Mechanisms underlying the temporal and selective induction of Ptf1a target genes

Abstract

The bHLH transcription factor Ptf1a is crucial for the generation of a GABAergic neuronal cell fate over an excitatory glutamatergic fate. Ptf1a is a component of a heterotrimeric transcription factor complex, which in addition to Ptf1a, is comprised of a commonly expressed bHLH E-protein such as E12 and Rbpj. Interaction with Rbpj is essential for the GABAergic cell fate inducing and glutamatergic repressing activities of Ptf1a. In the absence of Rpbj binding, Ptf1a maintains its proneural activity, however, the induced neurons express marker genes indicative of a glutamatergic excitatory neuronal cell fate. In this thesis, the temporal and selective induction of target genes by Ptf1a was further analyzed as well several Ptf1a mutants characterized with respect to the neurotransmitter inducing properties using X. laevis as a model system. A point mutation within the C-terminus of Ptf1a that leads to the induction of a mixed glutamatergic and GABAergic neuronal transmitter phenotype was identified and characterized (Ptf1T243A). BiFC analysis revealed that the mixed neuronal transmitter phenotype is not due to an impairment of the interaction with Rbpj or E12, but may be due to enhanced interaction with Prdm13. The global temporal transcriptional program induced Ptf1a was also studied in pluripotent embryonic cells by RNAseq. While some Ptf1a direct target genes such as prdm13, are induced within three hours (early genes), others, including neurog2 and gad1a are induced only after 12 h (late genes). To gain insight if chromatin accessibility plays a role in the delayed activation, Brg1, which is one of the catalytic subunits of the chromatin remodeling BAF complex, was knocked down. Brg1 was found to be required for Ptf1a-induced neuronal differentiation as shown by the loss of neural-specific tubulin. Only a subset of late induced Ptf1a target genes required Brg1 with most Brg1 dependent target genes being indirect Ptf1a target genes. Furthermore, ATAC-seq suggests that target gene activation by Ptf1a is likely independent of chromatin accessibility. Taken together, these data suggest that Ptf1a acts as a pioneer transcription factor to activate its target genes.