1 2 3 4 5 6 7 8 9 EED orchestration of heart maturation through interaction with HDACs is H3K27me3- 10 independent 11 12 Shanshan Ai1*, Yong Peng1*, Chen Li1, Fei Gu2, Xianhong Yu1, Yanzhu Yue1, Qing Ma2, 13 Jinghai Chen2, Zhiqiang Lin2, Pingzhu Zhou2, Huafeng Xie3, Terence W. Prendiville2,†, Wen 14 Zheng1, Yuli Liu1, Stuart H. Orkin3,4,5, Da-Zhi Wang2,4, Jia Yu6 15 William T. Pu2,4,7§ & Aibin He1,7§ 16 17 18 19 20 21 1Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, 22 Beijing 100871, China 23 2Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 24 02115, USA 25 3Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric 26 Oncology, Dana-Farber Cancer Institute, 27 4Harvard Stem Cell Institute, Harvard University, 1350 Massachusetts Avenue, Suite 727W, 28 Cambridge, MA 02138, USA. 29 5Howard Hughes Medical Institute, Boston, MA 02115, USA. 30 6Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical 31 Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical 32 Sciences, Peking Union Medical College, Beijing 100005, China. 33 7Co-senior author 34 35 *Contributed equally to this work. 36 †Present address: Department of Paediatric Cardiology, Our Lady's Children's Hospital 37 Crumlin, Dublin 12, Ireland. 38 39 40 41 §Correspondence: Aibin He ([email protected]) or William T. Pu 42 ([email protected])
43
44 45 46
- 1 - 47 ABSTRACT 48 In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have 49 been performed, gene repression is associated with PRC2 trimethylation of H3K27 50 (H3K27me3). However, it is uncertain whether PCR2 writing of H3K27me3 is mechanistically 51 required for gene silencing. Here we studied PRC2 function in postnatal mouse 52 cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after 53 each cell cycle. EED (Embryonic Ectoderm Development) inactivation in the postnatal heart 54 (EedCKO) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in EedCKO was 55 not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. 56 EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. 57 HDAC overexpression normalized EedCKO heart function and expression of derepressed 58 genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive 59 mechanism that is essential for normal heart function. Our results further illustrate that organ 60 dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring. 61
62
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63 INTRODUCTION 64 Normal developmental maturation of organ function requires precise transcriptional 65 regulation of gene expression. This transcriptional regulation depends upon the interplay of an 66 array of epigenetic regulators, which shape the epigenetic landscape by repositioning 67 nucleosomes and depositing covalent modifications on histones. Since the chromatin 68 landscape is established through a series of steps, each contingent upon normal completion of 69 prior steps, transient disruption, through environmental mishaps or genetic mutations, might be 70 anticipated to break the normal sequence and irreversibly impact organ development and 71 function. Whether or not this is the case is presently unknown, and the answer has clear 72 therapeutic implications for diseases that involve epigenetic changes. 73 Development and function of the heart is vulnerable to epigenetic insults, as mutation of 74 epigenetic regulators causes both structural heart disease and cardiomyopathy in humans and 75 in experimental model systems (He et al., 2012; Zaidi et al., 2013; Hang et al., 2010; Delgado- 76 Olguin et al., 2012; Lickert et al., 2004; Montgomery et al., 2007). Recent work has highlighted 77 the critical role of epigenetic silencing of ectopic transcriptional programs in normal heart 78 development and function (He et al., 2012; Delgado-Olguin et al., 2012; Montgomery et al., 79 2007; Trivedi et al., 2010). One class of epigenetic repressors are the histone deacetylases 80 (HDACs), which remove activating histone acetylation marks to repress gene expression. 81 HDACs consist of four classes (classes I, IIa, IIb, and IV) on the basis of their domain structure 82 and expression pattern (Laugesen and Helin, 2014; Chang and Bruneau, 2012). Zhao and 83 colleagues reported that HDACs target both actively transcribed and repressed genes to reset 84 chromatin state for subsequent complex-dependent transcriptional outcome (Wang et al., 85 2009). Consistent with this, class I HDACs such as HDAC1 and HDAC2 are frequently 86 subunits of multi-protein complexes, such as Sin3, NuRD and CoREST. Inactivation of 87 HDAC1/2 caused abnormal heart growth and function that was linked to ectopic expression of 88 slow twitch skeletal muscle genes (Montgomery et al., 2007). Class II HDACs were found to 89 repress cardiac MEF2 transcription factor activity and their genetic inactivation caused 90 pathological cardiac hypertrophy (Zhang et al., 2002). 91 Another class of epigenetic repressors is polycomb repressive complex 2 (PRC2), 92 comprising the core subunits EED, SUZ12, and either EZH1 or EZH2. Canonically, PRC2 93 represses gene transcription by catalyzing trimethylation of histone H3 on lysine 27 94 (H3K27me3) (Cao et al., 2002). However, whether or not H3K27me3 deposition is sufficient to
- 3 - 95 account for PRC2-mediated transcriptional repression in all contexts remains uncertain. 96 Margueron and colleagues reported that elevated EZH2 and H3K27me3 levels are a 97 consequence of high proliferation of cells in tumorigenesis, arguing that perturbation of this 98 equilibrium leads to irreversible transcriptional change (Wassef et al., 2015). In PRC2-null 99 ESCs as well as other cell types, most genes that are marked by H3K27me3 remain repressed 100 even after inactivation of PRC2 components, suggesting redundant repressive mechanisms. 101 Furthermore, most studies of PRC2 function have been performed in actively cycling cells, 102 whereas the majority of cells in adult mammals cycle slowly, and some cells, such as adult 103 cardiomyocytes (CMs), are largely post-mitotic. Since cell cycle activity mandates re- 104 deposition of histone marks (Margueron et al., 2009; Hansen et al., 2008), in mitotic cells 105 writing activity of epigenetic complexes may overshadow other functions that are important in 106 non-proliferating cells. 107 The role of PRC2 in the heart has been studied by cardiac-specific inactivation of EZH2 or 108 EED during early cardiac development. This resulted in disruption of H3K27me3 deposition, 109 derepression of non-cardiomyocyte gene programs, and lethal heart malformations (He et al., 110 2012; Delgado-Olguin et al., 2012). Here, we investigated the function of EED in postnatal 111 heart maturation. Postnatal CMs have largely exited the cell cycle (Bergmann et al., 2009; 112 Bergmann et al., 2015; Senyo et al., 2013; Ali et al., 2014; Alkass et al., 2015; Soonpaa et al., 113 2015; Hirai et al., 2016) and express little EZH2 protein (He et al., 2012), but nonetheless 114 contain abundant H3K27me3. Thus, the postnatal heart affords a unique opportunity to 115 investigate the mechanism by which EED represses gene expression in post-mitotic cells and 116 to harness this knowledge to determine whether the dysregulated chromatin landscape can be 117 rewired to restore gene expression and heart function. We found that EED silences the slow- 118 twitch myofiber gene program to orchestrate heart maturation by complexing with and 119 stimulating HDAC deacetylase activity. Surprising, de-repression of the majority of genes in 120 EED deficient cardiomyocytes occurred without loss of H3K27me3, pointing to important EED 121 repressive activity that is independent of its role in PRC2-mediated H3K27 trimethylation.
