Egr-1 Induces DARPP-32 Expression in Striatal Medium Spiny Neurons via a Conserved Intragenic Element

Abstract

DARPP-32 (dopamine and adenosine 3Ј, 5Ј-cyclic monophosphate cAMP-regulated phosphoprotein, 32 kDa) is a striatal-enriched protein that mediates signaling by dopamine and other first messengers in the medium spiny neurons. The transcriptional mechanisms that regulate striatal DARPP-32 expression remain enigmatic and are a subject of much interest in the efforts to induce a striatal phenotype in stem cells. We report the identification and characterization of a conserved region, also known as H10, in intron IV of the gene that codes for DARPP-32 (Ppp1r1b). This DNA sequence forms multiunit complexes with nuclear proteins from adult and embryonic striata of mice and rats. Purification of proteins from these complexes identified early growth response-1 (Egr-1). The interaction between Egr-1 and H10 was confirmed in vitro and in vivo by super-shift and chromatin immunoprecipitation assays, respectively. Importantly, brain-derived neurotrophic factor (BDNF), a known inducer of DARPP-32 and Egr-1 expression, enhanced Egr-1 binding to H10 in vitro. Moreover, overexpression of Egr-1 in primary striatal neurons induced the expression of DARPP-32, whereas a dominantnegative Egr-1 blocked DARPP-32 induction by BDNF. Together, this study identifies Egr-1 as a transcriptional activator of the Ppp1r1b gene and provides insight into the molecular mechanisms that regulate medium spiny neuron maturation.

Serotonin2C receptor stimulation inhibits cocaine-induced Fos expression and DARPP-32 phosphorylation in the rat striatum independently of dopamine outflow

“…Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Proteins were then revealed as previously described (Maitre et al, 2011) with affinity purified rabbit antibodies (Cell Signaling Technology, CST, Beverly, MA, USA), previously used in numerous immunoblotting studies (Beaulieu et al, 2004;Tang and Bezprozvanny, 2004;Atkins et al, 2005;Belkhiri et al, 2005;Tropea et al, 2008;Danielli et al, 2010;Keilani et al, 2012;Cathala et al, 2014). Specifically, total anti-DARPP-32 antibody (#2306; 1/1500 dilution) and phosphorylation state-specific antibodies against DARPP-32 phosphorylated at threonine 34 (#2304 and #5393, 1/250 dilution) and threonine 75 residues (#2301; 1/250 dilution) were used.…”

Induction of DARPP-32 by Brain-Derived Neurotrophic Factor in Striatal Neurons In Vitro Is Modified by Histone Deacetylase Inhibitors and Nab2

“…4d). Induction of DARPP-32 by BDNF in vitro also requires an increase in Egr-1, aka NGF1-A, which binds to a sequence contained in a conserved region of the DARPP-32 gene to which we refer as H10 [7]. Induction of Egr-1 by BDNF plus VPA was equivalent to that of BDNF alone (Fig.…”

“…The double-stranded DNA fragments, aka H10 [7] (5′-AGCCGCCCACACTGTTCCTTTCC-3′) were labeled with ATP [γ- 32 P] and T4 polynucleotide kinase, and purified with Illustra Microspin G-25 columns (GE Healthcare). Each EMSA reaction (25–30 µl) contained 5–10 µg nuclear proteins, 2 µg Poly (dI-dC), 1 pmole DNA probe in 1× binding buffer [10 mM HEPES pH = 7.9, 30 mM KCl, 1.2% glycerol, 0.5 mM DTT, 5 mM MgCl2, 0.2 mM PMSF, 0.1 mM EDTA pH = 8].…”

“…Brain-derived neurotrophic factor (BDNF) is a major regulator of MSN phenotype during development and in the adult, and multiple molecular mechanisms via which it regulates ppp1r1b transcription have been well characterized [4]–[7]. Many factors that contribute to neuronal differentiation and plasticity, including BDNF [8], lead to alterations of chromatin structure via core histone modifications, and inhibition of histone deacetylase (HDAC) activity promotes this process [9].…”

Cellular Prion Protein (PrPc) and Hypoxia: True to Each Other in Good Times and in Bad, in Sickness, and in Health

“…These emerged at least 29.1 million years ago in the common stem lineage of extant Catherrini, as determined by own sequence screening (see Table 1 for accession numbers). Binding of Egr-1 to a conserved intron sequence and consecutive regulation of gene expression has been demonstrated in mouse motor spiny neurons (Keilani et al, 2012). Egr-1 is a transcription factor that is rapidly induced by hypoxia, can directly bind to HIF-1α promoter region and trans -activate it (Sperandio et al, 2009), but it can also function independently of HIF-1 α (Yan et al, 1999).…”