The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages.

Circadian regulation of gene expression is prevalent and plays critical roles in cell differentiation. However, its roles in the reprogramming of differentiated cells remain largely unknown. Here, we found that one of the master circadian regulators PER1 promoted virus-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to induced neurons (iNs) and induced pluripotent stem cells (iPSCs). Unexpectedly, PER1 achieved this by repressing inflammatory activation of contaminating macrophages in the MEF culture, rather than by directly modulating the reprogrammability of MEFs. More specifically, we found that transduced viruses activated inflammatory genes in macrophages, such as Tnf encoding TNFα, one of the central inflammatory regulators and an autocrine activator of macrophages. TNFα inhibited iN reprogramming, whereas a TNFα inhibitor promoted iN reprogramming, connecting the inflammatory responses to iN reprogramming. In addition, macrophages were induced to proliferate and mature by non-macrophage cells serving as feeders, which also supported up-regulation of TNFα in macrophages without virus transduction. Furthermore, the 2 inflammatory responses were repressed by the circadian regulator PER1 in macrophages, making reprogrammability dependent on time-of-day of virus transduction. Similar results were obtained with iPSC reprogramming, suggesting a wide occurrence of macrophage-mediated inhibition of cell reprogramming. This study uncovers mechanistic links between cell reprogramming, bystander inflammatory macrophages, and circadian rhythms, which are particularly relevant to in vivo reprogramming and organoid formation incorporating immune cells.

Circadian Regulation of Bone Remodeling.

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments.

Circadian Regulation of Macrophages and Osteoclasts in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) represents one of the best examples of circadian fluctuations in disease severity. Patients with RA experience stiffness, pain, and swelling in afflicted joints in the early morning, which tends to become milder toward the afternoon. This has been primarily explained by the higher blood levels of pro-inflammatory hormones and cytokines, such as melatonin, TNFα, IL-1, and IL-6, in the early morning than in the afternoon as well as insufficient levels of anti-inflammatory cortisol, which rises later in the morning. Clinical importance of the circadian regulation of RA symptoms has been demonstrated by the effectiveness of time-of-day-dependent delivery of therapeutic agents in chronotherapy. The primary inflammatory site in RA is the synovium, where increased macrophages, T cells, and synovial fibroblasts play central roles by secreting pro-inflammatory cytokines, chemokines, and enzymes to stimulate each other, additional immune cells, and osteoclasts, ultimately leading to cartilage and bone erosion. Among these central players, macrophages have been one of the prime targets for the study of the link between circadian rhythms and inflammatory activities. Gene knockout experiments of various core circadian regulators have established that disruption of any core circadian regulators results in hyper- or hypoactivation of inflammatory responses by macrophages when challenged by lipopolysaccharide and bacteria. Although these stimulations are not directly linked to RA etiology, these findings serve as a foundation for further study by providing proof of principle. On the other hand, circadian regulation of osteoclasts, downstream effectors of macrophages, remain under-explored. Nonetheless, circadian expression of the inducers of osteoclastogenesis, such as TNFα, IL-1, and IL-6, as well as the knockout phenotypes of circadian regulators in osteoclasts suggest the significance of the circadian control of osteoclast activity in the pathogenesis of RA. More detailed mechanistic understanding of the circadian regulation of macrophages and osteoclasts in the afflicted joints could add novel local therapeutic options for RA.

Circadian Regulation in Tissue Regeneration.

Circadian rhythms regulate over 40% of protein-coding genes in at least one organ in the body through mechanisms tied to the central circadian clock and to cell-intrinsic auto-regulatory feedback loops. Distinct diurnal differences in regulation of regeneration have been found in several organs, including skin, intestinal, and hematopoietic systems. Each regenerating system contains a complex network of cell types with different circadian mechanisms contributing to regeneration. In this review, we elucidate circadian regeneration mechanisms in the three representative systems. We also suggest circadian regulation of global translational activity as an understudied global regulator of regenerative capacity. A more detailed understanding of the molecular mechanisms underlying circadian regulation of tissue regeneration would accelerate the development of new regenerative therapies.

Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD.

Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.

Regulation of embryonic stem cell self-renewal and pluripotency by leukaemia inhibitory factor.

