Embryonic stem cell–based mapping of developmental transcriptional programs

Mazzoni, Esteban O.; Mahony, Shaun; Iacovino, Michelina; Morrison, Carolyn A.; Mountoufaris, George; Closser, Michael; Whyte, Warren A.; Young, Richard A.; Kyba, Michael; Gifford, David K.; Wichterle, Hynek

doi:10.1038/nmeth.1775

Cited by 75 publications

(70 citation statements)

References 15 publications

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“…All antibodies used had all been previously used for ChIP and validated for their specificity. Immunoprecipitations were with mouse anti-ASCL1 (BD Pharmingen) (Castro et al 2006), rabbit anti TCF3 (Santa Cruz, sc-349) (Lin et al 2010), rabbit anti-OLIG2 (Millipore, AB9610) (Mazzoni et al 2011), rabbit anti-MAX (Santa Cruz, sc-197) (Rahl et al 2010), goat anti-NFI (Santa Cruz, sc-30918) (Pjanic et al 2011;Martynoga et al 2013), goat anti-SOX2 (Santa Cruz sc-17320) (Chen et al 2008), rabbit anti-SOX9 (Millipore, AB5535) (Mead et al 2013), and goat anti-SOX21 (R&D systems, AF3538) (Matsuda et al 2012). Primers used for ChIP-PCR are listed in Supplemental Table S5.…”

Section: Chromatin Immunoprecipitationmentioning

confidence: 99%

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Mateo

Berg²,

Haeussler

et al. 2014

Genome Res.

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The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.

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Section: Chromatin Immunoprecipitationmentioning

confidence: 99%

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Mateo

Berg²,

Haeussler

et al. 2014

Genome Res.

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“…To address the predicted repressor program functionally, we used a doxycycline-inducible transgenic mESC system Mazzoni et al, 2011) to express ectopically Nkx2.2, Nkx6.1 and Olig2 individually, or in pairwise combinations: Nkx2.2 and Nkx6.1 (Nkx6.1-2A-Nkx2.2), and Olig2 and Nkx6.1 (Nkx6.1-2A-Olig2). Samples were subjected to global analysis of transcriptional activity by RNA-seq 12 h post-Dox induction, and targeted analysis of a subset of genes by microfluidic-based RT-qPCR (Fluidigm) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A direct fate exclusion mechanism by Sonic hedgehog-regulated transcriptional repressors

et al. 2015

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Sonic hedgehog (Shh) signaling patterns the vertebrate spinal cord by activating a group of transcriptional repressors in distinct neural progenitors of somatic motor neuron and interneuron subtypes. To identify the action of this network, we performed a genome-wide analysis of the regulatory actions of three key ventral determinants in mammalian neural tube patterning: Nkx2.2, Nkx6.1 and Olig2. Previous studies have demonstrated that each factor acts predominantly as a transcriptional repressor, at least in part, to inhibit alternative progenitor fate choices. Here, we reveal broad and direct repression of multiple alternative fates as a general mechanism of repressor action. Additionally, the repressor network targets multiple Shh signaling components providing negative feedback to ongoing Shh signaling. Analysis of chromatin organization around Nkx2.2-, Nkx6.1-and Olig2-bound regions, together with co-analysis of engagement of the transcriptional activator Sox2, indicate that repressors bind to, and probably modulate the action of, neural enhancers. Together, the data suggest a model for neural progenitor specification downstream of Shh signaling, in which Nkx2.2 and Olig2 direct repression of alternative neural progenitor fate determinants, an action augmented by the overlapping activity of Nkx6.1 in each cell type. Integration of repressor and activator inputs, notably activator inputs mediated by Sox2, is probably a key mechanism in achieving cell type-specific transcriptional outcomes in mammalian neural progenitor fate specification.

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“…The most important experimental section, which sets this protocol apart from previous work The antibody is key to any ChIP experiment and sufficient controls need to be carried out to show its specificity for the epitope of interest (see guidelines by Landt et al 36 ). If no ChIP-grade antibody is available, the introduction of corresponding epitope-tagged fusion proteins may be a legitimate alternative as these proteins can occupy endogeneous binding sites 37 . In this case, uninjected embryos are best to use as a negative control rather than a ChIP with non-specific serum.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide Snapshot of Chromatin Regulators and States in <em>Xenopus</em> Embryos by ChIP-Seq

Gentsch

Patrushev

Smith

2015

JoVE

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The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by nextgeneration sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

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Embryonic stem cell–based mapping of developmental transcriptional programs

Cited by 75 publications

References 15 publications

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

A direct fate exclusion mechanism by Sonic hedgehog-regulated transcriptional repressors

Genome-wide Snapshot of Chromatin Regulators and States in <em>Xenopus</em> Embryos by ChIP-Seq

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