Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.
The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.
BackgroundNeuromyelitis optica (NMO) is a devastating inflammatory disorder of the optic nerves and spinal cord characterized by frequently recurring exacerbations of humoral inflammation. NMO is associated with the highly specific NMO-IgG biomarker, an antibody that binds the aquaporin-4 water channel. Aquaporin-4 is present on glial endfeet in the central nervous system (CNS). In humans, the NMO-IgG portends more frequent exacerbations and a worse long-term clinical outcome.MethodsWe tested the longer-term outcome of mice with EAE injected with NMO-IgG and followed them for 60 days. Clinical exams and pathology of the spinal cord and optic nerves were compared to mice that received control human IgG.ResultsPassively transferred human NMO-IgG leads to more severe neurology disability over two months after onset of disease. Clinical worsening is associated with an increased concentration of large demyelinating lesions primarily to subpial AQP4-rich regions of the spinal cord.ConclusionsNMO-IgG is pathogenic in the context of EAE in mice.
Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing transposons and genes, but the mechanisms that establish and maintain these domains are not fully understood. Here we use an inducible heterochromatin formation system combined with a CRISPR-based genetic screen to investigate the requirements for the establishment and maintenance of heterochromatin in mouse embryonic stem cells (mESCs). We show that DNA sequence-independent and histone H3 lysine 9 methylation (H3K9me)-dependent heterochromatin can be inherited for a limited number of cell divisions in mESCs but becomes stable upon differentiation. We provide evidence that the increased stability of heterochromatin in differentiated cells results from the downregulation of one or more enzymes that erase H3K9me and DNA methylation. Moreover, we show that in addition to components of the H3K9 and DNA methylation pathways, heterochromatin maintenance requires DHX9 and other RNA processing proteins. DHX9 is an RNA/DNA helicase with previously described roles in preventing genomic instability resulting from transcription-associated replication stress. We found that deletion of DHX9 results in defective heterochromatin inheritance and is associated with increased transcription of major satellite repeats, accumulation of R-loops, and loss of H3K9me. Our findings define the requirements for the establishment and epigenetic inheritance of mammalian heterochromatin and suggest that R-loops and replication stress lead to epigenetic instability.
9The polarized structure of axons and dendrites in neuronal cells depends in part on RNA 10 localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal 11 projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By 12 physically dissecting projections from cell bodies of primary rat hippocampal neurons and 13 sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical 14 branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that 15 escape debranching. They lack ribosome occupancy, sequence conservation, and known 16 localization signals, and their function, if any, is not known. Nonetheless, their enrichment in 17 projections has important implications for our understanding of the mechanisms by which RNAs 18 reach distal compartments of asymmetric cells. 19 20 42The interplay between intron retention and neuronal RNA localization has been studied in several 43 individual cases (Chen et al., 2008; Bell et al., 2010; Buckley et al., 2011; Khaladkar et al., 2013; 44 Ortiz et al., 2017; Sharangdhar et al., 2017). In this work, our aim was to systematically identify 45 localized RNAs in primary rat hippocampal neurons by sequencing total RNA (rRNA depleted) as 46 opposed to polyadenylated (polyA+) RNA, with a particular focus on the repertoire of projection-47 localized introns (both retained and excised). Our analyses identify hundreds of transcripts with 48 retained introns. Unexpectedly, we also found a set of free circular introns localized to distal 49 neuronal projections. 50 Results 51 Experimental Design and Validation 52 To physically separate cellular projections from cell bodies, we cultured dissociated primary rat 53 hippocampal cells on membranes with 1 µm diameter pores (Poon et al., 2006). These cultures 54 are a mixture of neuronal and glial cells; we add a DNA replication inhibitor to block cell division 55 and prevent dividing glia from overgrowing post-mitotic neurons. We refer to the projections 56 as "neuro-glial" projections because both neuronal (Map2-immunopositive) and non-neuronal 57 (Gfap/Vimentin-immunopositive) projections extend through the pores and continue growing on 58 the underside of the membrane, whereas cell bodies and nuclei are restricted to the top surface 59 (Figure 1A and Figure 1-Figure Supplement 1). Lysates prepared by scraping the underside are 60 highly enriched for projections ("projection" samples), while lysates prepared from the top surface 61 comprise whole cells with nuclei and projections ("whole cell" samples). 62 To capture both adenylated (polyA+) and non-adenylated (polyA-) long RNAs in our lysates, 63 we prepared rRNA-depleted total RNAseq libraries (mean insert size~200 nt) from five biological 64 replicates (10 samples total). The RNAseq libraries were subjected to paired-end sequencing 65 (100-125 nt reads) on the Illumina platform to obtain 30-80 million mate pairs per sample. 66 We generated addit...
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