Genetics: Published Articles Ahead of Print, published on September 7, 2012 as 10.1534/genetics.112.145128 Activity-dependent human brain coding / non-coding gene regulatory networks Leonard Lipovich*, Fabien Dachet§, Juan Cai§, Shruti Bagla§, Karina Balan§, Hui Jia*, and Jeffrey A. Loeb*§ *Center for Molecular Medicine and Genetics; §Department of Neurology, Wayne State University School of Medicine, Detroit, MI 48202 RUNNING TITLE: lncRNA networks in human brain KEY WORDS: long non-coding RNA (lncRNA) epilepsy human brain transcriptome BDNF AUTHOR TO WHOM CORRESPONDENCE SHOULD BE ADDRESSED: Dr. Jeffrey A. Loeb Department of Neurology Wayne State University 421 E. Canfield St. 3122 Elliman Detroit, MI 48201 Phone: (313) 577-9827 Fax: (313) 577-7552 E-mail: [email protected] Copyright 2012. ABSTRACT While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The BDNF gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long non-coding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and non- coding human transcription using a custom lncRNA microarray identified 1288 differentially-expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional eight were adjacent to or overlapping with differentially-expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, comprising an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF- BDNFOS co-expression that was not achieved with a single depolarization. RNAi- mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-mRNA pairs validated the effect of chronic neuronal activity on the transcriptome, and implied various lncRNA regulatory mechanisms. LncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. INTRODUCTION The availability of mammalian genome sequences has made it possible to delineate the boundaries and structures of all genes in a genome and has demonstrated an abundance of non-protein-coding transcriptional units that rivals the numbers of known protein-coding genes (reviewed in CARNINCI and HAYASHIZAKI 2007). Complex and potentially functional regulatory relationships between protein-coding and non-coding genes, including non-coding RNA genes that are poorly conserved across different species, have recently been delineated (ENGSTROM et al. 2006; KATAYAMA et al. 2005). These long non-coding RNA (lncRNA) genes can be defined by four fundamental criteria: encoding transcripts that lack any open reading frames (ORFs) greater than 100 amino acids or possessing protein database homologies (DINGER et al. 2008); being within the known range of lengths of mammalian mRNAs; support by transcript-to- genome alignments from cDNA data; and absence of matches to any known non- coding-RNA classes. Functionally, lncRNAs can have regulatory effects on coding mRNAs through a number of mechanisms, including those involving endogenous antisense lncRNA transcripts that repress their sense-strand protein-coding partners (KATAYAMA et al. 2005; YU et al. 2008). Endogenous lncRNAs can also have catalytic roles, as exemplified by the TERC telomerase RNA, and by the RNAse P and MRP RNAs required for processing of other RNAs. LncRNAs essential to nuclear architecture include NEAT1 and NEAT2. Nuclear hormone receptors, homeobox transcription factors, tumor suppressors, and immune regulators are all endogenously modulated by lncRNAs (reviewed in LIPOVICH et al. 2010). Numerous lncRNAs are transcribed in the vicinity of known protein-coding genes, and regulate those known genes through epigenetic mechanisms. Regulation of protein-coding genes by overlapping, or nearby-encoded, lncRNAs is central in cancer, cell cycle, and reprogramming (reviewed in LIPOVICH et al. 2010; LOEWER et al. 2010; OROM et al. 2010). LncRNAs encoded in an antisense orientation to, and overlapping with, known protein-coding genes are particularly abundant, and the small number of antisense lncRNAs characterized to date is replete with novel functions. Endogenous antisense lncRNAs are essential in mammalian X-inactivation (TIAN et al. 2010); can directly regulate tumor suppressors; function through dicer-independent mechanisms; and may be rapidly evolving or not conserved, raising the potential for new regulation of old genes over evolutionary time (LIPOVICH et al. 2010). RNAi and overexpression of lncRNAs in cell lines