Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases.

Markus Kwik,Emanuele Bernardinelli,Ulrich Koller,Greta Zara,Markus Paulmichl,Charity Nofziger,Wolfgang Patsch,Boris Tichy,Silvia Dossena,Stefan Hainzl,Markus Steiner,Serge Weis,Selma M Soyal,Jan Oppelt

doi:10.3390/ijms22073296

Abstract

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson’s disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.

Highlights

Alternative splicing and/or promoter usage occur in the great majority of mammalian genes and are key mechanisms in transcriptional regulation and generation of protein diversity [1,2,3]
We used an effective transcriptional activation system consisting of dCas9 fused to a potent tripartite activator called VPR encoding herpes simplex virus protein 16 (VP64), the activation domain of the p65 subunit of nuclear factor NF-κB and the replication and transcription activator (RTA) of the γ-herpesvirus family [33]
To verify lentiviral transduction of SH-SY5Y cells, dCas9-VPR encoding transcripts were amplified by qRT

Summary

Introduction

Alternative splicing and/or promoter usage occur in the great majority of mammalian genes and are key mechanisms in transcriptional regulation and generation of protein diversity [1,2,3]. Transcription from an alternative promoter located in intron 2 of the reference gene (RG) [13] generates a 75 kDa protein that shows subtle differences in co-activation selectivity compared to the reference protein [14]. We discovered CNS-specific PPARGC1A mRNAs that are transcribed from a novel promoter located ~583 kbp upstream of the reference promoter. These transcripts are more abundant than RG mRNAs in human post-mortem brain samples and are partially conserved in rodents [10]

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