LIF (leukaemia inhibitory factor) is a key cytokine for maintaining self-renewal and pluripotency of mESCs (mouse embryonic stem cells). Upon binding to the LIF receptor, LIF activates three major intracellular signalling pathways: the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3), PI3K (phosphoinositide 3-kinase)/AKT and SHP2 [SH2 (Src homology 2) domain-containing tyrosine phosphatase 2]/MAPK (mitogen-activated protein kinase) pathways. These pathways converge to orchestrate the gene expression pattern specific to mESCs. Among the many signalling events downstream of the LIF receptor, activation and DNA binding of the transcription factor STAT3 plays a central role in transducing LIF's functions. The fundamental role of LIF for pluripotency was highlighted further by the discovery that LIF accelerates the conversion of epiblast-derived stem cells into a more fully pluripotent state. In the present review, we provide an overview of the three major LIF signalling pathways, the molecules that interact with STAT3 and the current interpretations of the roles of LIF in pluripotency.

Immunofluorescence analysis of myogenic differentiation.

Skeletal muscle is a highly regenerative tissue that can efficiently recover from various damages caused by injuries and excessive exercises. In adult muscle, stem cells termed satellite cells are mitotically quiescent but activated upon muscle damages to enter the cell cycle as myogenic precursor cells or myoblasts. After several rounds of cell cycles, they exist the cycle and fuse to each other to form multinucleated myotubes, and eventually mature to become contractile myofibers. Satellite cells can be readily isolated from mouse skeletal muscle with enzymatic digestion and magnetic separation with antibodies against specific surface markers. C2C12 cells are an immortalized mouse myoblast cell line that is commercially available and more readily expandable than primary myoblasts. Both primary myoblasts and C2C12 cells have been extensively used as useful in vitro models for myogenic differentiation. Proper examination of this process requires monitoring specific protein expression in subcellular compartments, which can be accomplished through immunofluorescence staining. This chapter describes the workflow for the isolation of satellite cells from mouse skeletal muscle and subsequent immunofluorescence staining to assess the proliferation and differentiation of primary myoblasts and C2C12 cells.

Reversible disassembly of somatic nucleoli by the germ cell proteins FRGY2a and FRGY2b.

Egg cytoplasm has the capability to reprogramme differentiated somatic nuclei, as shown by nuclear transplantation in animal cloning. The nucleoli of donor nuclei are rapidly disassembled on injection into interphase eggs and are correctly reassembled when donor transcription initiates in the early embryos of frogs and mammals, recapitulating the physiological nucleolar dynamics of early embryogenesis. This is one of the most remarkable structural reorganizations of somatic nuclei in nuclear cloning. Despite the long history of nuclear cloning, almost nothing is known about the molecular mechanism of nucleolar disassembly in egg cytoplasm. Here we show that the Xenopus germ cell proteins FRGY2a and FRGY2b reversibly disassemble somatic nucleoli in egg cytoplasm, independently of continuing ribosomal RNA transcription. The carboxy-terminal domain of FRGY2a, which localizes to the nucleoli, is sufficient for nucleolar disassembly in transfected cells. Our results show that a single protein fragment can trigger reversible disassembly of the complex nucleolar structure.

Per1/Per2-Igf2 axis-mediated circadian regulation of myogenic differentiation.

Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter-enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.

Novel role of nucleostemin in the maintenance of nucleolar architecture and integrity of small nucleolar ribonucleoproteins and the telomerase complex.

Nucleostemin (NS) is a nucleolar protein involved in the regulation of cell proliferation. Both overexpression and knockdown of NS increase the activity of the tumor suppressor protein p53, resulting in cell cycle arrest. In addition, NS regulates processing of pre-rRNA and consequently the level of total protein synthesis. Here, we describe a previously uncharacterized function of NS in the maintenance of the tripartite nucleolar structure as well as the integrity of small nucleolar ribonucleoproteins (snoRNPs). NS is also necessary to maintain the telomerase complex which shares common protein subunits with the H/ACA box snoRNPs. First, immunofluorescence microscopy and electron microscopy demonstrated that knockdown of NS disorganized the nucleolar architecture, in particular, the dense fibrillar component where snoRNPs are localized. Second, gel filtration chromatography and immunoprecipitation indicated that NS depletion leads to dissociation of the components of snoRNPs and the telomerase complex. Third, NS depletion reduced both telomerase activity and the cellular level of pseudouridine, an H/ACA snoRNP-mediated modification of rRNA and other RNAs that are important for their folding and stability. These morphological, biochemical and functional studies demonstrate that NS plays an important role to maintain nucleolar structure and function on a more fundamental level than previously thought.

