Cut&Run-seq of MUTZ3 cells to investigate changes in a translocated enhancer following CRISPR treatment
Ontology highlight
ABSTRACT: We examined the effects of targeting the GATA2 super-enhancer on EVI1 expression in MUTZ3. To that end, we conducted genome editing with CRISPR and assessed H3K27 acetylation with Cut&Run. The protocol described by the Henikoff group was used to generate these data.
Project description:Open chromatin regions were analyzed by ATAC-seq in MUTZ3 and MOLM1 cell lines (both inv(3) AML) to characterize the GATA2 super-enhancer region. ATAC-seq was performed as described (Buenrostro et al., 2013) with a modification in the lysis buffer to reduce mitochondrial DNA contamination.
Project description:The capacity of dendritic cells (DC) to migrate from peripheral organs to lymph nodes (LN) is an important event in the initiation of a T cell-mediated immune response. Previously it was shown that the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; ABCB1) and the 2 multidrug resistance protein 1 (MRP1; ABCC1) play a role in both human and murine DC migration. Here we show that a more recently discovered family-member, MRP4 (ABCC4) is expressed on both epidermal and dermal human skin DC and contributes to the migratory capacity of DC. Pharmacological inhibition of MRP4 activity or down-regulation through RNAi in DC resulted in reduced migration of DC from human skin explants and of in vitro generated Langerhans cells. The responsible MRP4 substrate remains to be identified as exogenous addition of MRP4’s known substrates PGE2, leukotriene B4 and D4 or cyclic nucleotides (all previously implicated in DC migration) could not restore migration. This notwithstanding, our data show that MRP4 is an important molecule, significantly contributing to human DC migration towards the draining lymph nodes, and thereby relevant for the initiation of an immune response and a possible target for immunotherapy. Keywords: cell type comparison, RNAi knockdown for MRP4 2 samples were analyzed to compare. Immature DC cultured from MUTZ3 (reference control) or from MUTZ3-shMRP4 cells
Project description:ChIP-seq was conducted to evaluate the effect of deleting a MYB binding site in the GATA2 super-enhancer or treating MUTZ3 cells with a MYB inhibitor. H3K27ac, MYB and p300 ChIP-seq datasets were generated.
Project description:Following a CRISPR enhancer scan covering the GATA2 super-enhancer region, the top sgRNAs were selected for further inspection. MUTZ3 cells were thus treated with the selected sgRNAs and the region of interested was subjected to amplicons sequencing (amplicon-seq). To that end, we used the Illumina PCR-based custom amplicon sequencing method using the TruSeq Custom Amplicon index kit (Illumina). The same experiment was conducted in K562 cells, which do not harbor an inv(3)/t(3;3), to investigate the role of MYB in this enhancer in other leukemia settings
Project description:Purpose: To characterize the genome-wide distribution of H3K79me2 in human leukemia cell lines treated with the Dot1l inhibitor EPZ004777 or control Methods: We performed Chip-seq for the H3K79me2 on the leukemia cell lines Mutz3, Loucy and Molm14 after 6 days in culture in the presence of 3uM EPZ004777 or DMSO control Results: H3K79me2 is completely erased from key target genes such as the HOXA cluster. Conclusions: Exposure of Mutz3, Loucy and Molm14 to 3uM EPZ004777 erases H3K79 methylation globally as well as on key loci ChIP-Seq for H3K79me2 on human leukemia cell lines exposed for 6 days to the Dot1l inhibitor EPZ004777
Project description:To uncover, in an unbiased fashion, which elements of the 18 kb translocated region control EVI1 transcription, we devised a CRISPR/Cas9-based enhancer scanning approach. We considered all possible sgRNA target sites containing a canonical Cas9 PAM site (NGG) on both strands of the minimal 18 kb translocated region. Deep-sequencing libraries were generated by PCR amplification of sgRNA guide strands using primers that tag the product with standard Illumina adapters and a 4 bp sample barcode in a 2 step-PCR protocol.
Project description:We investigated whether GATA2 enhancer-driven transcription of EVI1 in inv(3)/t(3;3) is reversible in leukemia cells. In primary inv(3)/t(3;3) AML, immature CD34+CD15- cells can be discriminated from more mature CD34-CD15- and CD34-CD15+ cells. Besides, we also investigated the effect of deleting a MYB binding site on these cells using CRISPR/Cas9.
Project description:To infer enhancers and super enhancers in Acute Myeloid Leukemia (AML) Cell lines with a 3q-aberration we determined regions enriched for H3K27AC, H3K4ME3, H3K4ME1, P300, and BRD4 in MOLM1. Additionally we determined regions enriched for P300 and BRD4 in the cell line Mutz3 which also harbors a 3q-aberration. As an control we performed Chip-Seq to determine enrichment for BRD4 in K562, which overexpresses the proto-oncogene EVI1, but has no apparent 3q-aberration. Ultimately, the ChipSeq experiments were utilized to infer which enhancer or super enhancer drives the overexpression of EVI1 in AMLs with a 3q-aberration. Finally, the effect of the compound JQ1 on the inferred super enhancers and the overexpression of EVI1 is tested by treating the cell line MOLM1 for 6 hours and determining the residual binding of BRD4.
Project description:This experiment aimed at investigating how O-GlcNac occupancy sites are impacted by RNA Polymerase II removal upon doxycycline stimulation. These human colon adenocarcinoma DLD-1 cells express OsTIR and a cassette encoding mini-AID (mAID) and fluorescent protein mClover (mAID+mClover) at the initiation site of the endogenous Rpb1 gene locus (POLR2A) (Nagashima 2019).