ABSTRACT: Cell differentiation and proliferation are mutually exclusive. Although differentiating neurons are recognized as post-mitotic non-dividing cells, some Rb- and Rb family (Rb, p107, and p130)-deficient differentiating neurons proliferate and form tumor. Here, we found that the acute inactivation of all Rb family in differentiating cortical excitatory neurons caused radial migration defect and S-phase progression but not cell division, whereas that in cortical progenitors caused the cell division of the differentiating neurons generated from Rb –/–; p107 –/–; p130 –/– (Rb-TKO) progenitors. Genome-wide DNA methylation analysis revealed that proximal promoters tended to become methylated during differentiation in vivo. DNA demethylation by DNA methyltransferase inhibitor allowed the acutely inactivated Rb-TKO differentiating neurons to undergo G2/M-phase progression. Our finding illustrate that cortical excitatory neurons epigenetically lose their proliferative potency after neurogenesis. 1 sample of the V/SVZ tissue and the CP tissue
Project description:Cell cycle deregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107, and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and form tumors when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb -/-; p107 -/-; p130 -/- (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated DNA double-strand break (DSB) repair pathway without increasing the tri-methylation of histone H4 at lysine 20 (H4K20M3), which is known to protect from DNA damage. The activation of DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly-born cortical neurons from progenitors epigenetically become protected from DNA damage and cell division in a Rb family-dependent manner. 3 samples of pCAG-control, pCAG-RbTKO, pMAP2-control, and pMAP2-RbTKO cells
Project description:Cell cycle deregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107, and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and form tumors when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb -/-; p107 -/-; p130 -/- (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated DNA double-strand break (DSB) repair pathway without increasing the tri-methylation of histone H4 at lysine 20 (H4K20M3), which is known to protect from DNA damage. The activation of DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly-born cortical neurons from progenitors epigenetically become protected from DNA damage and cell division in a Rb family-dependent manner.
Project description:Cell differentiation and proliferation are mutually exclusive. Although differentiating neurons are recognized as post-mitotic non-dividing cells, some Rb- and Rb family (Rb, p107, and p130)-deficient differentiating neurons proliferate and form tumor. Here, we found that the acute inactivation of all Rb family in differentiating cortical excitatory neurons caused radial migration defect and S-phase progression but not cell division, whereas that in cortical progenitors caused the cell division of the differentiating neurons generated from Rb â??/â??; p107 â??/â??; p130 â??/â?? (Rb-TKO) progenitors. Genome-wide DNA methylation analysis revealed that proximal promoters tended to become methylated during differentiation in vivo. DNA demethylation by DNA methyltransferase inhibitor allowed the acutely inactivated Rb-TKO differentiating neurons to undergo G2/M-phase progression. Our finding illustrate that cortical excitatory neurons epigenetically lose their proliferative potency after neurogenesis. 4 samples of the V/SVZ (ventricular/subventricular zone) tissue and 4 samples of the CP (cortical plate) tissue
Project description:Cell differentiation and proliferation are mutually exclusive. Although differentiating neurons are recognized as post-mitotic non-dividing cells, some Rb- and Rb family (Rb, p107, and p130)-deficient differentiating neurons proliferate and form tumor. Here, we found that the acute inactivation of all Rb family in differentiating cortical excitatory neurons caused radial migration defect and S-phase progression but not cell division, whereas that in cortical progenitors caused the cell division of the differentiating neurons generated from Rb –/–; p107 –/–; p130 –/– (Rb-TKO) progenitors. Genome-wide DNA methylation analysis revealed that proximal promoters tended to become methylated during differentiation in vivo. DNA demethylation by DNA methyltransferase inhibitor allowed the acutely inactivated Rb-TKO differentiating neurons to undergo G2/M-phase progression. Our finding illustrate that cortical excitatory neurons epigenetically lose their proliferative potency after neurogenesis.
Project description:Cell differentiation and proliferation are mutually exclusive. Although differentiating neurons are recognized as post-mitotic non-dividing cells, some Rb- and Rb family (Rb, p107, and p130)-deficient differentiating neurons proliferate and form tumor. Here, we found that the acute inactivation of all Rb family in differentiating cortical excitatory neurons caused radial migration defect and S-phase progression but not cell division, whereas that in cortical progenitors caused the cell division of the differentiating neurons generated from Rb –/–; p107 –/–; p130 –/– (Rb-TKO) progenitors. Genome-wide DNA methylation analysis revealed that proximal promoters tended to become methylated during differentiation in vivo. DNA demethylation by DNA methyltransferase inhibitor allowed the acutely inactivated Rb-TKO differentiating neurons to undergo G2/M-phase progression. Our finding illustrate that cortical excitatory neurons epigenetically lose their proliferative potency after neurogenesis.
