Mechanisms driving the delayed activation of target genes

Nanostring and RNA-seq data from Ptf1a overexpressing animal caps indicate that Ptf1a activates its direct target genes at different time points. Interestingly, by comparing the induced target genes with a list of genes being activated by Ptf1a in the presence of the translational inhibitor CHX (Schneider-Poetsch et al., 2010), the time of late target gene induction could be detected at a 12 h timepoint after gene induction. This indicates that certain events have to take place leading to the activation of first delayed target genes.

Interestingly, delayed target gene activation correlates with the timing of tubb2b activation. As tubb2b is a marker for post-mitotic neurons (Oschwald et al., 1991), indicating that the timing of delayed target gene activation may correlate with the exit of the cell cycle. Considering that direct target genes having their onset after 12 h of Ptf1a induction and are highly enriched for functions in neuronal specification, while early activated genes are more associated with regulation of transcription and the regulation of the cell cycle, would support such a mechanism. Together this would mean that cell cycle escape triggers the events leading to the activation of late target genes.

However, how cell cycle escape can be associated with the delayed expression of Ptf1a late target genes need to be further analyzed. It has been reported that the length of the cell cycle plays an essential role in neuronal differentiation, as an elongation of the cell cycle is required for inducing the onset of differentiation by proneural bHLH transcription factors (Lange et al., 2009; Lange and Calegari, 2010). Furthermore, the activity of many bHLH transcription factors involved in promoting neuronal differentiation including Neurog2 or Neurod4, are highly regulated by the cell cycle through Cdk-mediated phosphorylation (Ali et al., 2011; Hindley et al., 2012; Hardwick and Philpott, 2015). As Ptf1a also contains residues that would make it a candidate for Cdk-dependent phosphorylation, a cell cycle dependent regulation of Ptf1a activity cannot be excluded.

Chromatin remodeling has been reported to regulate transcription during development since actively transcribed genes are associated with accessible chromatin as well as activating histone marks and demethylated DNA (Yao and Jin, 2014; Hsieh and Zhao, 2016; Yao et al., 2016). However, the findings in ATAC-seq that Ptf1a target genes are located within rather closed chromatin regions suggests that chromatin remodeling plays a minor role in the delayed activation of Ptf1a target genes. This is further supported by the fact that the presence of open chromatin marks is not sufficient for inducing transcription (Hontelez et al., 2015) and that high levels of DNA methylation, which is associated with transcriptional silencing, does not affect the transcription of target genes in the early embryo (Bogdanovic et al., 2012). Considering that especially late Ptf1a target genes need Brg1 to be expressed, while induction of most direct early Ptf1a target genes is not affected, BAF complex mediated chromatin remodeling still has an influence on late target gene activation. The finding that Brg1 is required for the activation of several of the late Ptf1a direct target genes would support an implement of cell cycle regulation in Ptf1a late target gene activation. This is further supported by Brg1 has been reported to be involved in the regulation of cell cycle exit via interaction with Geminin (Seo et al., 2005). This could also explain that early Ptf1a target genes are mostly unaffected by a Brg1 knockdown, because as those genes are mostly involved in transcriptional and cell cycle regulation, their activity is required before cell cycle exit, which is regulated by Brg1. Nevertheless, chromatin remodeling

alone cannot explain the delayed activation of late target genes, indicating that several mechanisms drive the delate activation of target genes. A potential mechanism might be the presence or absence of a certain Co-factor or repressor regulating the transcription of those genes. Such a possibility would be supported by findings that the onset of late Ptf1a target gene activation is preponed in presence of CHX (Hedderich, 2012).

Together, these indications lead to a model that Ptf1a induces at least two waves of target gene activation. The first one is activated nearly immediately and mostly controls the transcription of genes controlling the differentiation of the cell to a neuronal cell fate, which leads to a cell cycle escape. An exception is Prdm13, which allows for an early bias of the progenitor cell to a GABAergic cell fate. At the time of cell cycle arrest, a second wave of target genes are activated, which further regulate the subtype specification and function of the induced neuron. Activation of these genes is regulated by several mechanisms.

BAF complex mediated chromatin remodeling is required for the activation of a subset of those genes, while another unknown mechanism like the presence an unknown Co-factor or repressor controls the expression of the other late target genes. This would mean that investigating the Ptf1a transcription factor complex at the early and the late time point would be interesting to identify which differences occur in the composition of these complexes between those two time points.