Post-mitotic role of nucleostemin as a promoter of skeletal muscle cell differentiation.

Nucleostemin (NS) is a nucleolar protein abundantly expressed in a variety of proliferating cells and undifferentiated cells. Its known functions include cell cycle regulation and the control of pre-rRNA processing. It also has been proposed that NS has an additional role in undifferentiated cells due to its downregulation during stem cell differentiation and its upregulation during tissue regeneration. Here, however, we demonstrate that skeletal muscle cell differentiation has a unique expression profile of NS in that it is continuously expressed during differentiation. NS was expressed at similar levels in non-proliferating muscle stem cells (satellite cells), rapidly proliferating precursor cells (myoblasts) and post-mitotic terminally differentiated cells (myotubes and myofibers). The sustained expression of NS during terminal differentiation is necessary to support increased protein synthesis during this process. Downregulation of NS inhibited differentiation of myoblasts to myotubes, accompanied by striking downregulation of key myogenic transcription factors, such as myogenin and MyoD. In contrast, upregulation of NS inhibited proliferation and promoted muscle differentiation in a p53-dependent manner. Our findings provide evidence that NS has an unexpected role in post-mitotic terminal differentiation. Importantly, these findings also indicate that, contrary to suggestions in the literature, the expression of NS cannot always be used as a reliable indicator for undifferentiated cells or proliferating cells.

Chromatin decondensation and nuclear reprogramming by nucleoplasmin.

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.

Promotion of Myoblast Differentiation by Fkbp5 via Cdk4 Isomerization.

Fkbp5 is a widely expressed peptidyl prolyl isomerase that serves as a molecular chaperone through conformational changes of binding partners. Although it regulates diverse protein functions, little is known about its roles in myogenesis. We found here that Fkbp5 plays critical roles in myoblast differentiation through two mechanisms. First, it sequesters Cdk4 within the Hsp90 storage complex and prevents the formation of the cyclin D1-Cdk4 complex, which is a major inhibitor of differentiation. Second, Fkbp5 promotes cis-trans isomerization of the Thr172-Pro173 peptide bond in Cdk4 and inhibits phosphorylation of Thr172, an essential step for Cdk4 activation. Consistent with these in vitro findings, muscle regeneration is delayed in Fkbp5-/- mice. The related protein Fkbp4 also sequesters Cdk4 within the Hsp90 complex but does not isomerize Cdk4 or induce Thr173 phosphorylation despite its highly similar sequence. This study demonstrates protein isomerization as a critical regulatory mechanism of myogenesis by targeting Cdk4.

Cry2 Is Critical for Circadian Regulation of Myogenic Differentiation by Bclaf1-Mediated mRNA Stabilization of Cyclin D1 and Tmem176b.

Circadian rhythms regulate cell proliferation and differentiation; however, little is known about their roles in myogenic differentiation. Our synchronized differentiation studies demonstrate that myoblast proliferation and subsequent myotube formation by cell fusion occur in circadian manners. We found that one of the core regulators of circadian rhythms, Cry2, but not Cry1, is critical for the circadian patterns of these two critical steps in myogenic differentiation. This is achieved through the specific interaction between Cry2 and Bclaf1, which stabilizes mRNAs encoding cyclin D1, a G1/S phase transition regulator, and Tmem176b, a transmembrane regulator for myogenic cell fusion. Myoblasts lacking Cry2 display premature cell cycle exit and form short myotubes because of inefficient cell fusion. Consistently, muscle regeneration is impaired in Cry2-/- mice. Bclaf1 knockdown recapitulated the phenotypes of Cry2 knockdown: early cell cycle exit and inefficient cell fusion. This study uncovers a post-transcriptional regulation of myogenic differentiation by circadian rhythms.

A primer on stem cell research.