122 RESULTS
123 Heart dysfunction in neonatal cardiac-inactivation of Eed 124 To understand the role of Eed in regulating cardiac gene expression during heart 125 maturation, we generated Eedfl/fl; Myh6Cre (EedCKO) mice, in which Myh6Cre specifically 126 inactivates the conditional Eedfl allele in CMs. Western blotting and qRTPCR demonstrated
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127 effective cardiac EED protein depletion that occurred between postnatal day (P) 0 and 5 (Fig. 128 1A and Figure 1 -- figure supplement 1). EedCKO mice were born at the expected Mendelian 129 frequency, suggesting a lack of embryonic lethality, but most died over the first 3 months of life 130 (Fig. 1B). By echocardiography, EedCKO mice had left ventricular dilatation and progressive 131 systolic dysfunction (Fig. 1C and Figure 1 -- figure supplement 2A). Histopathological 132 examination confirmed massive cardiomegaly (Fig. 1D-F). The expression level of the heart 133 failure marker Nppa, encoding atrial natriuretic factor, was strongly up-regulated (Figure 1 -- 134 figure supplement 2B). EedCKO mice that survived to 2 months of age had substantial CM 135 hypertrophy and cardiac fibrosis (Figure 1 -- figure supplement 2C-F). These results show that 136 Eed is essential for neonatal heart maturation, and that its inactivation in CMs causes lethal 137 dilated cardiomyopathy.
138 Global distribution of H3K27me3 on the promoters/enhancers of derepressed genes 139 EED is a central, non-redundant subunit of PRC2, an enzyme that canonically represses its 140 target genes by depositing repressive H3K27me3 epigenetic marks (Margueron et al., 2009; 141 Hansen et al., 2008). As expected, cardiac Eed inactivation globally decreased H3K27me3 142 level in CMs (Fig. 1G). Genome-wide H3K27me3 occupancy also showed the anticipated 143 global reduction of H3K27me3 signal (Fig. 1H). Accordingly, Eed inactivation reduced the 144 number of regions enriched for H3K27me3 across the genome from 49991 to 7095 (Fig. 1I and 145 Supplementary file 1). Of the 7095 H3K27me3-occupied regions in EedCKO CMs, 5009 were 146 also observed in wild-type CMs and 2086 were not (Fig. 1I). These 2086 new peaks had low 147 intensity, and their centers were distributed as follows: 1040 (49.86%) in intergenic regions, 148 192 (9.20%) in promoter regions, 721 (34.56%) in introns, 98 (4.70%) in exons, and 35 149 (1.68%) in 5' and 3' UTR. 150 To gain insights into the mechanisms underlying the dilated cardiomyopathy phenotype, we 151 performed gene expression profiling of dissociated, purified, 8-week-old CMs using RNA-seq. 152 We identified 863 upregulated and 392 downregulated genes in EedCKO (fold change > 1.5 or 153 <0.67 and adjusted P-value < 0.05; Fig. 1J and Supplementary file 1). Remarkably, we found 154 that slow-twitch myofiber genes, such as Tnnt1, Tnni1, Myh7, Mybpc1, Myl9, Myl7, Myl4, 155 Mybpc2 and Acta1, were significantly upregulated in EedCKO heart (Fig. 1J). Indeed, the 156 upregulated genes were significantly enriched for gene ontology terms related to skeletal 157 muscle genes (Fig. 1K). Normal cardiac function requires proper expression of cardiac 158 sarcomere genes and repression of skeletal muscle and slow-twitch myofiber genes (Frey et
- 5 - 159 al., 2000; Montgomery et al., 2007; Ding et al., 2015), suggesting that abnormal expression of 160 these genes contributes to weak cardiac contraction in EedCKO mice. 161 To dissect the relationship between the differentially expressed genes and H3K27me3, 162 H3K27ac, and EED occupancy, we performed chromatin immunoprecipitation followed by high 163 throughput sequencing (ChIP-seq) in purified, 2 month-old CMs. CMs were over 95% pure by 164 immunofluorescence microscopy (Fig. 1 -- figure supplement 2G). Markedly reduced 165 H3K27me3 in EedCKO CMs demonstrated highly efficient EED inactivation (Fig. 1 -- figure 166 supplement 2G). PCR of genomic DNA from purified CMs, as well as review of the RNA-seq 167 track view of the Eed locus, further indicated both efficient gene inactivation by Myh6-Cre, 168 which is CM-specific, as well as high CM purity (Fig. 1 -- figure supplement 2H,I). ChIP-seq 169 signals were consistent high CM purity. For example, at Myh6, expressed in CMs, showed 170 robust signal for the active mark H3K27ac and little signal from the repressive mark 171 H3K27me3 (Figure 1 -- figure supplement 2J). On the other hand, Vim, expressed in non-CMs, 172 lacked H3K27ac and was enriched for H3K27me3 (Figure 1 -- figure supplement 2K). 173 About half of the upregulated genes (49.71%), including 7 of 14 upregulated skeletal 174 muscle genes, were occupied by EED at their TSSs in WT. EED occupancy at a subset of the 175 upregulated genes was confirmed by ChIP-qPCR (Figure 1 -- figure supplement 2L). Unlike 176 upregulated genes, only 8.42% of downregulated genes were bound by EED and H3K27me3. 177 These findings suggest that gene upregulation (derepression) rather than downregulation is 178 the predominant, direct effect of EED deficiency, and that repression of skeletal muscle genes 179 is a key direct role of EED in adult heart. 180 We evaluated the relationship between H3K27me3 occupancy and gene upregulation in 181 EedCKO. First, we quantitatively measured H3K27me3 signal within ± 500 bp of TSS on EED 182 target genes in control and EedCKO, stratified by H3K27me3 signal in wild type (Figure 1 -- 183 figure supplement 2M). This analysis showed that EED inactivation reduced the median 184 H3K27me3 signal only of the quartile of genes with highest H3K27me3 occupancy in wild-type. 185 Surprisingly, the median H3K27me3 signal in the remaining three quartiles of upregulated 186 genes was not significantly altered by EED inactivation. Consistent with this analysis, the 187 aggregate H3K27me3 signal at the promoters of the 863 upregulated genes was not reduced 188 in EedCKO, while it was lower in EedCKO at the unchanged and downregulated genes (Figure 1 - 189 - figure supplement 2N). To further corroborate these findings, we divided upregulated genes 190 into three clusters (Fig. 1J): those in which upregulation was coupled to H3K27me3 reduction
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191 (Cluster C1), those with low H3K27me3 signal (C2), and those with no change in H3K27me3 192 signal (C3). Cluster C1, which matches the canonical view in which gene derepressed is 193 association with loss of H3K27me3 (Margueron et al., 2009; Hansen et al., 2008; Boyer et al., 194 2006), contained only 252 of 863 upregulated genes (29%) in EedCKO. In comparison, 4731 of 195 10024 expressed genes (47%) had reduced promoter H3K27me3, indicating lack of 196 enrichment of H3K27me3 loss among upregulated genes. Again, the expected relationship 197 between gene upregulation and H3K27me3 loss did not hold for the majority of upregulated 198 genes, as those in Clusters 2 (303 genes) and 3 (308 genes) were not linked to loss of 199 H3K27me3 (Fig. 1L). Togerther, these analyses indicate that the majority of gene upregulation 200 that occurs with Eed inactivation occurs without loss of H3K27me3. These conclusions were 201 robust to the normalization method used for H3K27me3 ChIP-seq analysis (see Materials and 202 Methods), indicating that it was not a result of global differences in H3K27me3 between WT 203 and EedCKO. Furthermore, we independently confirmed retention of H3K27me3 at promoters of 204 selected upregulated genes by ChIP-qPCR (Figure 1 -- figure supplement 2O). 205 Overall, these results indicate that upregulation of most genes in EedCKO, including the 206 derepressed skeletal muscle genes, was not accompanied by loss of H3K27me3, as expected 207 by the canonical model of EED repression via "writing" of repressive H3K27me3 marks 208 (Margueron et al., 2009; Hansen et al., 2008).
