Here we identify ZNF335/NIF1 as a central regulator of mammalian neurogenesis and neuronal differentiation. A splice donor/missense mutation of ZNF335 results in an extremely small brain in humans, and genetic ablation leads to early embryonic lethality in mice, while Emx1-Cre driven knockout leads to virtual absence of cortical structure. Loss of Znf335 causes premature cell cycle exit of progenitors, precociously depleting the progenitor pool. Znf335 is a part of a H3K4 methyltransferase complex and associates with the promoters of many key developmental genes to affect H3K4me3 as well as expression levels of target genes. A critical downstream target of Znf335 is REST/NRSF (master regulator of neurogenesis) representing a pathway critical for this neurogenetic function. Beyond its effects on progenitor cell proliferation, Znf335 also has essential effects on cell fate and cell morphology (and ultimately survival).
Despite the profound phenotype of ZNF335 mutation in humans, the mutation we describe is almost certainly hypomorphic. Overexpression of the human mutation can only partially rescue Znf335 deficiency, and conditional ablation of Znf335 in mouse cortex results in loss of essentially the entire cortex. Thus, null mutations in ZNF335 in humans are presumably embryonically lethal as in mice, illustrating the utility of unusual, partial loss-of-function mutations in humans to elucidate essential early embryonic functions of such genes.
This study provides direct insight into the function of TrxG complex proteins in embryonic neurogenesis. The interaction of Znf335 with proteins of the H3K4 methyltransferase complex suggests roles for Znf335 in the regulation, targeting, or stability of the complex. Epigenetic regulation causes programming of gene expression, and specific histone methylation can further orchestrate gene regulation in a cell type and tissue dependent manner. Mutations in neural specific chromatin regulatory complexes, nBAF, have been shown to affect proliferation and are linked with microcephaly (Hoyer et al., 2012
). Thus, this interaction provides a potential parallel for the broad effects of the ZNF335 mutation on human patients, the large number of genes and developmental processes altered by Znf335
knockdown, as well as the embryonic lethality in Znf335
-null mice, especially since knockouts of other histone methyltransferases are also lethal embryonically (Glaser et al., 2009
Loss of Znf335 alters expression levels of many key genes--including DLX homeobox genes (early brain development), Neurogenin, Nfib, Olig1, Math1, REST/NRSF, Co-REST 2 (neurogenesis)--among many other genes important for dendritic branching, cell adhesion, and signaling. Changes in these genes could explain phenotypes seen in the patients and correlate with abnormal neurogenesis evident in mouse models and account for the virtual absence of cortical neurons in the Znf335 CKO. Genes whose expression changes upon Znf335 deficiency could be primary targets of Znf335, or secondary targets of other regulatory genes downstream of Znf335 such as REST/NRSF, but revealing Znf335 as a critical regulator of gene expression essential for proper neuronal development.
Znf335 also regulates differentiation and gene transcription in postmitotic neurons. Znf335 deficiency blocks normal expression of ‘canonical’ neuronal marker genes such as NeuN and Mef2C, which could be either a secondary effect of premature and improper neurogenesis or may hint at a role of Znf335 in regulating cell identity, survival, and activity of mature neurons. ZNF335 regulates a variety of non- REST/NRSF targets that are important for the final stages of neuronal differentiation, such as genes regulating dendritic branching, and ion channels, which may suggest roles of ZNF335 in other neuron specific transcriptional complexes.
Genetic causes of microcephaly continue to grow in diversity, and include proteins involved in vesicle trafficking, mitotic spindle organization, and DNA repair (Thornton and Woods, 2009
). Premature neuronal fate specification, with consequent loss of progenitor cells, could be a frequent cellular mechanism resulting in microcephaly (Lehtinen and Walsh, 2011
). ZNF335 deficiency causes additional feature of neuronal degeneration, making it strikingly different, and more severe, than other microcephaly syndromes, which are typically compatible with postnatal survival and in many cases some intellectual function. Thus our data reveal ZNF335
as a unique type of microcephaly gene, and provides evidence of a new upstream regulator of the balance between progenitor cell division and differentiation.