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Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1

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ABSTRACT

Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. However, regulatory networks through which this occurs are not well defined. Here, we identified Geminin-associated chromatin locations in embryonic stem cells and Geminin- and Zic1-associated locations during neural fate acquisition at a genome-wide level. We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to demonstrate that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment.

No MeSH data available.


Geminin over-expression during neural cell fate acquisition promotes the expression of Gmnn-associated genes with Gmnn-dependent histone acetylation.(A) Clustering of a subset of Gmnn-associated genes that also exhibit Gmnn-dependent H3K9ac, by comparison with their expression levels in ES cells, embryonic CNS, and adult cortex. (B) Locations of Gmnn association or Gmnn-dependent H3K9ac are shown for several genes in (A) (WashU Epigenome Browser). (C,D) ES cells were transfected and selected to overexpress a Gmnn cDNA construct, and (C) qRTPCR and (D) immunoblotting demonstrate increased Gmnn expression levels at the mRNA and protein level. (E) Levels of expression of four Gmnn-associated genes were defined on days 1–3 of neural fate acquisition, with versus without Gmnn over-expression (Gmnn OE). Gene expression levels on each day of NE fate acquisition are expressed relative to ES = 1.0 and p-values shown (student’s t-test) compare expression with versus without Gmnn OE on each day of the NE fate acquistion: ** =< 0.01, * =< 0.05, ns = not significant. Error bars represent standard deviation for a representative qPCR performed in triplicate.
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f4: Geminin over-expression during neural cell fate acquisition promotes the expression of Gmnn-associated genes with Gmnn-dependent histone acetylation.(A) Clustering of a subset of Gmnn-associated genes that also exhibit Gmnn-dependent H3K9ac, by comparison with their expression levels in ES cells, embryonic CNS, and adult cortex. (B) Locations of Gmnn association or Gmnn-dependent H3K9ac are shown for several genes in (A) (WashU Epigenome Browser). (C,D) ES cells were transfected and selected to overexpress a Gmnn cDNA construct, and (C) qRTPCR and (D) immunoblotting demonstrate increased Gmnn expression levels at the mRNA and protein level. (E) Levels of expression of four Gmnn-associated genes were defined on days 1–3 of neural fate acquisition, with versus without Gmnn over-expression (Gmnn OE). Gene expression levels on each day of NE fate acquisition are expressed relative to ES = 1.0 and p-values shown (student’s t-test) compare expression with versus without Gmnn OE on each day of the NE fate acquistion: ** =< 0.01, * =< 0.05, ns = not significant. Error bars represent standard deviation for a representative qPCR performed in triplicate.

Mentions: Our previous work suggested that Geminin over-expression promoted, while Geminin knockdown inhibited, histone acetylation at neural gene promoters29. Therefore, we defined profiles of histone H3 lysine 9 acetylation (H3K9ac) at gene promoters with and without Gmnn knockdown during NE fate acquisition, using an ES line that enables doxycycline-inducible knockdown of Gmnn29. These represent a subset of NE genes at which Gmnn levels control a transcriptionally active epigenetic status (Supplementary Table S4). One quarter of the Gmnn-associated genes in NE (24.5%) also exhibited Gmnn-dependent promoter acetylation (Fig. 3H). Genes that underwent Gmnn-dependent acetylation were associated with cell differentiation, development, and positive regulation of biological processes; the majority (64%) were more highly expressed in the embryonic CNS than in ES cells and this subset was enriched for development and neural development-related GO terms (Fig. 3I; Supplementary Fig. S5). Likewise, the subset of genes that are both Gmnn-associated in NE and undergo Gmnn-dependent histone acetylation exhibits predominantly enriched expression in embryonic CNS (73% of genes). This gene set contains transcription factors with later roles in neurogenesis (e.g. Nhlh1, Hes1, Dll3, Ebf2, Pax6, Vax1) and regional control of neural plate patterning or neuronal differentiation (e.g. Dlx1, Dbx1, Irx3, Lhx5, Pou4f1, Pax2, and Pax7) (Fig. 4A).


Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1
Geminin over-expression during neural cell fate acquisition promotes the expression of Gmnn-associated genes with Gmnn-dependent histone acetylation.(A) Clustering of a subset of Gmnn-associated genes that also exhibit Gmnn-dependent H3K9ac, by comparison with their expression levels in ES cells, embryonic CNS, and adult cortex. (B) Locations of Gmnn association or Gmnn-dependent H3K9ac are shown for several genes in (A) (WashU Epigenome Browser). (C,D) ES cells were transfected and selected to overexpress a Gmnn cDNA construct, and (C) qRTPCR and (D) immunoblotting demonstrate increased Gmnn expression levels at the mRNA and protein level. (E) Levels of expression of four Gmnn-associated genes were defined on days 1–3 of neural fate acquisition, with versus without Gmnn over-expression (Gmnn OE). Gene expression levels on each day of NE fate acquisition are expressed relative to ES = 1.0 and p-values shown (student’s t-test) compare expression with versus without Gmnn OE on each day of the NE fate acquistion: ** =< 0.01, * =< 0.05, ns = not significant. Error bars represent standard deviation for a representative qPCR performed in triplicate.
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f4: Geminin over-expression during neural cell fate acquisition promotes the expression of Gmnn-associated genes with Gmnn-dependent histone acetylation.(A) Clustering of a subset of Gmnn-associated genes that also exhibit Gmnn-dependent H3K9ac, by comparison with their expression levels in ES cells, embryonic CNS, and adult cortex. (B) Locations of Gmnn association or Gmnn-dependent H3K9ac are shown for several genes in (A) (WashU Epigenome Browser). (C,D) ES cells were transfected and selected to overexpress a Gmnn cDNA construct, and (C) qRTPCR and (D) immunoblotting demonstrate increased Gmnn expression levels at the mRNA and protein level. (E) Levels of expression of four Gmnn-associated genes were defined on days 1–3 of neural fate acquisition, with versus without Gmnn over-expression (Gmnn OE). Gene expression levels on each day of NE fate acquisition are expressed relative to ES = 1.0 and p-values shown (student’s t-test) compare expression with versus without Gmnn OE on each day of the NE fate acquistion: ** =< 0.01, * =< 0.05, ns = not significant. Error bars represent standard deviation for a representative qPCR performed in triplicate.
Mentions: Our previous work suggested that Geminin over-expression promoted, while Geminin knockdown inhibited, histone acetylation at neural gene promoters29. Therefore, we defined profiles of histone H3 lysine 9 acetylation (H3K9ac) at gene promoters with and without Gmnn knockdown during NE fate acquisition, using an ES line that enables doxycycline-inducible knockdown of Gmnn29. These represent a subset of NE genes at which Gmnn levels control a transcriptionally active epigenetic status (Supplementary Table S4). One quarter of the Gmnn-associated genes in NE (24.5%) also exhibited Gmnn-dependent promoter acetylation (Fig. 3H). Genes that underwent Gmnn-dependent acetylation were associated with cell differentiation, development, and positive regulation of biological processes; the majority (64%) were more highly expressed in the embryonic CNS than in ES cells and this subset was enriched for development and neural development-related GO terms (Fig. 3I; Supplementary Fig. S5). Likewise, the subset of genes that are both Gmnn-associated in NE and undergo Gmnn-dependent histone acetylation exhibits predominantly enriched expression in embryonic CNS (73% of genes). This gene set contains transcription factors with later roles in neurogenesis (e.g. Nhlh1, Hes1, Dll3, Ebf2, Pax6, Vax1) and regional control of neural plate patterning or neuronal differentiation (e.g. Dlx1, Dbx1, Irx3, Lhx5, Pou4f1, Pax2, and Pax7) (Fig. 4A).

View Article: PubMed Central - PubMed

ABSTRACT

Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. However, regulatory networks through which this occurs are not well defined. Here, we identified Geminin-associated chromatin locations in embryonic stem cells and Geminin- and Zic1-associated locations during neural fate acquisition at a genome-wide level. We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to demonstrate that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment.

No MeSH data available.