Unknown,Transcriptomics,Genomics,Proteomics

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ATAC-seq of HEK293 cells with Alu-motif targeted dCas9GFP

ABSTRACT: HEK293 cells were selected and subcloned for doxycyclin (DOX) and trimethoprim (TMP) inducible dCas9GFP and a guide RNA molecule targeting an EGA enriched Alu-motif (EEA-g1). Cells were treated with DOX and TMP for three days to induce dCas9GFP expression and ATAC-seq samples were collected from treated and non-treated cells to analyse the effect of targeting the motif with dCas9.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Shintaro Katayama

PROVIDER: E-MTAB-6195 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

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ATAC-seq of HEK293 cells with Alu-motif targeted dCas9GFP

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