Abstract A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.
ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.
Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.
In the context of the human airway, interleukin-17A (IL-17A) signaling is associated with severe inflammation, as well as protection against pathogenic infection, particularly at mucosal surfaces such as the airway. The intracellular molecule Act1 has been demonstrated to be an essential mediator of IL-17A signaling. In the cytoplasm, it serves as an adaptor protein, binding to both the intracellular domain of the IL-17 receptor as well as members of the canonical nuclear factor kappa B (NF-κB) pathway. It also has enzymatic activity, and serves as an E3 ubiquitin ligase. In the context of airway epithelial cells, we demonstrate for the first time that Act1 is also present in the nucleus, especially after IL-17A stimulation. Ectopic Act1 expression can also increase the nuclear localization of Act1. Act1 can up-regulate the expression and promoter activity of a subset of IL-17A target genes in the absence of IL-17A signaling in a manner that is dependent on its N- and C-terminal domains, but is NF-κB independent. Finally, we show that nuclear Act1 can bind to both distal and proximal promoter regions of DEFB4, one of the IL-17A responsive genes. This transcriptional regulatory activity represents a novel function for Act1. Taken together, this is the first report to describe a non-adaptor function of Act1 by directly binding to the promoter region of IL-17A responsive genes and directly regulate their transcription.
To evaluate whether subretinal or intravitreal injection of human CD34+ bone marrow-derived stem cells (BMSC) can have protective effects on retinal degeneration that may be enhanced by coadministration of exosomes harvested from human bone marrow mesenchymal stem cells (MSCs).Human CD34+ cells were harvested from the mononuclear cell fraction of bone marrow using magnetic beads and labeled with EGFP. Exosomes were harvested from cultured human MSCs under hypoxic conditions. Royal College of Surgeons (RCS) 3-weeks-old rats, immunosuppressed with cyclosporine A, received subretinal or intravitreal injection of CD34+ cells (50,000 cells), CD34+ cells with exosomes (50,000 cells+10 µg), exosomes alone (10 µg), or PBS. Retinal function was examined using electroretinography (ERG), and the eyes were harvested for histologic and immunohistochemical analysis.The b-wave amplitude of ERG at 2 weeks after injection was significantly higher in eyes with subretinal or intravitreal CD34+ BMSC alone or in combination with exosomes when compared to PBS injected eyes or untreated contralateral eyes. At 4 weeks after injection, the ERG signal decreased in all groups but eyes with subretinal CD34+ BMSCs alone or combined with exosomes showed partially preserved ERG signal and preservation of the outer nuclear layer of the retina near the injection site on histology when compared to eyes with PBS injection. Immunohistochemical analysis identified the human cells in the outer retina. Subretinal or intravitreal exosome injection had no effect on retinal degeneration when administered alone or in combination with CD34+ cells.Both subretinal and intravitreal injection of human CD34+ BMSCs can provide functional rescue of degenerating retina, although the effects were attenuated over time in this rat model. Regional preservation of the outer retina can occur near the subretinal injection site of CD34+ cells. These results suggest that CD34+ cells may have therapeutic potential in retinal degeneration.
OBJECTIVES/GOALS: To identify the role of sialoglycans in the mechanisms underlying cancer-associated fibroblast-derived exosomes (CAFEX) immuno-regulatory properties. The central hypothesis is that CAFEX manipulates the immune response to allow immunoevasion and glycomic approaches can identify the signaling factors involved. METHODS/STUDY POPULATION: Cancer associated fibroblasts (CAFs) were isolated from primary head and neck tumors, expanded, characterized and cryopreserved prior to experimentation and isolation of CAFEX. Sialoglycan expression was determined using lectin-specific staining of cells and bead-captured CAFEX in combination with flow cytometry analysis. Siglec expression and expression of M2-macrophage markers were also determined by flow cytometry analysis and cytokine bead arrays. Inhibition studies involved either the enzymatic removal of cell-surface sialoglycans or alternatively, a specific small molecule analog inhibitor of sialoglycan transferases. RESULTS/ANTICIPATED RESULTS: Both CAFs and CAFEX expressed abundant levels of cell-surface sialoglycans. CAFEX induced an M2-macrophage phenotype in primary monocytes, based on surface marker expression and cytokine secretion profiles. The induction of the M2 phenotype in macrophages was attenuated upon the removal of sialoglycans from the surface of CAFEX either by enzymatic removal or via a small molecule inhibitor. CAFEX were also able to directly bind members of the Siglec family, which are sialoglycan specific lectin receptors expressed on immune cells, including macrophages. DISCUSSION/SIGNIFICANCE: Collectively, these studies suggest that surface presentation of sialoglycans by CAFEX may induce an immunosuppressive phenotype in monocytes/macrophages. Consequently, this may be a novel mechanism by which cells within the tumor bed facilitate immunoevasion during tumor progression.
The ability of neurons to modulate gene expression in response to changing environmental conditions, both during development and throughout life, is necessary for proper brain function. Although the mechanisms responsible for transducing extracellular signals into changes in gene expression remain poorly characterized, S-nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols, has been shown to be critical. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is essential for gene expression during neuronal development, however few nuclear targets have been identified to date. Progress in this field of research has been hampered by the technical difficulties associated with the detection of S-nitrosylated nuclear proteins. Here, I took a novel approach by enriching for nuclear proteins of rat cortical neurons before inducing S-nitrosylation with nitrosocysteine (CysNO) and isolating S-nitrosylated proteins using S-nitrosothiol Resin Assisted Capture (SNORAC). Mass spectrometry analysis identified 614 S-nitrosylated nuclear proteins, including potential site(s) of S-nitrosylation for 67% of targets. 612 of these proteins are novel potential targets of S-nitrosylation in cortical neurons and 131 have not been shown to be S-nitrosylated in any system. Two hits, the transcription factor CREB and the histone binding protein RBBP7, were further studied and I confirmed endogenous S-nitrosylation in depolarized cortical neurons for both. In addition, I showed that CREB is S-nitrosylated at cysteines 300/310/337 located within the DNA binding domain, whereas RBBP7 is S-nitrosylated at cysteine 166, which is in a WD-repeat region that regulates protein-protein interactions. Overall, this work comprehensively identifies, for the first time, the nuclear proteins that undergo S-nitrosylation in neurons and highlights S-nitrosylation of CREB and RBBP7 as a candidate mechanism by which NO regulates gene expression in mammalian cells.
