Precise base editing without unintended indels in human cells and mouse primary myoblasts.

Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.

Betaine, a component of Lycium chinense, enhances muscular endurance of mice and myogenesis of myoblasts.

Sarcopenia is a disease characterized by the loss of muscle mass and function that occurs mainly in older adults. The present study was designed to investigate the hypothesis that water extract of Lycium chinense (WELC) would improve muscle function and promote myogenesis for sarcopenia. We investigated the effect of water extracts of L. chinense on muscular endurance function and myogenesis to examine its efficacy in sarcopenia. Intake of WELC-containing cheese enhanced the muscular endurance function of mice in treadmill endurance tests. In addition, the cross-sectional areas of muscle fibers in the gastrocnemius muscle of L. chinense-fed mice were greater than that of control mice. Furthermore, WELC and its key component marker substance betaine promoted myogenesis of myoblasts by increasing the expression of the myogenic protein myosin heavy chain 3 (Myh3) and myotube formation. Taken together, our results suggest that L. chinense may potentially be useful in the development of preventive and therapeutic agents for sarcopenia, as well as in providing basic knowledge on myogenesis and muscular functions.

Paternal bias expression of Igf2as is enhancer-dependent on the imprinting cluster of Igf2, H19 and Nctc1 in muscle cells.

Igf2, H19, and Nctc1 are linked co-regulated genes on distal mouse chromosome 7. This locus is an important model both for studying mechanisms of monoallelic expression and for elucidating the role of cis-regulatory elements - enhancers and insulators - in organizing chromatin and gene expression across a large domain. In this study we characterize regulated expression of the Igf2 antisense transcript (Igf2as) in primary muscle cells. We demonstrate that Igf2as is imprinted (expressed only from the paternal chromosome). We also show that Igf2as expression during differentiation follows the same patterns as Igf2 and H19. Moreover, this expression is dependent upon the same shared enhancer element. Thus, our work shows that the imprinted cluster includes Igf2as in addition to H19, Igf2, and Nctc1.

The Igf2/H19 muscle enhancer is an active transcriptional complex.

In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long non-coding RNA, Nctc1. Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function.

Extracellular annexins and dynamin are important for sequential steps in myoblast fusion.

Myoblast fusion into multinucleated myotubes is a crucial step in skeletal muscle development and regeneration. Here, we accumulated murine myoblasts at the ready-to-fuse stage by blocking formation of early fusion intermediates with lysophosphatidylcholine. Lifting the block allowed us to explore a largely synchronized fusion. We found that initial merger of two cell membranes detected as lipid mixing involved extracellular annexins A1 and A5 acting in a functionally redundant manner. Subsequent stages of myoblast fusion depended on dynamin activity, phosphatidylinositol(4,5)bisphosphate content, and cell metabolism. Uncoupling fusion from preceding stages of myogenesis will help in the analysis of the interplay between protein machines that initiate and complete cell unification and in the identification of additional protein players controlling different fusion stages.

Promoter cross-talk via a shared enhancer explains paternally biased expression of Nctc1 at the Igf2/H19/Nctc1 imprinted locus.

Developmentally regulated transcription often depends on physical interactions between distal enhancers and their cognate promoters. Recent genomic analyses suggest that promoter-promoter interactions might play a similarly critical role in organizing the genome and establishing cell-type-specific gene expression. The Igf2/H19 locus has been a valuable model for clarifying the role of long-range interactions between cis-regulatory elements. Imprinted expression of the linked, reciprocally imprinted genes is explained by parent-of-origin-specific chromosomal loop structures between the paternal Igf2 or maternal H19 promoters and their shared tissue-specific enhancer elements. Here, we further analyze these loop structures for their composition and their impact on expression of the linked long non-coding RNA, Nctc1. We show that Nctc1 is co-regulated with Igf2 and H19 and physically interacts with the shared muscle enhancer. In fact, all three co-regulated genes have the potential to interact not only with the shared enhancer but also with each other via their enhancer interactions. Furthermore, developmental and genetic analyses indicate functional significance for these promoter-promoter interactions. Altogether, we present a novel mechanism to explain developmental specific imprinting of Nctc1 and provide new information about enhancer mechanisms and about the role of chromatin domains in establishing gene expression patterns.

Induction of Per1 expression following an experimentally induced epilepsy in the mouse hippocampus.

The Period1 (Per1) is a clock-oscillating gene product that plays an essential role in the generation and modulation of circadian rhythm in the suprachiasmatic nucleus (SCN) of hypothalamus. However, Per1 is also expressed in many other brain regions including cerebral cortex, hippocampus, and amygdala, suggesting that Per1 may be involved in the broader cellular functions in addition to the rhythm regulation. In this study, we found that chemical or electrical seizure-inducing stimulations regulate Per1 expression. Treatments with electric convulsive shock (ECS) or kainic acid (KA) robustly up-regulated the expressions of per1 mRNA and protein in the hippocampal formation and cerebral cortex. In consistent, we found that neuronal depolarization or KA treatment increased per1 mRNA expression in cultured primary cortical neurons. Because it has been demonstrated that Per family molecules contribute to the regulation of stress-induced cell death, we also explored the effect of Per1 overexpression on the survival of cultured neurons. However, neither basal, staurosporine- nor KA-induced neuronal death was affected by forced expression of Per1. Collectively, these results suggest that the Per1 expression is neuronal activity- and epileptogen-dependent, although its functional significance is remained to be explored.

Induction of neuronal apoptosis by expression of Hes6 via p53-dependent pathway.