209 Elevated H3K27ac at genes upregulated in EedCKO 210 Our data suggested that EED repressed gene expression in the postnatal heart through 211 mechanisms other than H3K27me3 deposition. Acetylation of H3K27 (H3K27ac) is linked to 212 gene activation and active enhancers (He et al., 2014; Rada-Iglesias et al., 2011; Ferrari et al., 213 2014). Increased H3K27ac was previously noted in embryonic stem cells with PRC2 loss of 214 function (Ferrari et al., 2014; Pasini et al., 2010). Immunoblotting of purified EedCKO CMs 215 showed that Eed inactivation markedly increased total histone H4 acetylation and acetylation 216 of histone H3 at K9 and K27 (Fig. 2A); H3K14ac showed a trend towards being higher in 217 EedCKO, although the difference was not statistically significant (p=0.074; Fig. 2A). This result 218 was unlikely to be an indirect result of cardiac dysfunction in EedCKO hearts, because a similar 219 effect was observed after acute, siRNA-mediated Eed knockdown in the HL-1 CM-like cell line 220 (Fig. 2B). 221 Genome-wide H3K27ac occupancy analysis by ChIP-seq in 2 month-old WT or EedCKO 222 purified CMs showed that Eed inactivation led to acquisition of 8161 new H3K27ac sites and
- 7 - 223 loss of 5950 sites, with 20799 sites in common (Fig. 2C and Supplementary file 1). These peak 224 changes were accompanied by a broad, genome-wide increase of H3K27ac signal strength 225 (Fig. 2D). Indeed, the aggregate H3K27ac signal at the promoters of the 863 genes 226 upregulated in EedCKO increased, whereas it was either unchanged or reduced for the genes 227 that were unchanged or downregulated, respectively (Fig. 2E). These results were validated by 228 ChIP-qPCR at the promoters of several slow twitch muscle genes that were upregulated in 229 EedCKO (Figure 2 -- figure supplement 1). Increased H3K27ac signal was also observed at 230 enhancer regions (H3K27ac peaks region in either WT or EedCKO) associated with these 231 upregulated genes (Fig. 2E). Next, we further examined the specific TSS regions for the 232 correlation of H3K27ac change with gene upregulation (Fig. 2F; rows ordered as in Fig. 1J). 233 Heatmap analysis of all 3 clusters and quantitative analysis in box plots for each cluster 234 confirmed that all 863 derepressed genes markedly gained H3K27ac levels (Fig. 2F,G). 235 Together these data suggest that EED is required to suppress H3K27ac, and that loss of this 236 function leads to abnormal H3K27ac accumulation, global increase in histone H3 and H4 237 acetylation, and aberrant gene upregulation.
238 Reintroduction of EED normalizes heart function and reinstates H3K27ac but not H3K27me3 239 Additional data from timed EED replacement studies further strengthened the relationship 240 between the EedCKO phenotype and H3K27ac rather than H3K27me3. EED has been 241 proposed to facilitate perpetuation of H3K27me3 marks by itself binding to H3K27me3, thereby 242 reinforcing deposition of H3K27me3 at existing sites (Margueron et al., 2009; Hansen et al., 243 2008). If this is the case, then ablation of EED should disrupt the H3K27me3 landscape, 244 preventing its restoration even if EED is later re-expressed. To test this model, we temporally 245 re-expressed EED in CMs by taking advantage of highly efficient, durable, and CM-selective 246 gene transfer using adeno-associated virus serotype 9 (AAV9) and the cardiac troponin T 247 promoter (Tnnt2) (Lin et al., 2014; Jiang et al., 2013). We developed AAV9-Tnnt2-EED 248 (abbreviated AAV9-EED) and validated that it drove cardiac expression of EED when delivered 249 to mice at P14 or P25 (Figure 3 -- figure supplement 1A-C). We evaluated the effect of delayed 250 EED re-expression at either P14 or P25 (Fig. 3A). Interestingly, delayed EED re-expression 251 was still able to rescue cardiac systolic function and cardiac hypertrophy, normalize expression 252 of myofiber and heart failure genes, and ameliorate cardiomegaly of EedCKO mutants (Fig. 3B- 253 D and Figure 3 -- figure supplement 1D). 254 Next we investigated the effect of delayed EED re-expression on the chromatin landscape.
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255 Delayed EED re-expression corrected the abnormally high global H3K27ac levels observed in 256 EedCKO (Fig. 3E). However, delayed EED re-expression did not correct the low H3K27me3 257 levels seen in EedCKO (Fig. 3E). This result was reinforced by genome-wide measurement of 258 H3K27me3 and H3K27ac chromatin occupancy by ChIP-seq. Whereas EED re-expression at 259 P14 and P25 both conferred phenotypic rescue and normalized the genome-wide distribution 260 of H3K27ac signal, P14 and P25 EED re-expression did not normalize H3K27me3 distribution 261 (Fig. 3F,G). 262 We then evaluated the effect of EED re-expression on the 863 genes derepressed in CKO- 263 luc by performing RNA-seq on purified CMs. Out of 863 genes that were upregulated in 264 EedCKO, 256 (66, 87 and 103 in Cluster 1, 2, and 3 respectively) were significantly 265 downregulated (Fig. 3F). This normalization of gene expression was associated with reduction 266 of H3K27ac, but not H3K27me3, near the TSS (Fig. 3F,G). Inspection of the Acta1 and Myl7 267 loci reinforced the finding that, at the upregulated slow twitch skeletal muscle genes, delayed 268 EED re-expression corrected abnormal deposition of H3K27ac but did not significantly affect 269 H3K27me3 (Fig. 3H). These data are consistent with the model that EED "reads" its own mark 270 to reinforce its "writing", so that proper writing cannot be restored after a period has elapsed 271 during which the mark decays. An alternative explanation is that CM PRC2 histone 272 trimethylase activity declines with age due to downregulation of EZH2 and lack of detectable 273 trimethylase activity from EZH1-containing complexes from adult CMs. Regardless of the 274 precise mechanism, our data show that phenotypic rescue and gene expression correction by 275 AAV-EED were not associated with normalization of H3K27me3. Rather, correction aligned 276 with normalization of H3K27ac epigenetic marks.
