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Genome-wide chromatin binding profiles for Zic1 and Geminin during neuroectodermal cell specification

ABSTRACT: Cell intrinsic factors that control neuroectoderm specification of early embryonic cells include the nucleoprotein Geminin (Gmnn) and the Zic family of zinc finger transcription factors. Gmnn modulates chromatin state to activate neural gene expression during neural cell fate acquisition, while Gmnn deficiency in the forming neural plate disrupts transcriptional programs that control neural cell specification, neural plate patterning and neurogenesis, resulting in neural tube defects. Likewise, Zic1 over-expression promotes neural gene expression, while heterozygous deletion of Zic1/4 leads to Dandy-Walker malformation, the most common congenital cerebellar malformation. During embryonic development, Geminin and Zic1 expression is enriched in neuroectoderm from gastrula stages, with broad expression in the forming CNS during post-gastrula stages, when neural tube closure and neurogenesis are initiated. To gain a greater understanding of the molecular events that regulate neural cell specification, here we used ChIP-seq to define genome-wide chromatin binding profiles for Gmnn in embryonic stem cells (ESCs) and for Gmnn and Zic1 during specification of ESCs into neuroectoderm.

ORGANISM(S): Mus musculus

PROVIDER: GSE77246 | GEO | 2016/12/01

SECONDARY ACCESSION(S): PRJNA309845

REPOSITORIES: GEO

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Project description:Transcriptional targets of neurogenin (Ngnr1) were identified by over-expression of an inducible form of neurogenin in Xenopus ectodermal explants. The effects of co-expressing the nucleoprotein geminin on Ngnr1-dependent target gene transactivation were defined. Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized both neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, geminin antagonized the ability of neural bHLH transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. Geminin is highly expressed in undifferentiated neuronal precursor cells but is downregulated prior to differentiation. Therefore, these data support a model whereby geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis. A dexamethasone-inducible (GR ligand binding domain fused) form of Xenopus neurogenin-related 1, NgnrGR, was over-expressed in Xenopus embryonic ectodermal explants, in the presence or absence of over-expressed geminin. Induction of Ngnr1 activity was used to define direct targets as previously described (EMBO J. 26(24): 5093-5108). The ability of geminin to suppress Ngnr1-dependent transactivation of its target gene programs was determined.

Project description:Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein Geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized both neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, Geminin antagonized the ability of neural bHLH transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, Geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. Geminin is highly expressed in undifferentiated neuronal precursor cells but is downregulated prior to differentiation. Therefore, these data support a model whereby Geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis. A mouse embryonic stem (ES) cell line for inducible knockdown of the small nucleoprotein Geminin was utilized. ES cells were used to generate neural precursor cells by monolayer culture in N2B27 media for 5 days, and doxycycline-inducible knockdown of Geminin was performed from day 3. Changes in gene expression resulting from Geminin knockdown were assessed by RNA sequencing. Three experimental replicates were generated for Geminin knockdown (plus Dox) with a corresponding no-Dox control. These were subjected to sequencing, and data were analyzed using TopHat and Cufflinks/Cuffdiff. Transcripts were considered as differentially expressed upon Gem knockdown if data met statistical significance cutoffs in Cuffdiff (sufficient sequence alignments were obtained for analysis and transcript had significant change in FPKM value (normalized transcript abundance; fragments per kb of exon per million fragments mapped) between the no Dox and plus Dox sample pairs) in at least two of the three replicates.

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Genome-wide chromatin binding profiles for Zic1 and Geminin during neuroectodermal cell specification

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