Hes6 is a member of hairy/enhancer of split (Hes) family that plays a role in the cell proliferation and differentiation. Recently, we found that Hes6 is involved in the regulation of cell proliferation via p53-dependent pathway. In addition to the proliferating regions, brain regions where early post-mitotic neurons are enriched also exhibited Hes6 and p53 mRNA expression. Because p53 is involved in the post-mitotic neuronal apoptosis, here we investigated whether Hes6 can influence the neuronal survival/death. Overexpression of wild-type Hes6 and its mutants induced the apoptosis of primary cultured cortical neurons. In addition, neuronal apoptosis by Hes6 overexpression was markedly blunted in p53(-/-) or Bax(-/-) cortical neurons, suggesting that these pro-apoptotic effects are mediated by p53- and Bax-dependent pathway. However, transactivation-defective mutants of Hes6 also enhanced neuronal apoptosis, suggesting that apoptogenic activity of Hes6 is not directly related to its role in the transcriptional regulation. We propose that Hes6 may play a significant role in the neuronal cell death and/or pathological neurodegeneration via activation of p53 signaling.

Hes6 controls cell proliferation via interaction with cAMP-response element-binding protein-binding protein in the promyelocytic leukemia nuclear body.

Hes6 is a basic helix-loop-helix transcription factor that functions in the differentiation of pluripotent progenitor cells and during tumorigenesis. However, the molecular mechanism for its function is largely unknown. Here we show that Hes6 is a component of the promyelocytic leukemia nuclear body (PML-NB) complex in the nuclei and that Hes6 inhibits cell proliferation through induction of p21 cyclin-dependent kinase inhibitor. We further show that Hes6 directly interacts with CREB-binding protein (CBP), one of the key components of PML-NB, via its basic domain. This association is critical for p21 induction through multiple mechanisms, including chromatin remodeling and p53 acetylation. Taken together, these results suggest that the Hes6-CBP complex in PML-NB may influence the proliferation of cells via p53-dependent and -independent pathways.

Impaired migration in the rostral migratory stream but spared olfactory function after the elimination of programmed cell death in Bax knock-out mice.

Rats and mice exhibit neurogenesis of olfactory bulb (OB) interneurons throughout adulthood. To homeostatically maintain stable neuron numbers, it is necessary to continuously remove a subset of OB neurons by programmed cell death (PCD). Here we demonstrate that Bax is critical for the elimination of OB neurons by showing that Bax-KO mice exhibit greatly reduced PCD in the OB. Despite the reduction of PCD, however, proliferation of progenitors and the size of the OB were virtually unaffected in Bax-knock-out (KO) mice. However, reducing PCD by Bax deletion affected the migration of a subset of adult-produced neurons by the disruption of glial tube formation as well as by premature detachment of neuroblasts from the migratory chain. Rescued cells aberrantly remained in the subventricular zone (SVZ)-rostral migratory stream (RMS), in which they differentiated into calretinin+ or GABA-expressing interneurons. Because of the migratory deficit, OB cell homeostasis involving new cell entry and PCD (neuronal turnover) was virtually absent in adult Bax-KO mice. Despite this, Bax-KO mice exhibited normal olfactory behaviors such as odor discrimination and olfactory memory which are thought to be influenced by adult neurogenesis. These results demonstrate that PCD is involved in the regulation of RMS migration and differentiation after OB neurogenesis, but that animals maintain normal olfactory function in the absence of PCD.

Anti-apoptotic function of thymosin-beta in developing chick spinal motoneurons.

Thymosin-betas (Tbetas) are water-soluble peptides abundantly present in the cytoplasm and extracellular compartment. The functions of Tbetas appear to be pleiotrophic, including actin-remodeling, wound healing, angiogenesis, etc. In the present study, we present the evidence that Tbetas have anti-apoptotic activity on developing chick motoneurons (MNs) in vivo. Using in ovo electroporation, we introduced three isoforms of Tbeta (Tbeta4, Tbeta10, and Tbeta15) and found the significantly diminished normal and limb bud removal (LBR)-induced programmed cell death. Such anti-apoptotic activity is independent of Tbeta's actin remodeling activity. On the other hand, overexpression of Tbetas substantially reduced early cell death initiation signal, such as phosphorylation of c-Jun. Collectively, these results suggest that Tbetas may prevent apoptosis of neurons via blockade of early apoptogenic signals independent of actin remodeling action.

Cloning and expression of cryptochrome2 in the bullfrog (Rana catesbeiana).

Cryptochromes (CRYs) are non-opsin photoactive pigments that have recently been implicated in circadian photo-entrainment in humans, mice and Drosophila. In order to study the mechanism of circadian rhythm in amphibians, we have cloned and characterized a Rana cryptochrome in the bullfrog. We isolated a cDNA of about 2.1 kb from a bullfrog brain cDNA library by screening with a partial cry2 cDNA probe obtained by RT-PCR using degenerate primers. The cloned Rana cry2 cDNA has a complete single open-reading frame consisting of 323 amino acids, and its deduced amino acid sequence has a high degree of homology with human and mouse CRY2. Interestingly, we also isolated two other cry2 cDNAs, which may be splicing variants. Rana cry2 is expressed in all tissues as a 2.2 kb transcript. It is particularly highly expressed in the brain and ovaries, and also showed seasonal variation in expression in ovarian tissue. To examine its involvement in circadian rhythm, we tested whether expression in brain tissue followed a light-dark cycle, and found that expression was higher in the dark than in the light. Rana cry2 should therefore, be useful for studying circadian rhythms in seasonally breeding wild animals.