Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes

<div><p>CRISPR/Cas9-based approaches have greatly facilitated targeted genomic deletions. Contrary to coding genes however, which can be functionally knocked out by frame-shift mutagenesis, non-coding RNA (ncRNA) gene knockouts have remained challenging. Here we present a universal ncRNA knockout approach guided by epigenetic hallmarks, which enables robust gene silencing even in provisionally annotated gene loci. We build on previous work reporting the presence of overlapping histone H3 lysine 4 tri-methylation (H3K4me3) and DNaseI hypersensitivity sites around the transcriptional start sites of most genes. We demonstrate that excision of this gene-proximal signature leads to loss of microRNA and lincRNA transcription and reveals ncRNA phenotypes. Exemplarily we demonstrate silencing of the constitutively transcribed MALAT1 lincRNA gene as well as of the inducible miR-146a and miR-155 genes in human monocytes. Our results validate a role of miR-146a and miR-155 in negative feedback control of the activity of inflammation master-regulator NFκB and suggest that cell-cycle control is a unique feature of miR-155. We suggest that our epigenetically guided CRISPR approach may improve existing ncRNA knockout strategies and contribute to the development of high-confidence ncRNA phenotyping applications.</p></div

Excision of H3K4me3/DNaseI signatures by dual guideRNA CRISPR vectors.

<p><b>A)</b> Primary human monocyte DNase- and ChIP-Seq (H3K4me3 and K3K27me3) coverages at the transcriptional start sites of the MALAT1, miR-146a and miR-155 ncRNA genes. Regions selected for targeted excision are marked (dashed lines). <b>B)</b> Genomic PCR with primers flanking the respective H3K4me3/DNaseI element to be excised. Besides the wild-type band (WT) a band of reduced size (ΔTSS) is detected after transfection of HEK293 cells with the respective ncRNA knockout CRISPR construct (KO) but not after transfection of a control CRISPR construct (Ctrl.). Position of the DNA ladder bands (2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp) is indicated to the left of each gel image. <b>C)</b> Sanger-sequencing of ΔTSS bands validates the deletion in the MALAT1, miR155 and miR146a promoter, respectively. Positions of guideRNAs (gRNA) and protospacer-adjacent motifs (PAM) are indicated.</p

Silencing ncRNA genes based on proximal H3K4me3/DNaseI signatures.

<p><b>A)</b> Schematic representation of the ncRNA knockout strategy. To abrogate transcription a signature consisting of H3K4me3 and DNaseI hyper-sensitivity (DNase HSS) signals overlapping at the gene proximal promoter is excised through two flanking guideRNAs. <b>B)</b> Cloning strategy to obtain a pX458 CRISPR vector construct simultaneously expressing two guideRNAs driven by repeated U6 promoters for knockout of a given ncRNA gene TSS signature. The depicted insert is generated by gene synthesis.</p

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host

Elevated NFκB p65 but not ERK1/2 activity on miR-146a and miR-155 knockout.

<p><b>A)</b> Representative FACS scatter plots showing a right-shift of 30 min LPS-stimulated (1 μg / ml) compared to mock-treated monocytes stained with phospho-p65 antibody (PE-channel). <b>B)</b> Representative histogram plots showing an increased right-shift of miR-146a and miR-155 deficient compared to control or MALAT1 deficient monocytes after 30 min LPS-stimulation (1 μg / ml) and staining with a phospho-p65 antibody (PE-channel). <b>C)</b> Fold change in phospho-p65 signal in monocytes stimulated with LPS (1 μg / ml) for 15, 30 or 100 min compared to mock-treatment (ctrl) in wild-type (WT) or the indicated ncRNA knockout (KO) cells. All fold-changes are relative to the respective WT mock control. <b>D-F)</b> Same as A-C) but with phospho-ERK1/2 staining (APC-channel). Statistical significance was determined by a one-way ANOVA test with multiple comparisons (* p<0.05).</p

Excision of H3K4me3/DNaseI signatures abolishes ncRNA expression.

<p><b>A)</b> Experimental overview. U937 monocytes are spin-lipofected with dual guideRNA CRISPR constructs and clonally expanded after single cell sorting of cells positive for GFP (co-expressed from the CRISPR vector). <b>B)</b> Genomic PCR showing homozygous deletion of the proximal promoter elements identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193066#pone.0193066.g002" target="_blank">Fig 2</a> of the MALAT1, miR155, miR146a genes after clonal expansion of monocytes transfected with the respective dual guideRNA CRISPR constructs (KO). Transfection of control CRISPR construct does not result in the deletion, respectively (WT). <b>C)</b> Real-time PCR analysis of IL1β mRNA expression in wild type (WT) and MALAT1, miR-146a or miR-155 TSS knockout monocytes after control-stimulation (mock) or activation with 1 μg / ml of LPS for 4h, 8h or 16h. Fold-changes compared to mock-stimulation of control cells are shown. <b>D)</b> Real-time PCR analysis of MALAT-1 expression in wild-type (WT) or in MALAT1 H3K4me3/DNaseI element deficient cell clones stimulated as in C). <b>E)</b> Northern blot with miR-146a, and miR-17 detection probes showing loss of miR-146a induction on stimulation with LPS (1 μg / ml) in monocytes deficient in the gene-proximal H3K4me3/DNaseI element (KO) but not in wild-type (WT) cell clones. <b>F)</b> Same as E) but with miR-155 gene proximal H3K4me3/DNaseI element excised and miR-155 instead of miR-146a detection.</p

Model summarizing the observed ncRNA knockout phenotypes.

