N-Myc and GCN5 Regulate Significantly Overlapping Transcriptional Programs in Neural Stem Cells

Here we examine the functions of the Myc cofactor and histone acetyltransferase, GCN5/KAT2A, in neural stem and precursor cells (NSC) using a conditional knockout approach driven by nestin-cre. Mice with GCN5-deficient NSC exhibit a 25% reduction in brain mass with a microcephaly phenotype similar to that observed in nestin-cre driven knockouts of c- or N-myc. In addition, the loss of GCN5 inhibits precursor cell proliferation and reduces their populations in vivo, as does loss of N-myc. Gene expression analysis indicates that about one-sixth of genes whose expression is affected by loss of GCN5 are also affected in the same manner by loss of N-myc. These findings strongly support the notion that GCN5 protein is a key N-Myc transcriptional cofactor in NSC, but are also consistent with recruitment of GCN5 by other transcription factors and the use by N-Myc of other histone acetyltransferases. Putative N-Myc/GCN5 coregulated transcriptional pathways include cell metabolism, cell cycle, chromatin, and neuron projection morphogenesis genes. GCN5 is also required for maintenance of histone acetylation both at its putative specific target genes and at Myc targets. Thus, we have defined an important role for GCN5 in NSC and provided evidence that GCN5 is an important Myc transcriptional cofactor in vivo

Loss of GCN5 reduces NSC proliferation and increases differentiation along the oligodendrocyte lineage.

<p>Proliferating 4A4+ cells in the ventricular zone (VZ) of E12.5 control (A), E12.5 <i>GCN5</i> KO (B), E14.5 control (C), and E14.5 <i>GCN5</i> KO (D). Notice than fewer cells are 4A4+ in the VZ of the KOs than in the control mice. Proliferating 4A4+ cells in the VZ and subventricular zone (SVZ) of E17.5 control and <i>GCN5</i> KO (E,F). There are fewer 4A4+ cells in the SVZ of the KOs than in the control mice. Some of the dividing 4A4+ cells at the VZ are indicated with an arrow, and the dividing 4A4+ cells at the SVZ are indicated with an arrowhead. Scale bar: A-F: 10 µm. Error bars are standard deviations.</p

GCN5 maintains histone acetylation at specific target genes and in a widespread manner in NSC.

<p>(A–C) ChIP assays were conducted on control and <i>GCN5</i> KO NSC lines at both N-Myc (A–B) and <i>GCN5</i> target genes (C). <i>GCN5</i> binding itself was undetectable at any target. “a” and “b” represent biological replicate sets of neurospheres isolated from littermate embryos. (D) Control and <i>GCN5</i> KO NSC were immunostained for pan-Ac-H4 (Red) and DAPI (Blue). Scale Bars are 100 µM.</p

Ontological Gene Clusters Downregulated in KOs (p values shown).

<p>Ontological Gene Clusters Downregulated in KOs (p values shown).</p

Transcription Factors Downregulated in Kos.

<p>Transcription Factors Downregulated in Kos.</p

Mice with <i>GCN5</i> KO NSC have impaired brain growth.

<p>(A) Homozygously <i>GCN5</i> floxed mice were crossed with homozygously floxed nestin-cre+ mice, producing knockout mice (homozygous nestin-cre+) at a slightly less than predicted ratio. The dashed lines represent the number predicted based on Mendelian ratios. (B–C) Brains at E14.5 and E17.5 were isolated with evidence of impaired brain growth in the <i>GCN5</i> KOs at both stages. (D–E) Adult <i>GCN5</i> KO brains were smaller in mass and size. Scale bars are 2 mm in each image. Error bars are standard deviations.</p

Ontological Gene Clusters Upregulated in KOs (p values shown).

<p>Ontological Gene Clusters Upregulated in KOs (p values shown).</p

GCN5 and N-Myc regulate overlapping program of gene expression in NSC.

<p>(A) RNAs were isolated from control and <i>GCN5</i> KO neurosphere lines as well as from control and N-<i>myc</i> KO neurosphere lines. The KO in each case was verified by RT-PCR including PPIA for the loading control. (B) Expression microarrays were conducted on the RNAs and analyzed for overlap between genes altered in the <i>GCN5</i> and N-<i>myc</i> KOs. Overlap is indicated in the Venn diagrams along with p values. (C) Expression changes of select genes as measured by array were validated by RT-PCR.</p