Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

RNA Modifications and RNA Metabolism in Neurological Disease Pathogenesis.

The intrinsic cellular heterogeneity and molecular complexity of the mammalian nervous system relies substantially on the dynamic nature and spatiotemporal patterning of gene expression. These features of gene expression are achieved in part through mechanisms involving various epigenetic processes such as DNA methylation, post-translational histone modifications, and non-coding RNA activity, amongst others. In concert, another regulatory layer by which RNA bases and sugar residues are chemically modified enhances neuronal transcriptome complexity. Similar RNA modifications in other systems collectively constitute the cellular epitranscriptome that integrates and impacts various physiological processes. The epitranscriptome is dynamic and is reshaped constantly to regulate vital processes such as development, differentiation and stress responses. Perturbations of the epitranscriptome can lead to various pathogenic conditions, including cancer, cardiovascular abnormalities and neurological diseases. Recent advances in next-generation sequencing technologies have enabled us to identify and locate modified bases/sugars on different RNA species. These RNA modifications modulate the stability, transport and, most importantly, translation of RNA. In this review, we discuss the formation and functions of some frequently observed RNA modifications-including methylations of adenine and cytosine bases, and isomerization of uridine to pseudouridine-at various layers of RNA metabolism, together with their contributions to abnormal physiological conditions that can lead to various neurodevelopmental and neurological disorders.

TDP-43 Regulates Coupled Dendritic mRNA Transport-Translation Processes in Co-operation with FMRP and Staufen1.

Tightly regulated transport of messenger ribonucleoprotein (mRNP) granules to diverse locations of dendrites and axons is essential for appropriately timed protein synthesis within distinct sub-neuronal compartments. Perturbations of this regulation lead to various neurological disorders. Using imaging and molecular approaches, we demonstrate how TDP-43 co-operates with two other RNA-binding proteins, FMRP and Staufen1, to regulate the anterograde and retrograde transport, respectively, of Rac1 mRNPs in mouse neuronal dendrites. We also analyze the mechanisms by which TDP-43 mediates coupled mRNA transport-translation processes in dendritic sub-compartments by following in real-time the co-movement of RNA and endogenous fluorescence-tagged protein in neurons and by simultaneous examination of transport/translation dynamics by using an RNA biosensor. This study establishes the pivotal roles of TDP-43 in transporting mRNP granules in dendrites, inhibiting translation inside those granules, and reactivating it once the granules reach the dendritic spines.

DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells.

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca2+ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca2+ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca2+-dependent physiological processes.

p38α MAPK disables KMT1A-mediated repression of myogenic differentiation program.

BACKGROUND: Master transcription factor MyoD can initiate the entire myogenic gene expression program which differentiates proliferating myoblasts into multinucleated myotubes. We previously demonstrated that histone methyltransferase KMT1A associates with and inhibits MyoD in proliferating myoblasts, and must be removed to allow differentiation to proceed. It is known that pro-myogenic signaling pathways such as PI3K/AKT and p38α MAPK play critical roles in enforcing associations between MyoD and transcriptional activators, while removing repressors. However, the mechanism which displaces KMT1A from MyoD, and the signals responsible, remain unknown. METHODS: To investigate the role of p38α on MyoD-mediated differentiation, we utilized C2C12 myoblast cells as an in vitro model. p38α activity was either augmented via overexpression of a constitutively active upstream kinase or blocked via lentiviral delivery of a specific p38α shRNA or treatment with p38α/ß inhibitor SB203580. Overexpression of KMT1A in these cells via lentiviral delivery was also used as a system wherein terminal differentiation is impeded by high levels of KMT1A. RESULTS: The association of KMT1A and MyoD persisted, and differentiation was blocked in C2C12 myoblasts specifically after pharmacologic or genetic blockade of p38α. Conversely, forced activation of p38α was sufficient to activate MyoD and overcome the differentiation blockade in KMT1A-overexpressing C2C12 cells. Consistent with this finding, KMT1A phosphorylation during C2C12 differentiation correlated strongly with the activation of p38α. This phosphorylation was prevented by the inhibition of p38α. Biochemical studies further revealed that KMT1A can be a direct substrate for p38α. Importantly, chromatin immunoprecipitation (ChIP) studies show that the removal of KMT1A-mediated transcription repressive histone tri-methylation (H3K9me3) from the promoter of the Myogenin gene, a critical regulator of muscle differentiation, is dependent on p38α activity in C2C12 cells. Elevated p38α activity was also sufficient to remove this repressive H3K9me3 mark. Moreover, ChIP studies from C2C12 cells show that p38α activity is necessary and sufficient to establish active H3K9 acetylation on the Myogenin promoter. CONCLUSIONS: Activation of p38α displaces KMT1A from MyoD to initiate myogenic gene expression upon induction of myoblasts differentiation.

Co-regulation of mRNA translation by TDP-43 and Fragile X Syndrome protein FMRP.

For proper mammalian brain development and functioning, the translation of many neuronal mRNAs needs to be repressed without neuronal activity stimulations. We have discovered that the expression of a subclass of neuronal proteins essential for neurodevelopment and neuron plasticity is co-regulated at the translational level by TDP-43 and the Fragile X Syndrome protein FMRP. Using molecular, cellular and imaging approaches, we show that these two RNA-binding proteins (RBP) co-repress the translation initiation of Rac1, Map1b and GluR1 mRNAs, and consequently the hippocampal spinogenesis. The co-repression occurs through binding of TDP-43 to mRNA(s) at specific UG/GU sequences and recruitment of the inhibitory CYFIP1-FMRP complex by its glycine-rich domain. This novel regulatory scenario could be utilized to silence a significant portion of around 160 common target mRNAs of the two RBPs. The study establishes a functional/physical partnership between FMRP and TDP-43 that mechanistically links several neurodevelopmental disorders and neurodegenerative diseases.

Amyloid core formed of full-length recombinant mouse prion protein involves sequence 127-143 but not sequence 107-126.

The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrP(C)) into its disease-causing isoform, PrP(Sc). This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high ß-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23-230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107-143), mPrP(107-126), and mPrP(127-143). Our results showed that the amyloid fibrils formed from mPrP(107-143) and mPrP(127-143), but not those formed from mPrP(107-126), were able to seed the amyloidogenesis of mPrP(23-230), showing that the segment residing in sequence 127-143 was used to form the amyloid core in the fibrillization of mPrP(23-230).