Stem cell research holds promise for new treatments for diseases such as Parkinson's and injuries such as those to the spinal cord. Yet stem cell research remains steeped in controversy. This article defines key terms and outlines some of the basic concepts related to stem cell research in order to clarify concepts and correct misconceptions. It also briefly discusses what scientists, particularly those at the University of Minnesota, are learning about adult and embryonic stem cells and their potential for research and treatment.

Nuclear remodeling assay in Xenopus egg extract.

Xenopus egg extract is an ideal material to identify nuclear remodeling activities important for nuclear cloning. Since the protocol for egg extract preparation was established more than 20 yr ago, egg extract has been widely used as a source for the purification of factors associated with a number of physiological activities. The eggs are large, easily obtained in large quantities, and are abundant in a variety of bioactive proteins. Many aspects of somatic nuclear remodeling observed in nuclear cloning are recapitulated in somatic nuclei incubated in egg extract. Our in vitro nuclear remodeling assay has proven effective for the purification of two novel nuclear remodeling activities, ISWI, a key player in the dissociation of TATA binding protein from chromatin, and FRGY2a and FRGY2b, two proteins capable of nucleolar disassembly. Here we outline our protocol of egg extract preparation and in vitro nuclear remodeling assay, as well as the purification method for FRGY2a and FRGY2b. Our in vitro nuclear remodeling assay in combination with egg extract will serve as a powerful tool with which to biochemically uncover molecular events involved in nuclear cloning.

Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells.

Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563.

Preservation of Epigenetic Memory During DNA Replication.

Faithful duplication of a cell's epigenetic state during DNA replication is essential for the maintenance of a cell's lineage. One of the key mechanisms is the recruitment of several critical chromatin modifying enzymes to the replication fork by proliferating cell nuclear antigen (PCNA). Another mechanism is mediated by the dual function of some histone modifying enzymes as both "reader" and "writer" of the same modification. This capacity allows for parental histones to act as a seed to copy the modification onto nearby newly synthesized histones. In contrast to the vast quantity of research into the maintenance of epigenetic memory, little is known about how the recruitment of these maintenance enzymes changes during stem cell differentiation. This question is especially pertinent due to the recent emphasis on cell reprogramming for regenerative medicine.

Mechanisms and Developmental Roles of Promoter-proximal Pausing of RNA Polymerase II.

RNA polymerase II (Pol II) temporarily stops transcription after synthesizing 30-50 bases, and resumes elongation only after stimulations by various signaling molecules and developmental cues. This phenomenon, called promoter-proximal pausing, is observed in 10-50% of the entire genes from Drosophila embryos to human cells. Release of paused Pol II is primarily mediated by the activated form of positive transcription elongation factor b (P-TEFb) initially sequestered in the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Many proteins and RNAs have been discovered and studied in detail to explain the process of the pausing and release of Pol II in relation to P-TEFb. At the functional level, promoter-proximal pausing regulates genes involved in stimulus-response and development in Drosophila. In mammalian stem cell biology, pausing is important for proliferation and signaling in embryonic stem cells and the formation of induced pluripotent stem cells. Other than this, however, little is known about the biological significance of pausing in mammalian cell differentiation. Further study on pausing mechanisms as well as its functions will contribute to the development of stem cell biology and its clinical applications.

Epigenetic regulation of open chromatin in pluripotent stem cells.

The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, alter, eventually, the epigenetic status of their target genes during the process of activating or suppressing the genes to acquire or maintain pluripotency. It has long been thought that the chromatin of pluripotent stem cells is open globally to enable the timely activation of essentially all genes in the genome during differentiation into multiple lineages. The current article reviews descriptive observations and the epigenetic machinery relevant to what is supposed to be globally open chromatin in pluripotent stem cells, including microscopic appearance, permissive gene transcription, chromatin remodeling complexes, histone modifications, DNA methylation, noncoding RNAs, dynamic movement of chromatin proteins, nucleosome accessibility and positioning, and long-range chromosomal interactions. Detailed analyses of each element, however, have revealed that the globally open chromatin hypothesis is not necessarily supported by some of the critical experimental evidence, such as genomewide nucleosome accessibility and nucleosome positioning. Greater understanding of epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem cells.