277 EED interacts with HDACs and enhances its deacetylase activity but is dispensable for HDAC 278 recruitment 279 Histone H3 and H4 acetylation, including H3K27ac, is regulated by a balance between 280 histone acetyltransferases and histone deacetylases (HDACs) (Stefanovic et al., 2014; Tie et 281 al., 2009; Wang et al., 2009). HDACs generally function as co-repressors, and class I and 282 class II HDACs have critical roles in regulating cardiac gene expression and function, including 283 suppression of skeletal muscle gene expression (Montgomery et al., 2007; Song et al., 2006; 284 Zhang et al., 2002; Trivedi et al., 2007). Although EedCKO hearts had elevated global histone 285 H3 and H4 acetylation, most strikingly inducing H3K27ac, total HDAC levels were not changed 286 (Figure 4 -- figure supplement 1A,B). Otte and colleagues showed that HDAC2 interacts with 287 EED and that EED co-immunoprecipitated protein complexes containing HDAC activity (van
- 9 - 288 der Vlag and Otte, 1999), suggesting that HDACs participate in EED-mediated gene 289 repression. These lines of evidence led us to hypothesize that EED represses its postnatal 290 cardiac target genes by interacting with HDACs to decrease their H3K27ac. To test this 291 hypothesis, we screened class I and class II HDACs (HDAC1-HDAC9) for interaction with 292 EED. Immunoprecipitation of FLAG-tagged HDAC1-HDAC9 co-precipitated HA-tagged EED, 293 with the exception of HDAC8 (Fig. 4A and Figure 4 -- figure supplement 1C). This was 294 confirmed by reciprocal immunoprecipitation experiments, in which HA-EED co-precipitated 295 HDAC1-HDAC9 except HDAC8 (Fig. 4A and Figure 4 -- figure supplement 1D). We further 296 confirmed the interaction of endogenous EED and HDAC2 in HL-1 CM-like cells (Fig. 4B). 297 To further approach the molecular function of this interaction on a genome-wide level, we 298 examined the genomic binding distribution of EED and HDAC2 in adult CMs. Nearly half of 299 HDAC2 peaks (4042 out of 9869, 41%) overlapped with EED peaks in WT, in line with their 300 biochemical interaction (Fig. 4C). HDAC2, H3K27me3, and H3K27ac signal at the entire set of 301 8625 EED occupied regions further confirmed this result (Fig. 4D). We identified three classes 302 of EED-bound genomic domains (Fig. 4D). A first class (E1, 5073 regions) was marked by 303 EED, HDAC2 and H3K27ac, but had little H3K27me3. This class was consistent with a 304 previous study that showing that HDACs mark not only inactive genes but also active genes 305 that are poised for transcriptional repression (Wang et al., 2009). A second class (E2, 1830 306 regions) had strong EED and H3K27ac signals, and H3K27me3 was present but weak. A third 307 class (E3, 1722 regions) was marked by strong and broad EED and H3K27me3, but little 308 H3K27ac. Together, these data suggest that EED may function in a complex involving HDACs, 309 independent of H3K27me3. 310 Overall, HDAC2 signals did not change in EED deficient CMs (Fig. 4E). This finding was 311 also confirmed by ChIP-qPCR measurement of HDAC2 or HDAC5 enrichment at promoters of 312 genes upregulated in EedCKO compared to control, revealing no significant difference between 313 HDAC2 or HDAC5 occupancy (Fig. 4F and Figure 4 -- figure supplement 1E). Given that 314 upregulated genes accounted for a small portion of EED occupied regions, we further analyzed 315 HDAC2 occupancy of genes upregulated, unchanged or downregulated in EedCKO. This 316 analysis reinforced that Eed inactivation did not change HDAC2 occupancy, indicating that 317 HDAC2 accumulation on genomic domains is independent of EED (Fig. 4G). Thus, elevated 318 acetylation on H3K27ac in EedCKO was unlikely due to decreased HDAC binding. 319 To identify mechanisms that account for increased H3K27ac at upregulated, EED-occupied
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320 genes in EedCKO CMs, we considered the possibility that EED biochemically regulates HDAC 321 activity. We measured the in vitro deacetylase activity of HDAC2 in the presence of increasing 322 amounts of recombinant EED protein, expressed and affinity purified from Sf21 cells using a 323 baculovirus expression system. EED protein did not contain detectable contaminating proteins 324 (Figure 4 -- figure supplement 2A). EED alone had no detectable deacetylase activity, but 325 when added to HDAC2 it augmented HDAC2's deacetylase activity by up to 3-fold (Fig. 4H). 326 Together, the aforementioned data all support a model in which EED represses a subset of its 327 target genes by stimulating HDAC. These results also raise the possibility that EED drives 328 transcriptional repression at least partially by modulating the activity of another class of 329 epigenetic repressors, HDACs.
