The Demethylase JMJD2C/KDM4C Localizes to H3K4me3 Positive Transcription Start Sites (ChIP-seq ESCs)
ABSTRACT: We have mapped binding sites for the histone demethylase, Jmjd2c/Kdm4c/Gasc1, in mouse embryonic stem cells (ESCs). ChIP-seq was performed using an antibody recognizing Jmjd2c. Chromatin was obtained from conditional Jmjd2c knockout ESCs cultured in the absence or presence of OHT to induce activation of Cre recombinase and loss of Jmjd2c expression.
The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell ...[more]
Project description:We have mapped binding sites for the histone demethylase, JMJD2C/KDM4C/GASC1, and the effect of JMJD2C depletion on H3K9me3 and H3K36me3 distributions in KYSE150 cells. The human esophageal carcinoma cell line, KYSE150, contains an amplification of the JMJD2C locus. ChIP-seq was performed using chromatin from control or JMJD2C-depleted KYSE150 cells and antibodies recognizing JMJD2C, H3K4me3, H3K9me3 or H3K36me3.
Project description:We have mapped binding sites for the histone demethylase, Jmjd2c/Kdm4c/Gasc1, in mouse embryonic fibroblasts (MEFs) and the impact of Jmjd2c depletion on H3K9me3 and H3K36me3 distributions. ChIP-seq was performed using antibodies recognizing Jmjd2c, H3K9me3 or H3K36me3. Chromatin was obtained from conditional Jmjd2c knockout MEFs cultured in the absence or presence of OHT to induce activation of Cre recombinase and loss of Jmjd2c expression.
Project description:We investigated the regions that are occupied by deltaNp63 in BxPC-3 and L3.6pl and identification of super enhancers in different pancreatic cancer cell lines. Thereby, we identified a group of 45 super enhancers that are associated with poorer prognosis and are highly dependent on deltaNp63.
Project description:Genome-wide occupancy of biotinylated Jmjd2b, Jmjd2c from mESCs, as well as occupancy of selected factors and histone marks from wild-type mESCs, Anti-GFP KD, Jmj2b KD and Jmjd2c KD mESCs genome To identify genome-wide binding target sites of Jmjd2b and Jmjd2c in the mESCs genome, and genome-wide binding sites for selected factors and histone marks from Anti-GFP KD, Jmjd2b KD and Jmjd2c KD mESCs
Project description:Jmjd2/Kdm4 H3K9-demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we uncover that Jmjd2c facilitates this process by stabilizing the assembly of Mediator-Cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2, independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and furthermore destabilizes loading of the Mediator and Cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c-G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with impact on timely gene activation. Overall design: Flag ChIP was performed for identification of Flag-Jmjd2c binding sites in mouse ES cells cultured in either naïve (2i/LIF) or primed (serum/LIF) conditions.
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).
Project description:We have mapped transcriptional changes after depletion of the histone demethylases JMJD2C/GASC1/KDM4C and JMJD2A/KDM4A alone or in combination in the esophageal squamous carcinoma cell line, KYSE150. The KYSE150 cell line contains an amplification of the JMJD2C locus. RNA was extracted from KYSE150 cells transfected with shRNAs targeting JMJD2C and/or JMJD2A. The experiment was performed in triplicates and expression levels analyzed using Affymetrix microarrays.
Project description:Global gene expression effects of silencing Jmjd1a and Jmjd2c genes We used microarrays to detail the global programme of gene expression after silencing Jmjd1a gene and Jmjd2c gene separately Keywords: dose response Overall design: three biological replicates each for Jmjd1a and Jmjd2c RNAi transcript levels were compared with six biological replicated of LUC RNAi