generate reproducible phenotypes, as we and others have shown (BERNARD et al. 2010; SHEIK MOHAMED et al. 2010). Hundreds of human lncRNAs bind the polycomb repressor complex 2 (PRC2), a key epigenetic negative regulator (KHALIL et al. 2009). Beyond high-throughput evidence of interactions with epigenetic factors, specific epigenetic roles of lncRNAs are beginning to be defined. Antisense lncRNAs actively and specifically modulate gene expression by serving as effectors of epigenetic changes at target loci (YU et al. 2008). These changes include antisense lncRNA- mediated epigenetic silencing of the sense-strand protein-coding gene promoter; such silencing can be abrogated by Argonaute-2-dependent, small-RNA-mediated suppression of the antisense lncRNA, resulting in “RNA activation” of the sense gene (MORRIS et al. 2008). Promoter-overlapping antisense lncRNAs can also be targeted by exogenous short RNAs that regulate sense gene expression, also via Argonaute (SCHWARTZ et al. 2008). Despite these promising examples, a majority of the thousands of other lncRNAs evident in transcriptome data still remain devoid of assigned functions. This abundance of lncRNAs, many of which are primate-specific, warrants a systematic assessment of whether they have functional, regulatory roles. Perhaps nowhere might this be more important than in the human brain that is composed of a diverse set of cell types connected through complex synaptic arrangements. The degree of synaptic activity in the brain can be translated into functional and structural changes through activity-dependent changes in gene expression (KATZ and SHATZ 1996). Although these changes can be effected through direct activation of synaptic genes, they can also be achieved through the release of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) that have direct effects on synaptic architecture and indirect effects by producing changes in gene expression (BINDER et al. 2001; ISACKSON et al. 1991). BDNF, a member of the nerve growth factor family, regulates the survival and differentiation of neuronal populations, axonal growth and pathfinding, dendritic growth and morphology and has been linked to many human brain disorders (reviewed in BIBEL and BARDE 2000; BINDER and SCHARFMAN 2004; HU and RUSSEK 2008). BDNF mRNA and protein are upregulated by seizure activity in animal models of epilepsy as well as in human brain tissues that display increased epileptic activities (BEAUMONT et al. 2012; ERNFORS et al. 1991; LINDVALL et al. 1994; NIBUYA et al. 1995). The genomic locus encoding BDNF is structurally complex and also encodes BDNFOS, a primate-specific lncRNA that is antisense to the coding BDNF gene (AID et al. 2007; LIU et al. 2006; PRUUNSILD et al. 2007). BDNF and BDNFOS form double-stranded duplexes, suggesting a potential for BDNFOS to post-transcriptionally regulate BDNF (PRUUNSILD et al. 2007). Antisense knockdown of BDNFOS, in fact, has recently been shown to increase BDNF expression in HEK293 cells and promotes neuronal outgrowth in vitro (Modarresi et al. 2012) BDNF binding to its receptors results in a diverse array of downstream signaling pathways including the activation of cyclic adenosine monophosphate response element binding protein (CREB), that in turn can also regulate BDNF by binding to a cognate site within the BDNF gene (SPENCER et al. 2008; TAO et al. 1998). Activation of CREB by phosphorylation at Serine 106 as a result of neuronal activity leads to changes in gene expression that cause reinforcement and stabilization of more active neuronal circuits (reviewed in HERDEGEN and LEAH 1998; KANDEL 2001; MATYNIA et al. 2002; WEST et al. 2002). Downstream from phosphorylated CREB (pCREB), immediate early genes (IEGs) have been shown to mediate long-lasting changes in neuronal structure and excitability. Upstream of CREB activation, several known signaling pathways are rapidly activated in response to neuronal activity (KANDEL 2001; reviewed in WEST et al. 2002), including CaMKinase IV, protein kinase A, and MAPK. We have recently observed a pattern of transcriptional activation in human brain regions where seizures start that strongly implicates sustained MAPK/CREB activation and downstream coding gene activations that could underlie layer specific changes