330 HDACs functions downstream of EED to direct normal heart maturation
331 Both class I and class II HDACs regulate cardiac gene expression, hypertrophy, and 332 function (Hohl et al., 2013; Hang et al., 2010; Trivedi et al., 2007; Kong et al., 2006; Kook et 333 al., 2003; Zhang et al., 2002). Notably, cardiac-restricted deletion of both HDAC1 and HDAC2 334 caused dilated cardiomyopathy accompanied by upregulation of genes encoding skeletal 335 muscle-specific contractile proteins (Montgomery et al., 2007). These findings converged with 336 our studies to suggest the hypothesis that EED inactivation caused cardiomyopathy and 337 upregulation of skeletal muscle myofiber genes through loss of HDAC1/2 activity. To test this 338 hypothesis, we asked if overexpression of HDAC1/2 ameliorated the EedCKO phenotype. 339 Accordingly, we developed AAV9-Tnnt2-HDAC1/2 (abbreviated AAV-HDAC1/2) to direct CM- 340 selective overexpression of HDAC1/2 (Figure 5 -- figure supplement 1A-C). Remarkably, AAV- 341 HDAC1/2 delivery to P3 EedCKO mice (CKO-HDAC1/2) normalized systolic dysfunction and 342 cardiomegaly, compared to control treatment with luciferase (CKO-luc; Fig. 5A-C). 343 Heterozygous littermates (Het; Eedfl/+; Myh6Cre) were used as unaffected controls. AAV- 344 HDAC1/2 also restored repression of slow twitch sarcomere genes Acta1 and Myh7, and 345 reduced expression of the heart failure marker Nppa (Fig. 5D). RNA-seq gene expression 346 measurements showed that out of 863 genes upregulated in EedCKO, 104 were downregulated 347 in the rescue group (adjusted p-value <0.05 and fold change (CKO-luc/CKO-HDAC1/2) >1.5; 348 Fig. 5E). Of these 104 genes, 39, 34 and 31 were found in Cluster 1, 2 and 3, respectively. 349 We measured the effect of AAV-HDAC1/2 on genome-wide chromatin occupancy of 350 H3K27me3 and H3K27ac by ChIP-seq (Fig. 5E). This revealed that the greater H3K27ac 351 signal observed at the TSS of most of the 863 genes upregulated in EedCKO was normalized by
- 11 - 352 AAV-HDAC1/2: the ratio of H3K27ac signal in CKO-luc to CKO-HDAC1/2 was ≥ 1.5 for 460 353 genes and 1-1.5 for 293 genes, while only 110 were not changed (Fig. 5E). This conclusion 354 was reinforced by comparison of H3K27ac signal between CKO-HDAC1/2 and CKO-luc (Fig. 355 5F). Within each of the three classes of regions (C1, C2, C3) defined previously based on 356 H3K27ac occupancy and change with Eed inactivation (Fig. 1J), AAV-HDAC1/2 significantly 357 reduced H3K27ac signal. Similar results were observed when all H3K27ac regions that 358 increased in EED loss of function were analyzed, rather than only the TSSs of upregulated 359 genes (Figure 5 -- figure supplement 1D-F). Normalization of H3K27ac was further supported 360 by calculating each upregulated gene’s change in H3K27ac at the TSS in Eed loss of function 361 (CKO-luc/het) compared to its change with AAV-HDAC1/2 rescue (CKO-luc/CKO-HDAC1/2; 362 Figure 5 -- figure supplement 1F, upper panel). For the 104 normalized genes, H3K27ac 363 occupancy was reduced towards control levels. In contrast, no similar change was observed 364 for H3K27me3 (Figure 5 -- figure supplement 1F, lower panel). 365 Notably, these changes in H3K27ac occurred despite lack of global H3K27ac level, as 366 measured by immunoblotting (Figure 5 -- figure supplement 1G). Thus, heart function 367 normalization was not dependent upon broad normalization of H3K27ac but rather on targeted 368 HDAC1/2 activity at specific loci. On the other hand, H3K27me3 was not strongly affected by 369 AAV-HDAC1/2 (Fig. 5E-G and Figure 5 -- figure supplement 1D-G), consistent with the 370 findings in EED rescue experiments that correction of gene expression and heart function was 371 independent of H3K27me3.
372 HDAC inhibition antagonized EedCKO rescue by EED re-expression
373 To further probe the requirement of HDAC for EED activity in CMs, we studied the effect of 374 HDAC inhibition on rescue of EedCKO by AAV-mediated EED re-expression. Neonatal EedCKO 375 mice were treated with AAV9-EED at P5. They were then treated daily with the broad spectrum
376 HDAC inhibitor suberanilohydroxamic acid (SAHA) or vehicle control. Het mice transduced with
377 AAV9-luc and then SAHA or vehicle served as additional controls. Consistent with previous 378 reports (Xie et al., 2014), SAHA treatment for 2 months did not adversely effect heart function 379 in the Het controls, nor did it perturb expression of 5 genes (Acta1, Myl9, Tnni1, Tbx15 and
380 Mybpc2) that were aberrantly upregulated in EedCKO. Interestingly, SAHA treatment appeared to
381 block heart function rescue by AAV9-EED re-expression. Two out of five mice died prematurely in
382 the EedCKO+EED+SAHA group and were lost to the study. Despite this potential survivor bias, heart
383 function rescue by AAV9-EED tended to impair AAV9-EED rescue in the remaining three mice,
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384 although this did not reach statistical significance (P=0.07; Fig. 6A,B). These data suggest that HDAC
385 activity is essential to suppress aberrant gene expression in EedCKO. In line with this, normalized
386 transcription of 4 out of 5 genes by EED re-expression in EedCKO was blocked by SAHA
387 treatment (Fig. 6C). The different effect of SAHA on normal hearts compared to AAV9-EED 388 rescue of EED deficiency suggests that multiple redundant repressive pathways operate in 389 normal heart development, making the HDAC pathway dispensible. However, in AAV-EED 390 rescue of EED deficiency, these redundancies are disrupted, perhaps as a result of 391 perturbations of the chromatin landscape, leaving heart development vulnerable to HDAC 392 inhibition. Collectively, these data indicate that normalization of HDAC activity by EED re- 393 expression was essential for rescue of heart failure in EedCKO. 394 Altogether, these data demonstrate that HDACs collaborate with EED to mediate postnatal 395 cardiac gene repression required for normal heart development and function. Furthermore, our 396 data indicate that EED functions genetically upstream of HDACs and support a model in which 397 EED maintains transcriptional repression of target genes in post-mitotic cardiomyocytes. Our 398 results demonstrate an essential mechanism of EED repression that is independent of its 399 requirement for PRC2 deposition of H3K27me3 (Fig. 7).
400 DISCUSSION 401 We found that postnatal loss of EED caused lethal dilated cardiomyopathy associated with 402 ecoptic expression of a slow skeletal muscle gene program. Surprisingly, de-repression of this 403 program was not associated with loss of H3K27me3 at these genes, but rather marked gain of 404 H3K27ac. Moreover, EED depletion caused global increase in histone H3 and H4 acetylation. 405 Our rescue experiments further demonstrated that phenotypic reversal was associated with 406 normalization of H3K27ac but not H3K27me3. Furthermore, we demonstrate a physical and 407 genetic interaction between EED and HDACs that mechanistically accounts, at least in part, for 408 the link between EED loss of function and aberrant H3K27ac accumulation. Together these 409 experiments highlight an H3K27me3-independent repressive activity of EED, and demonstrate 410 that disease caused by aberrant epigenetic regulation can be reversed through epigenetic 411 rewiring.
