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.

c- and N-myc regulate neural precursor cell fate, cell cycle, and metabolism to direct cerebellar development.

Separate murine knockout (KO) of either c- or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre causes microcephaly. The cerebellum is particularly affected in the N-myc KO, leading to a strong reduction in cerebellar granule neural progenitors (CGNP) and mature granule neurons. In humans, mutation of N-myc also causes microcephaly in Feingold Syndrome. We created a double KO (DKO) of c- and N-myc using nestin-cre, which strongly impairs brain growth, particularly that of the cerebellum. Granule neurons were almost absent from the Myc DKO cerebellum, and other cell types were relatively overrepresented, including astroglia, oligodendrocytes, and Purkinje neurons. These findings are indicative of a profound disruption of cell fate of cerebellar stem and precursors. DKO Purkinje neurons were strikingly lacking in normal arborization. Inhibitory neurons were ectopic and exhibited very abnormal GAD67 staining patterns. Also consistent with altered cell fate, the adult DKO cerebellum still retained a residual external germinal layer (EGL). CGNP in the DKO EGL were almost uniformly NeuN and p27KIP1 positive as well as negative for Math1 and BrdU at the peak of normal cerebellar proliferation at P6. The presence of some mitotic CGNP in the absence of S phase cells suggests a possible arrest in M phase. CGNP and NSC metabolism also was affected by loss of Myc as DKO cells exhibited weak nucleolin staining. Together these findings indicate that c- and N-Myc direct cerebellar development by maintaining CGNP and NSC populations through inhibiting differentiation as well as directing rapid cell cycling and active cellular metabolism.

c-myc and N-myc promote active stem cell metabolism and cycling as architects of the developing brain.

myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre impairs mouse brain growth and mutation of N-myc also causes microcephaly in humans in Feingold Syndrome. To further define myc function in NSC and nervous system development, we created a double KO (DKO) for c- and N-myc using nestin-cre. The DKO mice display profoundly impaired overall brain growth associated with decreased cell cycling and migration of NSC, which are strikingly decreased in number. The DKO brain also exhibits specific changes in gene expression including downregulation of genes involved in protein and nucleotide metabolism, mitosis, and chromatin structure as well as upregulation of genes associated with differentiation. Together these data support a model of nervous system tumorigenesis in which excess myc aberrantly locks in a developmentally active chromatin state characterized by overactive cell cycling, and metabolism as well as blocked differentiation.

N-Myc regulates a widespread euchromatic program in the human genome partially independent of its role as a classical transcription factor.

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and methylated at lysine 4 is N-Myc-dependent. However, Myc regulation of transcription, even of genes it directly binds and at which it is required for the maintenance of active chromatin, is generally weak. Thus, Myc has a much more potent ability to regulate large domains of euchromatin than to influence the transcription of individual genes. Overall, Myc regulation of chromatin in the human genome includes both specific genes, but also expansive genomic domains that invoke functions independent of a classic transcription factor. These findings support a new dual model for Myc chromatin function with important implications for the role of Myc in cancer and stem cell biology, including that of induced pluripotent stem cells.

N-myc coordinates retinal growth with eye size during mouse development.

Myc family members play crucial roles in regulating cell proliferation, size, differentiation, and survival during development. We found that N-myc is expressed in retinal progenitor cells, where it regulates proliferation in a cell-autonomous manner. In addition, N-myc coordinates the growth of the retina and eye. Specifically, the retinas of N-myc-deficient mice are hypocellular but are precisely proportioned to the size of the eye. N-myc represses the expression of the cyclin-dependent kinase inhibitor p27Kip1 but acts independently of cyclin D1, the major D-type cyclin in the developing mouse retina. Acute inactivation of N-myc leads to increased expression of p27Kip1, and simultaneous inactivation of p27Kip1 and N-myc rescues the hypocellular phenotype in N-myc-deficient retinas. N-myc is not required for retinal cell fate specification, differentiation, or survival. These data represent the first example of a role for a Myc family member in retinal development and the first characterization of a mouse model in which the hypocellular retina is properly proportioned to the other ocular structures. We propose that N-myc lies upstream of the cell cycle machinery in the developing mouse retina and thus coordinates the growth of both the retina and eye through extrinsic cues.