Targeted attenuation of elevated histone marks at SNCA alleviates α‐synuclein in Parkinson's disease
2021; Springer Nature; Volume: 13; Issue: 2 Linguagem: Inglês
10.15252/emmm.202012188
ISSN1757-4684
AutoresSubhrangshu Guhathakurta, Jin-Il Kim, Levi Adams, Sambuddha Basu, Min Kyung Song, Evan Adler, Goun Je, Mariana Bernardo Fiadeiro, Yoon‐Seong Kim,
Tópico(s)Autism Spectrum Disorder Research
ResumoArticle11 January 2021Open Access Source DataTransparent process Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease Subhrangshu Guhathakurta Subhrangshu Guhathakurta Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Jinil Kim Jinil Kim Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Nexmos, Yongin-Si, South Korea Search for more papers by this author Levi Adams Levi Adams Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA Search for more papers by this author Sambuddha Basu Sambuddha Basu Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Min Kyung Song Min Kyung Song Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA Search for more papers by this author Evan Adler Evan Adler Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Goun Je Goun Je Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Mariana Bernardo Fiadeiro Mariana Bernardo Fiadeiro Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Yoon-Seong Kim Corresponding Author Yoon-Seong Kim [email protected] orcid.org/0000-0001-6246-5477 Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA Search for more papers by this author Subhrangshu Guhathakurta Subhrangshu Guhathakurta Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Jinil Kim Jinil Kim Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Nexmos, Yongin-Si, South Korea Search for more papers by this author Levi Adams Levi Adams Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA Search for more papers by this author Sambuddha Basu Sambuddha Basu Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Min Kyung Song Min Kyung Song Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA Search for more papers by this author Evan Adler Evan Adler Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Goun Je Goun Je Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Mariana Bernardo Fiadeiro Mariana Bernardo Fiadeiro Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Search for more papers by this author Yoon-Seong Kim Corresponding Author Yoon-Seong Kim [email protected] orcid.org/0000-0001-6246-5477 Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA Search for more papers by this author Author Information Subhrangshu Guhathakurta1, Jinil Kim1,2, Levi Adams1,3, Sambuddha Basu1, Min Kyung Song1,3, Evan Adler1, Goun Je1, Mariana Bernardo Fiadeiro1 and Yoon-Seong Kim *,1,3 1Burnett School of Biomedical Sciences, UCF College of Medicine, University of Central Florida, Orlando, FL, USA 2Nexmos, Yongin-Si, South Korea 3Robert Wood Johnson Medical School Institute for Neurological Therapeutics, Rutgers Biomedical and Health Sciences, Piscataway, NJ, USA *Corresponding author. Tel: +1 732 235 6499; Fax: +1 732 235 4773; E-mail: [email protected] EMBO Mol Med (2021)13:e12188https://doi.org/10.15252/emmm.202012188 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Epigenetic deregulation of α-synuclein plays a key role in Parkinson’s disease (PD). Analysis of the SNCA promoter using the ENCODE database revealed the presence of important histone post-translational modifications (PTMs) including transcription-promoting marks, H3K4me3 and H3K27ac, and repressive mark, H3K27me3. We investigated these histone marks in post-mortem brains of controls and PD patients and observed that only H3K4me3 was significantly elevated at the SNCA promoter of the substantia nigra (SN) of PD patients both in punch biopsy and in NeuN-positive neuronal nuclei samples. To understand the importance of H3K4me3 in regulation of α-synuclein, we developed CRISPR/dCas9-based locus-specific H3K4me3 demethylating system where the catalytic domain of JARID1A was recruited to the SNCA promoter. This CRISPR/dCas9 SunTag-JARID1A significantly reduced H3K4me3 at SNCA promoter and concomitantly decreased α-synuclein both in the neuronal cell line SH-SY5Y and idiopathic PD-iPSC derived dopaminergic neurons. In sum, this study indicates that α-synuclein expression in PD is controlled by SNCA’s histone PTMs and modulation of the histone landscape of SNCA can reduce α-synuclein expression. Synopsis Histone posttranslational modifications play a major role in the regulation of α-synuclein expression in Parkinson’s disease (PD). Locus-specific editing of H3K4me3 at the SNCA promoter reverts the deregulated expression of α-synuclein in neurons in the context of PD. α-synuclein expression is controlled by epigenetic regulation. H3K4me3 is heavily enriched at the SNCA promoter in PD patient brains. Locus-specific editing of H3K4me3 reduces neuronal α-synuclein expression in PD. The paper explained Problem High levels of α-synuclein protein and its aggregation in the substantia nigra pars compacta (SNpc) of midbrain region are considered as the main culprit for degeneration of dopamine producing neurons in Parkinson’s disease. The main problem remains the lack of understanding about the molecular mechanisms how this protein gets expressed more in PD patients and how that initiates misfolding which leads to neurodegeneration. Several hypotheses have been put forward to understand α-synuclein’s aggregation behavior. However, there remain caveats in our understanding about dysregulated expression of this protein. Epigenetic regulations are one of the key mechanisms which regulate gene expression. However, how epigenetic factors underlying fine-tuning of α-synuclein expression get deregulated in the disease remains to be elucidated. Results We observed the transcription-promoting histone post-translational modification (PTM), H3K4me3, is exclusively enriched at the α-synuclein gene promoter in post-mortem brain samples of PD patients. The high levels of H3K4me3 at the promoter were positively correlated with higher α-synuclein protein levels in the SN of the brain. We have developed a novel CRISPR/dCas9-based SunTag-JARID1A system which effectively reduced H3K4me3 enrichment from the SNCA gene promoter and concomitantly decreased the protein levels both in neuronal SHSY5Y cells as well as in dopaminergic neurons derived from PD patients’ iPSCs. These results implicate the importance of H3K4me3 in regulation of α-synuclein in PD. Impact Epigenetic factors confer the first line of regulation for gene expression. Histone PTMs are the most important regulators for tissue-specific gene expression. We identified a histone PTM, H3K4m3, gets deregulated in PD which in turn perturbs α-synuclein expression in the patients. Genomic locus-specific editing of this epigenetic mark in cultured human neurons from patients significantly reduced α-synuclein levels. The impact of this novel approach indicates that H3K4me3 may serve as a molecular target to slow down synucleinopathy-mediated dopaminergic neuronal degeneration in PD. Introduction Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease affecting nearly one million people worldwide. In the USA alone, 60,000 patients are diagnosed with PD each year (Parkinson’s Foundation). PD is a late-onset disease that destroys 70–80% of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain before motor symptoms are typically noticeable (Morrish et al, 1998; Postuma et al, 2010; Heisters, 2011). The disease is usually idiopathic, but cases with known genetic components account for around 10% of reported cases (Gasser, 2009). Of note, α-synuclein is one of the primary proteins linked with PD, and it has been identified as playing a role in both genetic and non-genetic cases. The first evidence of α-synuclein involvement in PD came from several familial studies that showed individuals harboring coding region mutations in SNCA, and the gene encoding α-synuclein, including A53T, E46K, and A30P, had early-onset disease with an autosomal dominant pattern of inheritance (Polymeropoulos et al, 1997; Kruger et al, 1999; Zarranz et al, 2004). Later, more familial SNCA mutations were identified including H50Q, G51D, A18T, and A29S, all of which have been shown to affect disease manifestation and aggregation of α-synuclein (Appel-Cresswell et al, 2013; Hoffman-Zacharska et al, 2013; Lesage et al, 2013). Moreover, familial PD cases with locus multiplications, such as duplication and triplication of SNCA, exhibited severe forms of the disease and early onset (Singleton et al, 2003; Chartier-Harlin et al, 2004). Duplication or triplication of SNCA could produce higher amounts of α-synuclein in neurons, which might account for the aggressive form of the disease observed in those patients (Miller et al, 2004; Fuchs et al, 2008). The central role of α-synuclein in the pathogenesis of idiopathic PD was supported by the discovery that aggregates of α-synuclein are major components of Lewy bodies, the proteinaceous intracytoplasmic inclusions in dopaminergic neurons found in post-mortem brains of PD patients (Spillantini