412 Gene de-repression in EedCKO was linked to gain of H3K27ac and not loss of H3K27me3 413 Previous studies of PRC2 inactivation, performed primarily in cultured cells such as 414 embryonic stem cells, have shown that many de-repressed genes are associated with loss of 415 H3K27me3 (Simon and Kingston, 2013; Simon and Kingston, 2009). The role of PRC2
- 13 - 416 methylation of H3K27me3 in gene repression was further reinforced by studies of Drosophila 417 imaginal discs in which wild-type histone H3 was replaced by a point mutation, H3K27R, that 418 can neither be methylated nor acetylated (Pengelly et al., 2013). The effect of this H3K27R 419 mutation resembled PRC2 inactivation, suggesting that in this system the major repressive 420 activity of PRC2 is trimethylation of H3K27. However, several lines of evidence from our study 421 indicate that an essential mechanism of PRC2 repression in adult CMs is independent of 422 H3K27me3. First, genes de-repressed by Eed inactivation had largely unchanged H3K27me3. 423 This result was robust to the method used to normalize the H3K27me3 ChIP-seq data, and 424 was independently validated at selected sites by ChIP-qPCR. Second, AAV-EED rescued 425 heart function without correcting global H3K27me3 levels or occupancy of upregulated gene 426 TSSs. Third, AAV-HDAC1/2 rescued heart function and re-established gene repression without 427 elevating H3K27me3. 428 Our data indicate that in adult CMs, PRC2 regulation of H3K27ac plays an essential role in 429 repression of a subset of genes. First, gene upregulation in EedCKO was associated with gain 430 of H3K27ac. Second, AAV-EED rescue correlated with restoration of H3K27ac rather than 431 H3K27me3. Third, AAV-HDAC1/2 rescued function of EedCKO hearts and normalized H3K27ac 432 but not H3K27me3. HDACs have multiple substrates, including other histone acetylation sites 433 as well as non-histone proteins. Our data do not exclude important effects of HDACs on 434 targets other than H3K27ac, but the close correlation between H3K27ac and cardiac rescue 435 strongly suggests that HDAC acts, at least in part, through H3K27ac. Depletion of essential 436 PRC2 subunits in ES cells and Drosophila was previously reported to increase H3K27ac 437 (Pasini et al., 2010; Tie et al., 2009; Ferrari et al., 2014), consistent with the genetic interplay 438 between PRC2 and histone acetylation that we observed in this study. Similarly, dominant 439 negative inhibition of PRC2 by expression of a point mutant of histone H3.3 (H3.3K27M) 440 globally increased H3K27ac (Herz et al., 2014). Furthermore, EED-HDAC2 interaction and 441 HDAC activity were previously reported to be essential for PRC2 repression of selected target 442 loci (van der Vlag and Otte, 1999). However, the global role of H3K27me3 versus H3K27ac in 443 EED-mediated repression in vivo has remained uncertain. Our study clarifies this relationship 444 in postnatal CMs, by demonstrating that gene de-repression downstream of EED loss of 445 function is closely tied to H3K27ac gain rather than to H3K27me3 loss. 446 As reviewed above, the canonical dogma is that PRC2 represses genes by depositing 447 H3K27me3. Why might our study have reached a different conclusion? One key difference is
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448 the mitotic state of the model systems: whereas the vast majority of studies of PRC2 have 449 been performed in actively cycling cells such as embryonic stem cells and developing 450 embryos, mature CMs have largely exited the cell cycle. Cycling cells require active rewriting 451 of histone marks, so that PRC2 inactivation is rapidly accompanied by widespread H3K27me3 452 loss. In contrast, adult CMs have relatively stable H3K27me3 and little detectable PRC2 453 methyltransferase activity. Under these conditions, there is likely a greater opportunity to 454 expose writing-independent activities of EED. Importantly, many cells of adult mammals are 455 either slowly cycling or terminally differentiated, and thus aspects of their epigenetic regulation 456 may more closely resemble adult CMs than cultured cells such as embryonic stem cells.
457 Remodeling the chromatin landscape to correct heart failure 458 Mutations of epigenetic regulators cause congenital heart disease, and abnormalities of the 459 chromatin landscape have been implicated in the pathogenesis of heart failure. (He et al., 460 2012; Zaidi et al., 2013; Hang et al., 2010; Delgado-Olguin et al., 2012; Lickert et al., 2004; 461 Montgomery et al., 2007) The chromatin landscape is not hardwired but rather dynamically 462 constructed through the sequential action of epigenetic regulators, suggesting that perturbation 463 of the normal sequence may irreversibly disrupt the chromatin landscape. Consistent with this 464 expectation, we found that transient loss of EED irrevocably altered the landscape of 465 H3K27me3. EED inactivation also globally increased H3K27ac levels, and gene de-repression 466 was associated with H3K27ac gain on TSS regions of de-repressed genes. This was closely in 467 line with the findings by Margueron and colleagues that transcription and histone modification 468 changes as a consequence of EZH2 loss are predominantly irreversible (Wassef et al., 2015). 469 Despite these irreversible changes to the epigenetic landscape, remarkably AAV-EED and 470 AAV-HDAC1/2 were able to successfully remodel the chromatin landscape to functionally 471 correct abnormalities of gene expression and organ function, even without correcting the 472 genome-wide distribution of modified histone marks. These results reveal that it is possible to 473 rehabilitate deregulated gene programs due to distorted chromatin landscapes, which 474 contribute to diseases such as heart failure and congenital heart disease. 475
476 ACKNOWLEGEMENTS 477 We thank H. Cheng from Institute of Molecular Medicine, Peking-Tsinghua Center for Life 478 Sciences, Peking University, B. Zhou from the Institute of Nutritional Sciences and Shanghai 479 Institute for Biological Sciences and Y. Zhang from the National Laboratory of Plant Molecular
- 15 - 480 Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for 481 Biological Sciences, Chinese Academy of Sciences for valuable suggestions. A.H. was 482 supported by a startup fund from Peking-Tsinghua Center for Life Sciences, Peking University, 483 the 1000 Youth Talents Program of China, the National Natural Science Foundation of China 484 (31571487), and a Scientist Development Grant from American Heart Association 485 (13SDG14320001). WTP was supported by funding from the National Heart Lung and Blood 486 Institute (U01HL098166 and HL095712), by an Established Investigator Award from the 487 American Heart Association, and by charitable donations from Karen Carpenter, Edward 488 Marram, and Gail Federici Smith. The content is solely the responsibility of the authors and 489 does not necessarily represent the official views of the funding agencies. 490
491 MATERIALS AND METHODS 492 Mice 493 All animal experiments were performed according to protocols (protocol #, Lsc-HeAB-1) 494 approved by the Institutional Animal Care and Use Committees of Peking University and 495 Boston Children's Hospital. Eedfl (Xie et al., 2014) and Myh6Cre (Agah et al., 1997) alleles were 496 described previously. "WT" denotes either Eedfl/fl; Myh6Cre– or Eedfl/+ genotypes. Mice were 497 injected with AAV (1x1011 viral particles/gram body weight) by intraperitoneal or intravascular 498 injection into pups and adults, respectively. Mice were intraperitoneally injected with SAHA 499 (Sigma, 25 mg/kg/day) for the period of time as indicated. Echocardiography was conducted in 500 either conscious or lightly anesthetized mice (isoflurane) using a Vevo 2100 imaging system 501 (VisualSonics, Inc). Adult cardiomyocytes were isolated using type II collagenase in the 502 Langendorff retrograde perfusion mode (O'Connell et al., 2007), and cardiomyocytes purity 503 was evaluated for by co-immunostaining for cardiomyocyte mark Troponin i 3 (TNNI3) and 504 DAPI. Isolated cardiomyocytes will be used only if more than 95% cells are positive for TNNI3.