<p>NFκB p65 phosphorylation data indicate a negative feed-back function of both miR-146a and miR-155 in TLR signaling, while ERK1/2 signaling is not affected. Promoting entry into the G2/M cell cycle phase is a function unique to miR-155.</p

Knockout of miR-155 affects monocyte cell cycle.

<p><b>A)</b> Gating scheme for determining cell fractions in G1/G0-, S- or G2/M-phase through propidium iodide staining. Representative data from LPS-treated wild-type (WT) or ncRNA knockout monocytes are shown. <b>B)</b> Quantification of percent-distribution of cell fractions in G1/G0-, S- or G2/M-phase in 4, 8 or 16 h mock or LPS (1 μg / ml) treated monocytes (upper and middle panels) and representation of individual mean- and replicate values for the 16 h time-point (lower panel). Data refer to wild-type (WT), MALAT1, miR-146a and miR-155 knockout U937 cells as indicated below the lower panel. Statistical significance was determined by one-way ANOVA with multiple comparisons (* p<0.05, ** p<0.01).</p

PPARα-mediated peroxisome induction compensates PPARγ-deficiency in bronchiolar club cells.

Despite the important functions of PPARγ in various cell types of the lung, PPARγ-deficiency in club cells induces only mild emphysema. Peroxisomes are distributed in a similar way as PPARγ in the lung and are mainly enriched in club and AECII cells. To date, the effects of PPARγ-deficiency on the overall peroxisomal compartment and its metabolic alterations in pulmonary club cells are unknown. Therefore, we characterized wild-type and club cell-specific PPARγ knockout-mice lungs and used C22 cells to investigate the peroxisomal compartment and its metabolic roles in the distal airway epithelium by means of 1) double-immunofluorescence labelling for peroxisomal proteins, 2) laser-assisted microdissection of the bronchiolar epithelium and subsequent qRT-PCR, 3) siRNA-transfection of PPARγand PPRE dual-luciferase reporter activity in C22 cells, 4) PPARg inhibition by GW9662, 5) GC-MS based lipid analysis. Our results reveal elevated levels of fatty acids, increased expression of PPARα and PPRE activity, a strong overall upregulation of the peroxisomal compartment and its associated gene expression (biogenesis, α-oxidation, β-oxidation, and plasmalogens) in PPARγ-deficient club cells. Interestingly, catalase was significantly increased and mistargeted into the cytoplasm, suggestive for oxidative stress by the PPARγ-deficiency in club cells. Taken together, PPARα-mediated metabolic induction and proliferation of peroxisomes via a PPRE-dependent mechanism could compensate PPARγ-deficiency in club cells

PPAR alpha-mediated peroxisome induction compensates PPAR gamma-deficiency in bronchiolar club cells

Despite the important functions of PPARγ in various cell types of the lung, PPARγ-deficiency in club cells induces only mild emphysema. Peroxisomes are distributed in a similar way as PPARγ in the lung and are mainly enriched in club and AECII cells. To date, the effects of PPARγ-deficiency on the overall peroxisomal compartment and its metabolic alterations in pulmonary club cells are unknown. Therefore, we characterized wild-type and club cell-specific PPARγ knockout-mice lungs and used C22 cells to investigate the peroxisomal compartment and its metabolic roles in the distal airway epithelium by means of 1) double-immunofluorescence labelling for peroxisomal proteins, 2) laser-assisted microdissection of the bronchiolar epithelium and subsequent qRT-PCR, 3) siRNA-transfection of PPARγand PPRE dual-luciferase reporter activity in C22 cells, 4) PPARg inhibition by GW9662, 5) GC-MS based lipid analysis. Our results reveal elevated levels of fatty acids, increased expression of PPARα and PPRE activity, a strong overall upregulation of the peroxisomal compartment and its associated gene expression (biogenesis, α-oxidation, β-oxidation, and plasmalogens) in PPARγ-deficient club cells. Interestingly, catalase was significantly increased and mistargeted into the cytoplasm, suggestive for oxidative stress by the PPARγ-deficiency in club cells. Taken together, PPARα-mediated metabolic induction and proliferation of peroxisomes via a PPRE-dependent mechanism could compensate PPARγ-deficiency in club cells.status: publishe