Regulating the dorsal neural tube expression of Ptf1a through a distal 3′ enhancer

Abstract

Generating the correct balance of inhibitory and excitatory neurons in a neural network is essential for normal functioning of a nervous system. The neural network in the dorsal spinal cord functions in somatosensation where it modulates and relays sensory information from the periphery. PTF1A is a key transcriptional regulator present in a specific subset of neural progenitor cells in the dorsal spinal cord, cerebellum and retina that functions to specify an inhibitory neuronal fate while suppressing excitatory neuronal fates. Thus, the regulation of Ptf1a expression is critical for determining mechanisms controlling neuronal diversity in these regions of the nervous system. Here we identify a sequence conserved, tissue-specific enhancer located 10.8 kb 3′ of the Ptf1a coding region that is sufficient to direct expression to dorsal neural tube progenitors that give rise to neurons in the dorsal spinal cord in chick and mouse. DNA binding motifs for Paired homeodomain (Pd-HD) and zinc finger (ZF) transcription factors are required for enhancer activity. Mutations in these sequences implicate the Pd-HD motif for activator function and the ZF motif for repressor function. Although no repressor transcription factor was identified, both PAX6 and SOX3 can increase enhancer activity in reporter assays. Thus, Ptf1a is regulated by active and repressive inputs integrated through multiple sequence elements within a highly conserved sequence downstream of the Ptf1a gene.

Positive autofeedback regulation of Ptf1a transcription generates the levels of PTF1A required to generate itch circuit neurons

“…These include a 2.3-kb autoregulatory (AR) enhancer located 13.4 kb upstream of Ptf1a (Masui et al 2007(Masui et al , 2008Meredith et al 2009) and another 1.2-kb dorsal neural tube (DNT) enhancer located 11 kb downstream from Ptf1a (Fig. 1A, top; Mona et al 2016). The AR enhancer is directly regulated by the PTF1 transcriptional activator complex, a trimeric complex comprising PTF1A, an E-protein partner such as E47 or HEB (TCF3 or TCF12), and RBPJ (Beres et al 2006;Hori et al 2008).…”

“…The spacing between these motifs is constrained to one to three DNA helical turns for the PTF1 trimer to bind and activate transcription. The DNT enhancer contains a highly conserved 132-bp region required for its function (Mona et al 2016). The assays used to demonstrate enhancer activity of a DNA sequence test sufficiency to direct tissue-specific gene expression but do not address their requirement in vivo.…”

“…The DNT enhancer was identified through direct tests of seven genomic regions downstream from the Ptf1a-coding region selected due to extensive sequence conservation across mammals (Mona et al 2016). Nevertheless, deletion of a 118-bp region within the DNT had no detectable consequence to Ptf1a transcription or to Ptf1a-dependent behaviors.…”

“…The Ptf1a enhancer mutant mouse lines were generated by pronuclear injection of Cas9 mRNA and sgRNA targeting previously identified enhancer regions of Ptf1a (Meredith et al 2009;Mona et al 2016) in the Transgenic Technology Center at University of Texas Southwestern. Three experimental strategies were used (Fig.…”

“…However, given the lack of sustained Ptf1a expression into the mature nervous system, the importance of positive autoregulation for this tissue was not clear. A second enhancer comprises a highly con-served sequence 3 ′ of Ptf1a that was defined as a dorsal neural tube specific enhancer (Mona et al 2016). Here we test the requirement for these enhancer sequences for generating the correct spatiotemporal expression of Ptf1a in the mouse neural tube.…”