505 Cell culture 506 HL-1 cardiomyocyte-like cells (Claycomb et al., 1998) were cultured and validated as 507 described previously (He et al., 2011), and determined as free of mycoplasma contamination. 508 Fully confluent HL-1 cells were transfected using RNAiMax (Life Technoligies) with a pool of 509 TriFECTa Dicer-Substrate siRNAs (DsiRNAs) (Integrated DNA Technologies) against Eed 510 (conserved in both mouse and rat). Scrambled DsiRNA was used as the control. Sequences 511 are provided (Supplementary file 2A). Cells were analyzed 72 hours after transfection.
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512 Transfection efficiency was verified to be more than 90% using fluorescently-labeled control 513 duplex as the indicator.
514 Western Blot 515 Cells were lysed in Nuclear Lysis Buffer (NLB, 50 mM Tris-Hcl pH8.0, 150 mM NaCl, 1% 516 Nonidet P-40 (NP40), 1 mM EDTA, and fresh 1 mM PMSF and protease inhibitor cocktail (PIC) 517 ) for 30 mins, and 1% SDS was added before homogenization on QIAshredder columns. Equal 518 amounts of proteins were resolved on 7.5% or 10% SDS-polyacrylamide gels and 519 immunoblotted with primary antibodies listed (Supplementary file 2B). EED antibody was a 520 kind gift from D. Reinberg (Margueron et al., 2008).
521 Protein-protein interactions 522 Co-immunoprecipitation was performed as described (He et al., 2012) with minor 523 modifications. Nuclei were isolated using Hypotonic Lysis Buffer (20 mM HEPES pH 7.5, 10 524 mM KCl, 1 mM EDTA, 0.1 mM Na3VO4, 0.1 mM 0.2% (vol/vol) NP-40, 10% (vol/vol) glycerol 525 plus PIC) and then resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% 526 NP-40, 1 mM EDTA, and fresh 1 mM PMSF and PIC). Lysates were treated with 20 U of 527 Benzonase nuclease (Stratagene) for 1 hr at 4°C. After centrifugation at 16,000 x g for 20 min 528 at 4°C, soluble materials were kept as nuclear extracts. Nuclear extracts were precleared with 529 Protein G beads before incubation with 5 µg of the indicated antibodies prebound to Protein G 530 beads or Anti-Flag Affinity gel (Sigma, A2220) or Anti-HA High Affinity Matrix (Roche, 531 12013819001) for 4 hrs or overnight at 4°C. After washing three times with lysis buffer, 532 precipitated proteins were recovered by elution in the sample buffer.
533 In vitro HDAC activity assay 534 In vitro deacetylation assay was performed using a colorimetric immunoassay for 535 deacetylated histones (Epigentek, P-4034-96). 50 ng recombinant HDAC2 (Active Motif 31343) 536 and 0-100 ng recombinant EED (Cayman Chemical 10628) or bovine serum albumin were 537 incubated at 37°C for 1 hr, and determined from the absorbance at 450 nm.
538 ChIP and ChIP-seq 539 Antibody-based ChIP was performed as described (He and Pu, 2010) using antibodies 540 listed (Supplementary file 2B). EED ChIP-seq was performed in adult cardiomyocytes using 541 bioChIP method as described previously (He et al., 2012). Briefly, BirA mice were injected 542 with AAV expressing the cardiomyocyte specific promoter cTNT driving EED in fusion with 543 FLAG and bio (fbio) epitope tags, where bio is 23 amino-acid sequence specifically biotinylated
- 17 - 544 by the enzyme BirA, and cardiomyocytes were harvested for bioChIP (ChIP pulldown through 545 streptavidin beads) as indicated. ChIP-qPCR values were expressed as fold-enrichment. 546 Illumina ChIP-seq libraries were prepared as described (He et al., 2014) from purified 547 cardiomyocytes, sequenced on an Illumina Hiseq 2500, and analyzed as described (He et al., 548 2014). Briefly, filtered reads were aligned to the July 2007 assembly of the mouse genome 549 (NCBI 37, mm9) using the Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2009) with default 550 settings. Reads with no more than 4% (2 bp) mismatches and uniquely mapped to reference 551 genome were kept for downstream analyses. The sequencing data are summarized here 552 (Supplementary file 3). 553 Peaks were identified using MACS2 (Zhang et al., 2008) (version 2.1.0), with the parameter 554 settings (--keep-dup=1; --broad). Peaks were assigned to the gene with the closest TSS within 555 100 kb. Proximal was defined within ±5 kb of a gene's TSS. Remaining regions were defined 556 as distal. Cardiac enhancers were identified by H3K27ac peaks using adult heart H3K27ac 557 ChIP-seq data (He et al., 2014). BigWig files normalized per 10 million aligned reads were 558 viewed using the Integrative Genomics Viewer (IGV) (version 2.3.59) (Robinson et al., 2011). 559 ChIP and input reads were normalized to 10 million total aligned reads for H3K27ac, EED, 560 and HDAC2. To reduce background signals for H3K27me3 in EedCKO vs WT we firstly 561 implemented a normalizing factor quantified through ChIP experiments (the relative levels of 562 ChIP derived DNA) to the reads intensities in EedCKO and WT (Ferrari et al., 2014). To further 563 exclude the bias due to normalization, we further chose the second normalization manner, 564 yielding the similar results (Kranz et al., 2013). Simply, H3K27me3 ChIP signals were 565 standardized using z-score transformation of ChIP reads by calculating mean and standard 566 deviation of each dataset without all peak regions, presumably called background vs input. 567 DANPOS (version 2.2.2) (Chen et al., 2013) was used to calculate H3K27me3 and H3K27ac 568 signals (ChIP reads minus input reads, kept only if the value>0) for aggregate plots and box 569 plots. H3K27ac and H3K27me3 signals were calculated at 20 bp intervals and plotted using R 570 (version 3.2.2) and ggplot2 (version 1.0.1). Genome-wide distributions of H3K27me3 and 571 H3K27ac signals were determined by counting ChIP signals within non-overlapping 1 kb wide 572 windows tiled over the mouse genome. The box plots were generated using the average ChIP 573 signals of ±5 kb of genes' TSS. 574 575 RNA expression 576 RNA was prepared from purified adult cardiomyocytes as described (He et al., 2012).
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577 mRNA sequencing library was prepared with Script-seq v2 (Illumina), and sequenced on 578 Illumina Hi-seq 2000 (PE100). The resulting sequences were mapped to the mouse genome 579 mm9 with STAR (Dobin et al., 2013). Transcripts per million reads (TPM) and Reads per 580 kilobase of exon per million fragments (RPKM) were generated for further quantification by 581 RSEM and Bowtie 2 (Langmead and Salzberg, 2012; Li and Dewey, 2011), and differentially 582 expressed genes were called with edgeR (v3.14.0) with the following criteria: adjusted P-value 583 < 0.05 and Fold change (FC) > 1.5 or < 0.67. The gene ontology (GO) enrichment analysis of 584 DEGs was performed using Metascape (Tripathi et al., 2015). The top 6 GO terms with P- 585 value < 0.001 in the ‘biological process’ category were used.
