cell cycle progression.

KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.

CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

CRISPR-Cpf1–mediated correction of Duchenne muscular dystrophy mutations in human cells and a mouse model. Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.