Genomic analysis of transcriptional networks directing progression of cell states during MGE development 06 Biological Sciences 0604 Genetics

Magnus Sandberg, Leila Taher, Jianxin Hu, Brian L. Black, Alexander Nord, John L.R. Rubenstein

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background: Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation. Methods: Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures. Results: We identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures. Conclusions: Tcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

Original languageEnglish (US)
Article number21
JournalNeural Development
Volume13
Issue number1
DOIs
StatePublished - Sep 14 2018

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Median Eminence
Biological Science Disciplines
Gene Regulatory Networks
Transcription Factors
GABAergic Neurons
Cholinergic Neurons
Clustered Regularly Interspaced Short Palindromic Repeats
Site-Directed Mutagenesis
Cholinergic Agents
Transcriptional Activation
Chromatin
In Situ Hybridization
Gene Expression
Mutation

Keywords

  • CRISPR engineering
  • LHX
  • Medial ganglionic eminence
  • MEIS
  • Neurogenesis
  • OCT
  • SOX
  • TCF12
  • Transcriptional network

ASJC Scopus subject areas

  • Developmental Neuroscience

Cite this

Genomic analysis of transcriptional networks directing progression of cell states during MGE development 06 Biological Sciences 0604 Genetics. / Sandberg, Magnus; Taher, Leila; Hu, Jianxin; Black, Brian L.; Nord, Alexander; Rubenstein, John L.R.

In: Neural Development, Vol. 13, No. 1, 21, 14.09.2018.

Research output: Contribution to journalArticle

Sandberg, Magnus ; Taher, Leila ; Hu, Jianxin ; Black, Brian L. ; Nord, Alexander ; Rubenstein, John L.R. / Genomic analysis of transcriptional networks directing progression of cell states during MGE development 06 Biological Sciences 0604 Genetics. In: Neural Development. 2018 ; Vol. 13, No. 1.
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abstract = "Background: Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation. Methods: Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures. Results: We identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures. Conclusions: Tcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.",
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AU - Hu, Jianxin

AU - Black, Brian L.

AU - Nord, Alexander

AU - Rubenstein, John L.R.

PY - 2018/9/14

Y1 - 2018/9/14

N2 - Background: Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation. Methods: Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures. Results: We identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures. Conclusions: Tcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

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KW - MEIS

KW - Neurogenesis

KW - OCT

KW - SOX

KW - TCF12

KW - Transcriptional network

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