Project description:Cell fate decisions during hematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. In order to gain insight about how these transcription factors regulate the activation of hematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from hemogenic endothelium to hematopoietic progenitors. Using a RUNX1-/- embryonic stem cell differentiation model expressing an inducible RUNX1 gene, we show that in the absence of RUNX1, SCL/TAL1, FLI1 and C/EBPM-NM-2 prime hematopoietic genes and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous new sites, initiating a local increase of histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of hematopoietic fate controlled by RUNX1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing hemogenic endothelium program but instead entails global reorganization of lineage-specific transcription factor assemblies. ChIPseq data from transcription factors Runx1, Fli-1, Scl/Tal1 and C/EBPM-NM-2, histone modification H3K9Ac as well as RNA Pol II obtained from differentiating murine hematopoietic cells
Project description:Oncogene-induced senescence is an anti-proliferative stress response program that acts as a fail-safe mechanism to limit oncogenic transformation and is regulated by the retinoblastoma protein (RB) and p53 tumor suppressor pathways. We identify the atypical E2F family member E2F7 as the only E2F transcription factor potently upregulated during oncogene-induced senescence, a setting where it acts in response to p53 as a direct transcriptional target. Once induced, E2F7 binds and represses a series of E2F target genes and cooperates with RB to efficiently promote cell cycle arrest and limit oncogenic transformation. Disruption of RB triggers a further increase in E2F7, which induces a second cell cycle checkpoint that prevents unconstrained cell division despite aberrant DNA replication. Mechanistically, E2F7 compensates for the loss of RB in repressing mitotic E2F target genes. Examination of E2F7 binding in either growing or senescent IMR90 cells with different hairpins.
Project description:Proprioception relies on two main classes of proprioceptive sensory neurons (pSNs). These neurons innervate two distinct peripheral receptors in muscle, muscle spindles (MSs) or Golgi tendon organs (GTOs), and synapse onto different sets of spinal targets, but the molecular basis of their distinct pSN subtype identity remains unknown. We used microarray analysis to compare gene expression profiles between MS- and GTO- innervating proprioceptors. We generated transgenic mice in which MS and GTO pSNs are labelled with different fluorescent proteins (see de Nooij et al., 2015 for details). We used Fluorescent Activated Cell Sorting (FACS) to isolate the MS and GTO pSN subsets from dissociated DRG from p7-10 transgenic mice. Neurons from multiple FACS experiments were pooled into three samples each for the MS and GTO pSN subset.
Project description:Transcription factor GATA1 binding in erythroblasts in the presence and absence of BET inhibitor JQ1, and BET protein BRD3 and BRD4 binding in erythroblasts in the presence and absence of GATA1. Inhibitors of Bromodomain and Extra-Terminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases yet their physiologic mechanisms remain largely unknown. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that while BRD3 occupied the majority of GATA1 binding sites, BRD2 and BRD4 were also recruited to a subset of GATA1-occupied sites. Functionally, BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Co-activation by BETs was accomplished both by facilitating genomic occupancy of GATA1 and subsequently supporting transcription activation. Using a combination of CRISPR/CAS9-mediated genomic engineering and shRNA approaches we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation, while depletion of BRD3 only affected erythroid transcription in the setting of BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and partially overlapping functions among BET family members. GATA1 null erythroblasts (G1E) conditionally expressing GATA1 as a GATA1-ER fusion protein were induced to express GATA1 by addition of 100nM estradiol for 24 hours. For GATA1 binding experiments this occurred in the absence or presence of 250nM JQ1. For BRD3 and BRD4 occupancy experiments G1E cells were compared to G1E cells with activated GATA1-ER fusion protein.
Project description:The expression of v5-tagged Hoxc9 is induced and ChIP-seq is used to profile genome-wide occupancy in differentiating motor neurons The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog signaling. Here, ChIP-seq is used to profile the genome-wide occupancy of Hoxc9 after five days of differentiation.