et al, 1997). In addition, a single-cell study using laser capture microdissection found significantly higher levels of α-synuclein transcripts in PD post-mortem brains (Grundemann et al, 2008). Together, these studies indicate regulation of the SNCA gene is important and elevated levels of α-synuclein could be a key factor in development of PD and other synucleinopathies. Numerous studies have investigated the make-up, formation, and propagation of α-synuclein aggregates in PD (Stefanis, 2012; Giraldez-Perez et al, 2014); however, little is known about the mechanisms underlying transcriptional or epigenetic deregulation of SNCA in PD. Except for two conflicting reports that investigated hypomethylation of the intron 1 CpG island of SNCA in PD and control post-mortem brain samples, studies of epigenetic factors regulating the α-synuclein gene in PD have been limited (Guhathakurta et al, 2017a; Guhathakurta et al, 2017b). The ENCODE database now allows for in-depth analysis of the epigenetic environment of genes (Consortium EP, 2012). And while the importance of histone post-translational modifications (PTMs) in regulation of gene expression has been established, to date, no studies have comprehensively investigated the potential role of histone PTMs in regulation of SNCA in PD (Guhathakurta et al, 2017a). In this study, we investigated the epigenetic environment of SNCA with special emphasis on histone PTMs that are potentially enriched in the regulatory region of the gene. Interestingly, we observed a significant increase in one histone PTM, histone H3 lysine 4 trimethylation (H3K4me3) at the SNCA promoter in post-mortem PD samples. This transcription-initiating histone mark is one of the major determinants of gene transcription (Barski et al, 2007). Reduction of H3K4me3 in neuronal cell lines and PD-derived induced pluripotent stem cell lines (iPSCs) using the dCas9-Suntag system-mediated locus-specific targeting approach decreased levels of α-synuclein. Results of this epigenetic investigation could open new avenues for therapeutic intervention to reduce α-synuclein expression by preventing enrichment of H3K4me3 in the SNCA gene. Results Analysis of histone architecture of human SNCA in post-mortem midbrain samples Human SNCA consists of six coding exons and two upstream non-coding exons and spans a 114-kb region in chromosome 4 (Guhathakurta et al, 2017a). We first searched in silico for any histone marks in the adult human brain substantia nigra (SN) region using the Roadmap Epigenomics Database (http://www.roadmapepigenomics.org/data/tables/adult). In total, the database lists seven histone PTMs in the SN region of the brain from two adult post-mortem samples: H3 lysine 27 trimethylation (H3K27me3), histone H3 lysine 36 trimethylation (H3K36me3), histone H3 lysine 4 monomethylation (H3K4me1), histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 9 trimethylation (H3K9me3), histone H3 lysine 9 acetylation (H3K9ac), and histone H3 lysine 27 acetylation (H3K27ac). However, for SNCA, not all seven histone PTMs were enriched in their cohort of tissues analyzed. H3K36me3, the histone mark associated with full-length transcription, was enriched throughout the gene body, ensuring SNCA is actively transcribed in the SN region. Most interestingly, we observed that H3K4me3, H3K27ac, and H3K27me3 were the three histone PTMs preferentially enriched in the primary regulatory regions of SNCA—the area ranging from approximately −1 kb to +1.5 kb of the transcription start site (TSS). This transcriptionally important region includes the promoter, its upstream regions, and the intron 1 area of the gene (chr4: 90,757,022–90,759,612 bp; GRCh37/hg19). Fig 1A and Appendix Fig S1 shows the distribution pattern of the three histone marks around the promoter region for exon 2, the first coding exon. Both H3K4me3 and H3K27ac are transcription-favoring histone marks. H3K4me3 is the principal mark associated with transcription initiation, and H3K27ac is an active enhancer-associated mark often enriched at the promoter. By contrast, H3K27me3 is associated with gene repression. Figure 1. Parkinson’s disease patients harbor high levels of H3K4me3 at the α-synuclein promoter A. Human SNCA gene contains six coding exons and two 5’ non-coding exons. The exons are represented by vertical colored boxes. The region from exon 1a to exon 2 (~2.5 kb) is scaled up to show the distribution of histone PTMs. Distribution of the histone PTMs from the SN region of one donor brain sample is shown. Peaks of three different histone PTMs, H3K4me3 (green), H3K27ac (blue), and H3K27me3 (black), at the regulatory region of SNCA were adopted from Roadmap Epigenomics Database. For a detailed view, please see Appendix Fig S1 where screenshot of the original figure is shown. The TSS is indicated by a red vertical bar and distances of exon 1a and 1b from the TSS are indicated. B. ChIP gel images showing the relative enrichment by H3K4me3 in controls (n = 9) and PD patients (n = 18). PCR amplified a 188-bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum. Mouse IgG (mIgG) was used as control and the bands were normalized by unbiased amplification from respective inputs. C. Relative intensities calculated from the gel images. Graph shows that H3K4me3 was significantly enriched at the upstream regulatory region of SNCA in PD compared to control subjects. ****P < 0.0001. Data were analyzed using non-parametric t-test followed by Mann–Whitney post hoc corrections. Two-tailed P-values were calculated for all. Data information: Data represent mean ± standard error of the mean. Download figure Download PowerPoint In SNCA, we observed both the transcription-promoting marks, H3K4me3 and H3K27ac, had sharp peaks around the TSS and surrounding areas, while H3K27me3 had an overall low-level distribution at the promoter and intron 1 areas. We next analyzed these three histone marks in our cohort of post-mortem midbrain tissue samples of PD and matched controls specifically from the SN region using chromatin immunoprecipitation (ChIP) (Fig 1B and C; Appendix Fig S2). We found that H3K4me3 was significantly enriched at the SNCA regulatory region in PD samples compared to controls (P < 0.0004; Fig 1B and C). On the other hand, no significant difference was observed for H3K27ac between control and PD subjects (Appendix Fig S2A and B). We also found relatively higher enrichment of H3K27me3 in PD; however, the difference in H3K27me3 between control and PD was less prominent and significant than the difference in H3K4me3 between the two groups (Appendix Fig S2C and D). Enhancer activity of the SNCA intron 4 region has been reported (Soldner et al, 2016; Guhathakurta et al, 2017a). As mentioned above, enhancer regions are often enriched by H3K27ac as shown in the Roadmap epigenetic data for SNCA (Soldner et al, 2016; Guhathakurta et al, 2017a). However, we did not find any significant difference in the enrichment of H3K27ac in the intron 4 enhancer region between control and PD subjects (Appendix Fig S3). Correlation between H3K4me3 levels and high levels of α-synuclein in PD patients We evaluated α-synuclein levels in midbrain SN tissues from control and PD subjects by Western blot analysis. The entire cohort of samples used for analyzing enrichment of H3K4me3 was used for α-synuclein expression. We found that α-synuclein was significantly higher in PD compared to controls (P < 0.05; Fig 2A and B). We then compared whether α-synuclein levels could be correlated with corresponding H3K4me3 enrichment. We calculated the median level of α-synuclein among all study subjects (0.43) and found that nine PD subjects and three controls had higher α-synuclein levels than the median. Interestingly, higher levels of α-synuclein in those subjects were significantly correlated with corresponding H3K4me3 enrichment at the gene promoter (Spearman correlation coefficient (R), 0.71, P = 0.01; Fig 2C). This further established that enrichment of H3K4me3 at the SNCA promoter could account for higher expression of α-synuclein. Figure 2. PD patients exhibit high expression of α-synuclein A. Western blot gel images showing α-synuclein (α-SYN) levels in SN tissues from control (n = 9) and PD subjects (n = 18). The numbers on top of each gel panel show the ID of each sample. B. The relative levels of α-synuclein between the two groups were evaluated statistically. PD subjects had moderately higher levels of α-SYN compared to controls. ID of each dot is shown in the graph. C. The normalized levels of α-synuclein in the entire cohort were divided into high (n = 12) and low levels. The cutoff value for determining the threshold was set at the median from all the subjects. The high levels of α-synuclein were plotted with corresponding H3K4me3 values of those subjects. The corresponding ID of each sample is shown next to each point in the graph. There was a significant correlation between high levels of α-synuclein and H3K4me3. Data information: Data represent mean ± standard error of the mean. Data in (B) were analyzed using non-parametric t-test followed by Mann–Whitney post hoc corrections and two-tailed P-values were calculated. Spearman rank correlation test was performed in (C). Two-tailed P-value was calculated with Fisher’s exact P. Download figure Download PowerPoint Relative enrichment of H3K4me3 in the neuronal population of post-mortem PD brain Cellular heterogeneity in the brain is a major obstacle when studying neuronal epigenomic architecture. We further investigated whether the observed difference in H3K4me3 between control and PD is maintained in neuronal populations in the SN region. We isolated 20,000 neuronal nuclei from the SN region of a subset of brain samples (6 control and 7 PD) using NeuN-based fluorescence-activated nuclei sorting (FANS). NeuN antibody is a widely used neuron-specific antibody that preferentially binds to Fox3 transcription factor in the neuronal nuclei (Mullen et al, 1992; Kim et al, 2009). We were able to amplify neuron-specific transcripts (NeuN, synaptophysin, and α-synuclein), but not for astrocytes (GFAP) from the isolated NeuN+ nuclei, confirming the purity of neuron-specific isolation (Fig 3A and B). As expected, neuronal nuclei isolated from equal amounts of tissue from the SN region yielded much higher numbers of NeuN+ nuclei in controls compared to PD (Fig 3). Using the same primer set used for whole-tissue ChIP of H3K4me3 at the SNCA promoter/intron 1 region, we found that NeuN+ nuclei from PD samples demonstrated significantly higher enrichment of H3K4me3 compared to controls (P = 0.015; Fig 3C and D). Figure 3. NeuN-positive neurons from the SN demonstrate high enrichment by H3K4me3 A. 20,000 NeuN labeled neuronal nuclei were collected by fluorescent activated nuclei sorting (FANS) from control (n = 6) and PD (n = 7) SN tissues. The NeuN-positive nuclei were labeled by anti-rabbit IgG secondary antibody tagged with Alexa fluor 488. The top and bottom panels show representative sort gating windows from an unlabeled and labeled patient sample, respectively. The left column represents size versus granularity gatings. The samples were then gated for singularity using forward scatter height (y-axis) versus forward scatter area (x-axis), and lastly, singularly gated nuclei were sorted based on NeuN positivity (x-axis; FL1 channel) versus side scatter (y-axis). The green rectangular quadrants on the right column of both panels represent the NeuN+ region, as unstained sample did not show any significant representation. Therefore, from each of the stained samples, 20,000 bright NeuN+ nuclei (green rectangle gate) were sorted and collected. B. Gel images from RT–PCR show purity of the sorted nuclei from a representative PD sample. Nuclear RNA was isolated, and cDNA was generated and pre-amplified (see Materials and Methods for details) before target-specific PCR. Neuron-specific genes (NeuN, synaptophysin) and astrocyte-specific gene (GFAP), α-synuclein, and GAPDH were amplified from the isolated nuclei. C. ChIP was performed on the equal number of isolated nuclei against H3K4me3 from all samples. Gel images represent the ChIP-based PCR amplification. The same primer pair was used to amplify the target region on SNCA as Fig 1. Mouse IgG was used as a control. D. The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD (n = 7) and controls (n = 6). Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 (P = 0.01) compared to controls. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann–Whitney post hoc corrections. Two-tailed P-values were calculated for all. Data information: Data represent mean ± standard error of the mean. Download figure Download PowerPoint Design of CRISPR/dCas9 SunTag-JARID1A system and its recruitment at the SNCA promoter As the histone mark H3K4me3 was significantly enriched at the SNCA promoter in PD patients and was correlated with high levels of α-synuclein in all study subjects, we aimed to investigate whether removal of H3K4me3 at the SNCA promoter affected α-synuclein levels. To this end, we searched for an epigenetic eraser that could efficiently remove trimethylation at lysine 4 from the histone H3 tail. Histone lysine demethylases (KDM) are specific “erasers” for methylated lysine residues on histones (Hyun et al, 2017). KDM5A, also known as JARID1A (Jumonji, AT-rich interactive domain, member 1A), has been shown to have specific demethylating activity at H3K4me3 (Klose et al, 2007; Horton et al, 2016). JARID1A contains several conserved domains including n- and c-terminal JmjN and JmjC domains, a DNA binding ARID domain, three PHD domains, a Zn2+ binding domain, and a PLU domain (Horton et al, 2016). The first 797 amino acids containing JmjN, ARID, PHD1, JmjC, and Zn2+ domains were identified as catalytic because overexpression of a recombinant construct containing these domains was sufficient to demethylate H3K4me3 in vitro (Horton et al, 2016). Based on this information, we selected JARID1A as the H3K4me3 demethylase for our next set of experiments. To recruit a JARID1A catalytic domain to the SNCA promoter, we used the CRISPR/dCas9 technology-based SunTag or SuperNova system by replacing the synthetic transcription activator, VP64, with the JARID1A catalytic domain (Fig 4A). Originally, the SunTag system was designed to activate gene expression (up to 300×) by recruiting multiple copies (10 or 22) of VP64 tagged with a single chain variable fragment (scFV) that specifically binds to a GCN4 peptide at the target gene promoter (Tanenbaum et al, 2014). Upon co-expression of a dead-Cas9 (dCas9) containing c-terminal repeats of 10 or 22 GCN4 peptides (10× or 22× GCN4) together with genomic locus-specific small guide RNAs (sgRNAs), target gene-specific accumulation of VP64 is achieved, ensuring robust transcriptional activation (Tanenbaum et al, 2014). We sub-cloned the scFV-sfGFP-JARID1A catalytic domain into a lentiviral system where the transgene is expressed under the CMV immediate enhancer element (Fig 4B). The entire sequence of this novel vector system is provided in Appendix Fig S15. Morita et al (2016) previously showed that to avoid steric hindrance between adjacent effector molecules and for proper recruitment of larger effectors such as TET1 catalytic domain, a minimum 22 amino acid spacer is necessary between each GCN4 unit. Therefore, we also used this dCas9-5xGCN4 system, which could recruit five JARID1A molecules at the SNCA promoter site when co-overexpressed with scFV-sfGFP-JARID1A and sgRNA (Fig 4A). We designed nine guide RNAs upstream of the TSS of SNCA where the peaks of H3K4me3 were relatively higher (Fig 4C). Guide RNA design was carried out using Broad Institute’s genomic perturbation platform to ensure selection of only those sequences that had 0 to 1 unintended potential off-targets elsewhere in the genome (see Materials and Methods for details). The list of sgRNAs with sequences and their relative locations are provided in Appendix Table S3. Figure 4. Design of CRISPR/dCas9 based SunTag-JARID1A system A. Schematic diagram shows how the SunTag-JARID1A system is recruited at the SNCA promoter. The dCas9-5xGCN4, scFV-JARID1A, and sgRNA plasmids are co-overexpressed in the cells. The dCas9-5xGCN4 is recruited to the SNCA promoter as directed by the specific sgRNA. Five scFV-JARID1A molecules in turn recognize the GCN4 polypeptide sequences of dCas9. Upon recruitment of the entire system, SunTag-JARID1A demethylates H3K4me3 at the target region. B. Structure of pLvx-scFV-sfGFP-JARID1A. The scFV-sfGFP catalytic domain of JARID1A was sub-cloned into a lentiviral vector. The distance between the 5’ and 3’ Long Terminal Repeats in the vector is 8.5 kb. The components of the plasmids are as follows: ψ, packaging signal; RRE, rev response element; cPPT, central polypurine tract; pCMV IE, immediate early cytomegalovirus promoter; scFV, single chain variable fragment; sfGFP, super folder GFP; JARID1A; catalytic domain of JARID1A; GB1, solubility tag protein; NLS, nuclear localization signal; WPRE, woodchuck hepatitis virus (WHP) post-transcriptional regulatory element. C. Relative positions of the nine sgRNAs. A ray diagram exhibits the locations of the nine sgRNAs in relation to the upstream regulatory regions of SNCA and the H3K4me3 and H3K27me3 peaks. The locations of the sgRNAs are as follows with respect to the TSS: sgA, 1,117 bp; sgB, 836 bp; sgC, 747 bp; sgD, 700 bp; sgE, 153 bp; sgF, 1,454 bp; sgG, 1,537 bp; sgH, 132 bp; sgI, 1,320 bp. The two non-coding exons (1A and 1B) along with the first coding exon (exon 2) are also shown. GRCh37/hg19 contig was used to describe the location of the gene and associated distribution of histone PTMs. The histone peaks shown were from midbrain regions of two adult post-mortem human samples. Download figure Download PowerPoint To determine whether dCas9-5xGCN4 and scFV-sfGFP-JARID1A could form a complex when expressed together, we transiently co-overexpressed these constructs in HEK293 cells and immunoprecipitated using anti-Cas9 antibody and further blotted against anti-GFP antibody (Appendix Fig S4). We observed that cells expressing both dCas9-5xGCN4 and scFV-sfGFP-JARID1A were successfully immunoblot
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