Differential regulation of the SMN2 gene by individual HDAC proteins

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. We sought to identify the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 genes. We used quantitative PCR to determine the effects shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.

Fig. 1

HDAC inhibition increases SMN-luciferase reporter mRNA levels. qRT-PCR was used to measure increases of SMN-luciferase mRNA following treatment with HDAC inhibitors. Fold increase of mRNA was normalized to GAPDH.

Fig. 2

shRNA silencing of individual HDACs increase SMN-luciferase activity and mRNA. (A) HDAC shRNAs increase SMN-luciferase activity levels. SMN2 reporter cells were treated for 48 h with shRNA. HDAC2 silencing increase luciferase activity the greatest at nearly 2-fold. HDAC5 knockdown appears to have no effect on luciferase activity (B) qRT-PCR was used to measure increases of SMN-luciferase mRNA following treatment with HDAC shRNA. Fold increase of mRNA was normalized to GAPDH.

Fig. 3

Quantitative PCR of endogenous splicing factors in response to HDAC inhibitor treatment in SMN2-luciferase reporter cells. SMN2-luciferase reporter cells were treated with 4 concentrations of HDAC inhibitors SAHA, M344, and MS-275. mRNA levels of splicing factors hTra2b (white bars), hnRNPA1 (gray bars), and SF2/ASF (dark gray bars). qRT-PCR was used to measure fold changes in transcript levels relative to DMSO carrier treated cells. GAPDH was used for normalization. (B) SMN2-luciferase reporter cells were treated with HDAC specific shRNA. mRNA levels of splicing factors hTra2b (white bars), hnRNPA1 (gray bars), and SF2/ASF (dark gray bars). qRT-PCR was used to measure fold changes in transcript levels relative to a non-silencing shRNA control. GAPDH was used for normalization.

Fig. 4

Quantitative chromosome immunoprecipitation of the SMN2-luciferase promoter. The SMN promoter was isolated from HDAC inhibitor and HDAC shRNA treated HEK293T SMN2-luciferase reporter cells using chromosome immunoprecipitation. Irrelevant rabbit IgG was used for a negative control. qPCR was used to accurate determine the percent of input DNA bound by anti-acetyl-H3 and anti-RNA Polymerase CTD antibodies.