14 research outputs found
An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability.
Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.Research in the authorsâ laboratories was supported by Bloodwise, The Wellcome Trust, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, the National Institute of Health Research, the Medical Research Council, the MRC Molecular Haematology Unit (Oxford) core award, a Weizmann-UK âMaking Connectionsâ grant (Oxford) and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research (100140) and Wellcome TrustâMRC Cambridge Stem Cell Institute (097922).This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.1146
Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain
The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfsâ71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals
Computational analysis GeCKO v2 library (human)
<b>Table 1 - Non-specific sgRNAs GeCKO v2 (human). </b>This table represents the total of 5,664 sgRNAs within the entire GeCKO v2 library (half-libraries A and B combined) that recognise at least two identical target sites (20 bp protospacer + NGG PAM) within the reference genome. For the analysis, sgRNA target sites were aligned against the human reference genome (GRCh38/hg19 - primary assembly) using BLAT (v36) after optimisation of the algorithm for short length queries (-tileSize=6, -stepSize=3). The resulting alignments were further filtered to only include perfect matches and unique targets (exclusion of hits on alternative assemblies). Analysis was performed using R (v3.2.3) in R studio (v1.0.136) using Ubuntu.<div><br></div><div><b>Table 2 - Flagged genes GeCKO v2 (human).</b> This table represents the total of 775 genes for which more than half the corresponding sgRNAs within the entire human GeCKO v2 library (half-libraries A and B combined) are non-specific, i.e. recognise at least two identical target sites (20 bp protospacer + NGG PAM) within the reference genome. A threshold of >50% non-specific sgRNAs per gene (e.g. 4 out of 6 for protein coding genes; 3 out of 4 for miRNA genes) was set in order to flag a group of sgRNAs. Analysis was performed using R (v3.2.3) in R studio (v1.0.136) using Ubuntu.</div
MicroRNA-mediated Down-Regulation of M-CSF Receptor Contributes to Maturation of Mouse Monocyte-Derived Dendritic Cells
HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing
Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy
MicroRNA-Mediated Down-Regulation of M-CSF Receptor Contributes to Maturation of Mouse Monocyte-Derived Dendritic Cells
Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCIIhi CD86hi DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation