Editing DNA Methylation in the Mammalian Genome

“STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mouse lines and breeding strategies B Mouse primary cortical neuron culture, EDU labeling, and neural ind…”

“Peaks are called using MACS (Zhang et al., 2008) with default settings.”

“Clustered regularly interspaced palindromic repeats (CRISPR), a type II bacterial adaptive immune system, has been modified to target the Cas9 nuclease to the desired genomic loci with sequence-specific guide RNAs for genome editing (Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013).”

“Clustered regularly interspaced palindromic repeats (CRISPR), a type II bacterial adaptive immune system, has been modified to target the Cas9 nuclease to the desired genomic loci with sequence-specific guide RNAs for genome editing (Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013).”

“Clustered regularly interspaced palindromic repeats (CRISPR), a type II bacterial adaptive immune system, has been modified to target the Cas9 nuclease to the desired genomic loci with sequence-specific guide RNAs for genome editing (Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013).”

“Mammalian DNA methylation at 5-cytosine plays critical roles in many biological processes, including genomic imprinting, cell fate determination, chromatin architecture organization, and regulation of gene expression (Bird, 2002; Jaenisch and Bird, 2003; Smith and Meissner, 2013).”

“…are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al., 2014; Ji et al., 2016; Phillips-Cremins et al., 2013), which, in turn, can form clusters of loops that contribute to topologically associating domains (TADs) (Dixon et al., 2012; Nora et al., 2012).”

“Genetic studies have revealed that DNA methylation is essential for mammalian development and adaptation to environmental signals (Jaenisch and Bird, 2003).”

“…activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).”

“Owing to the advancement in sequencing technologies, single-nucleotide resolution methylation maps for many types of human and mouse cells and tissues have been depicted (Lister et al., 2009, 2013).”

“…activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).”

“The role of MyoD as a master regulator for muscle development was initially defined by the observations that demethylation of DNA in fibroblasts by 5-Aza (5-Aza-2’-deoxycytidine) treatment resulted in activation of MyoD and subsequent myoblast conversion and myotube formation (Constantinides et al., 1977; Davis et al.…”

“DNA methylation is established by two de novo DNA methyltransferases (Dnmt3a/b) and is maintained by Dnmt1 (Smith and Meissner, 2013).”

“…are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al., 2014; Ji et al., 2016; Phillips-Cremins et al., 2013), which, in turn, can form clusters of loops that contribute to topologically associating domains (TADs) (Dixon et al., 2012; Nora et al., 2012).”

“To test whether endogenous Tet activity was required to regulate BDNF expression upon neuronal activity stimulation, we treated DIV3 neurons with 2-hydroxygluterate, a competitive inhibitor for α-ketoglutarate-dependent dioxygenases including Tet enzymes (Xu et al., 2011).”

“Abnormal DNA methylation has been observed in cancer and neurological disorders (Robertson, 2005).”

“…activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).”

“…activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).”

“Transcriptional enhancers normally interact with their target genes through the formation of DNA loops (Gibcus and Dekker, 2013; Gorkin et al., 2014; Kagey et al., 2010), which typically are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al.”

“…activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).”

“Owing to the advancement in sequencing technologies, single-nucleotide resolution methylation maps for many types of human and mouse cells and tissues have been depicted (Lister et al., 2009, 2013).”

“Interestingly, methylation of the DNA recognition site of CTCF has been reported to block CTCF binding (Bell and Felsenfeld, 2000; Wang et al., 2012).”

“240 Cell 167, 233–247, September 22, 2016 (Phillips and Corces, 2009).”

“DNA replication-independent active demethylation has been proposed to operate in postmitotic neurons (Guo et al., 2011; Martinowich et al., 2003).”

“DNA replication-independent active demethylation has been proposed to operate in postmitotic neurons (Guo et al., 2011; Martinowich et al., 2003).”

“For instance, treatment of neurons with KCl has been shown to de-silence promoter IV of BDNF and induce BDNF expression associated with demethylation of some methylated CpGs in the promoter region (Chen et al., 2003; Martinowich et al., 2003).”

“, 2010), which typically are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al., 2014; Ji et al., 2016; Phillips-Cremins et al., 2013), which in turn can form clusters of loops that contribute to topologically associating domains (TADs) (Dixon et al.”

“Because the neurons were post-mitotic, the dCas9-Tet1-mediated demethylation of the promoter sequences was likely the result of active demethylation as has been proposed previously (Wu and Zhang, 2014).”

“Importantly, these maps have allowed for the identification of differentially methylated regions (DMRs) at base pair resolution during different stages of normal development as well as disease (De Jager et al., 2014; Landau et al., 2014).”

“These observations are consistent with the results obtained when these CTCF sites were deleted (Dowen et al., 2014), and support the notion that methylation of the CTCF binding site interferes with its insulator function.”

“also reported that dCas9 fused with Dnmt3a can be used to methylate two human gene promoters (Vojta et al., 2016), and Xu et al.”

“Transcriptional enhancers normally interact with their target genes through the formation of DNA loops (Gibcus and Dekker, 2013; Gorkin et al., 2014; Kagey et al., 2010), which typically are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al.”

“DMRs upstream of mouse MyoD gene were defined based on human/mouse genome homology (Schultz et al., 2015).”

“Importantly, the same group of dCas9-Tet1/gRNAs did not induce Npas4 expression (Figure S4F), another neuronal activity-inducible gene (Lin et al., 2008).”

“Interestingly, methylation of the DNA recognition site of CTCF has been reported to block CTCF binding (Bell and Felsenfeld, 2000; Wang et al., 2012).”

“Emerging data suggest that higher-order chromatin structures confer epigenetic information during development and are frequently altered in cancer (Ji et al., 2016; Narendra et al., 2015).”

“Mouse lines and breeding strategies—Tet1 mutant mice were previously generated in our lab (Dawlaty et al., 2011).”

“The PiaggyBac-dCas9-Tet1 and -dCas9-Dnmt3a were cloned by ligation of PCR-amplified dCas9-NLS-Tet1 or dCas9-NLS-Dnmt3a from FUWconstructs withmodified PiggyBac transposon vector (Wilson et al., 2007) with NheI and EcoRI.”

“Importantly, these maps have allowed for the identification of differentially methylated regions (DMRs) at base pair resolution during different stages of normal development as well as disease (De Jager et al., 2014; Landau et al., 2014).”

“, 2010), which typically are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al., 2014; Ji et al., 2016; Phillips-Cremins et al., 2013), which in turn can form clusters of loops that contribute to topologically associating domains (TADs) (Dixon et al.”

“Transcriptional enhancers normally interact with their target genes through the formation of DNA loops (Gibcus and Dekker, 2013; Gorkin et al., 2014; Kagey et al., 2010), which typically are constrained within larger CTCF-mediated loops called insulated neighborhoods (Dowen et al.”

“…our manuscript, Vojta et al. also reported that dCas9 fused with Dnmt3a can be used to methylate two human gene promoters (Vojta et al., 2016), and Xu et al. and Choudhury et al. reported that dCas9 fused with Tet1 can be used to demethylate gene promoters (Choudhury et al., 2016; Xu et al., 2016).”

“The role of MyoD as a master regulator for muscle development was initially defined by the observations that demethylation of DNA in fibroblasts by 5-Aza (5-Aza-2’-deoxycytidine) treatment resulted in activation of MyoD and subsequent myoblast conversion and myotube formation (Constantinides et al., 1977; Davis et al.…”

“…our manuscript, Vojta et al. also reported that dCas9 fused with Dnmt3a can be used to methylate two human gene promoters (Vojta et al., 2016), and Xu et al. and Choudhury et al. reported that dCas9 fused with Tet1 can be used to demethylate gene promoters (Choudhury et al., 2016; Xu et al., 2016).”

“Fibroblast-to-myoblast conversion assay—Myoblast conversion assay was described previously (Constantinides et al., 1977).”

“Dynamic DNA methylation has been proposed to decode neuronal activities (Sweatt, 2013).”

“5 embryos and cultured for 2 days in vitro (DIV2) following a well-established experimental procedure for producing primary neuronal culture (Ebert et al., 2013).”

“5 mESC (C57BL/6 3 129S4/SvJae) Brambrink et al., 2006 N/A Dazl-Snrpn-GFP V6.”

“Immunostaining procedures for tissue sections were previously described (Wu et al., 2014a).”

“…Alterations of CpG Methylation State To assess whether the dCas9-Tet1 and -Dnmt3a fusion constructs would induce demethylation or de novo methylation, respectively, of specific sequences, we utilized a methylation reporter system previously developed in our laboratory (Stelzer et al., 2015).”

“To compare the methylation editing efficacy and effective range by dCas9-Tet1/Dnmt3a with TALE-based methods, we chose two previously reported loci edited by TALE-based method (Bernstein et al., 2015; Maeder et al., 2013) and designed a single gRNA targeting dCas9Tet1/Dnmt3 to the same site bound by the TALE-Tet1/Dnmt…”

“Six muscle-specific DMRs have been described within the 50-kb upstream region of MyoD gene (Schultz et al., (legend continued on next page) Cell 167, 233–247, September 22, 2016 239 2015), and DMR-5 overlaps with a known distal enhancer of MyoD (Brunk et al., 1996) as shown in Figure 4A.”

“…NCBI Gene Expression Omnibus NCBI GEO: GSE83890 Experimental Models: Organisms/Strains Mouse: C57Bl/6J Jackson Laboratories RRID: IMSR_JAX:000664 Mouse: B6; Tet1 KO mouse Dawlaty et al., 2011 N/A Mouse: B6; Dlk1-Dio3 IG-DMR-Snrpn-GFP Stelzer et al., 2016 N/A Experimental Models: Cell Lines V6.”

“In glioma cells that harbor mutations in the IDH1 gene, hypermethylation of insulated neighborhood anchor sites occurs, which leads to loss of CTCF binding at those anchors and activation of oncogenes that were previously silent in the intact neighborhoods (Flavahan et al., 2016).”