Characterization of Multiple Exon 1 Variants and Neuron-specific Transcriptional Control of Mammalian HuD

Abstract

The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models. Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD. Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.