586 Quantitative PCR 587 Real time PCR was used to measure relative ChIP enrichment or gene expression. 588 Quantitative PCR was performed using Power Sybr Green Master Mix (Life Technologies). 589 Primer sequences are listed here (Supplementary file 2C). ChIP-qPCR values were expressed 590 as fold-enrichment.
591 Statistical analysis 592 Student’s two-tailed t-test was used to determine the significance of differences between 593 two groups in all qPCR assays. For bar charts, data is presented as mean ± SEM. For violin 594 plots, the center line indicates the median. Wilcoxon-Mann-Whitney test was used to compare 595 aggregate curves and violin-bar plots. Statistical significance was indicated with: *, P<0.05; **, 596 P<0.01; ***, P<0.001. 597
598 COMPETING FINANCIAL INTERESTS 599 The authors declare no competing financial interests.
600 ACCESSION CODES 601 The high throughput data used in this study are available through the Cardiovascular 602 Development Consortium Server at https://b2b.hci.utah.edu/gnomex/. Sign in as guest and go 603 to experiment #410R. The data have also been deposited at 604 GEO,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gxeviggwnrqbjmt&acc=GSE7377 605 1.
606 607 608
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808 FIGURE LEGENDS 809 Figure 1. Neonatal cardiomyocyte inactivation of Eed caused lethal dilated 810 cardiomyopathy. A. EED protein expression in WT and cardiac EedCKO (CKO, Myh6- 811 Cre+;Eedf/f) on postnatal day 0 (P0) and 5 (P5). Quantification shows relative EED protein 812 normalized to GAPDH loading control. Several splice isoforms of EED were detected. * 813 indicates a non-specific band that is larger than full length EED's predicted molecular weight. 814 B. Kaplan-Meier survival curve of WT and EedCKO mice. C. Heart function was measured by 815 echocardiography as fractional shortening (FS%) at 2 months of age. See Fig. 1 -- figure 816 supplement 2A for FS% at earlier time points. D-F. Cardiac dilatation and hypertrophy were 817 observed by heart to body weight ratio (D), gross morphology (E), and histology (F) in WT and 818 EedCKO at 2 months of age. Representative hearts are shown. Bar = 1 mm. G. Immunoblotting 819 for H3K27me3 in cardiomyocytes from WT and EedCKO at 2 months of age. H. Genome-wide 820 distribution of H3K27me3 ChIP-seq signals in WT and EedCKO purified cardiomyocytes. ChIP- 821 seq signal was measured in 1 kb windows across the genome. The signal distribution is 822 displayed as a violin plot. Yellow lines denote the median value. I. Venn diagram showing the 823 distribution of H3K27me3 peaks in WT and EedCKO heart. J. Heat map of RNA transcript levels 824 of differentially expressed genes (fold-change >1.5 or <0.67 and adjusted P-value <0.05) are 825 shown in the left heatmap. Expression values for each gene were row scaled. Selected 826 contractile myofiber and heart failure marker genes are shown in red and black, respectively. 827 Right heatmap shows H3K27me3 and EED ChIP-seq signal at the transcriptional start site 828 (TSS) of the differentially expressed gene on the same row. Gene expression, H3K27me3 and 829 EED ChIP-seq studies were performed on purified cardiomyocytes at 2 months of age. Rows 830 were ordered by k-means clustering on H3K27me3 and EED ChIP-seq signal into 3 clusters, 831 C1-C3. K. Gene Ontology analysis of genes differentially expressed genes between WT and 832 EedCKO. The top 6 significant terms are shown.L. Box plots showing H3K27me3 and signals in 833 these 3 clusters as shown in G. A, C, D, Student’s t-test; H, L, Wilcoxon-Mann-Whitney test. *, 834 P<0.05; **, P<0.01; ***, P<0.001, NS, not significant. Numbers in bars indicate independent 835 biological replicates. 836
837 Figure 2. Eed depletion induced globally elevated histone acetylation. A. Global 838 upregulation of histone H3 and H4 acetylation at different lysine residues in isolated adult 839 cardiomyocytes from 2 month old WT and EedCKO hearts. Histone levels were measured by
- 29 - 840 immunoblotting and further quantified by normalization to total histone H3. B. Acute EED 841 depletion increased H3K27ac levels in HL-1 cardiomyocyte-like cells. Fully confluent HL-1 cells 842 were transfected with TriFECTa DsiRNAs against Eed (si-Eed) or scrambled sequence 843 negative control (si-NC). Protein levels were measured by quantitative immunoblotting. Arrow, 844 EED band. Asterisk, non-specific band. C. Venn diagram showing the overlap of H3K27ac 845 ChIP-seq peaks in isolated cardiomyocytes from WT and EedCKO hearts at 2 months of age. D. 846 Genome-wide distribution of H3K27ac signals. The violin plot displays H3K27ac ChIP-seq 847 signals in 1 kb windows across the genome. Yellow horizontal lines denote median values. E. 848 Aggregation plots of H3K27ac ChIP-seq signals at ± 5 kb of TSS (upper row) or at distal 849 regions (lower row) of genes that were up-regulated, down-regulated, or unchanged by Eed 850 inactivation. F-G. Heat map (F) and box plots (G) of H3K27ac levels at TSS of differentially 851 expressed genes. The row order and clustering is the same as in Fig. 1J. A, B, Unpaired 852 Student’s t-test; D,G, Wilcoxon-Mann-Whitney test. **, P<0.01; ***, P<0.001; NS, not 853 significant. 854 855 Figure 3. Delayed re-expression of EED rescued heart function and normalized 856 H3K27ac but not H3K27me3. A. Schematic of the experimental design. AAV9 expressing 857 EED or luciferase (luc) in cardiomyocytes was injected at P14, or P25 to control or EedCKO 858 mice. Lines represent the temporal pattern of EED expression. B-E. Heterozygous (Het; 859 Eedfl/+; Myh6Cre+) and EedCKO mice were injected with AAV-luc or AAV-EED at indicated ages. 860 At 2 months of age, heart function (FS%) was measured by echocardiography (B), and 861 expression of Myh7 and Acta1, two slow-twitch myofiber genes aberrantly expressed in 862 EedCKO, were measured by qRTPCR (C). Representative images showing gross morphology 863 of hearts at 2 months of age (D). Cardiomegaly of CKO-luc hearts was abrogated by EED 864 replacement at P14 or P25. Isolated cardiomyocytes were immunoblotted to measure 865 expression of virally delivered EED, and global levels of H3K27ac and H3K27me3 (E). Graphs 866 show quantitation of global H3K27ac and H3K27me3 levels, normalized to histone H3. F. 867 Heatmaps of RNA expression, H3K27me3 and H3K27ac ChIP signals at ± 5 kb of TSS of 863 868 upregulated genes from EedCKO mice injected with AAV-luc or AAV-EED at P14 or P25. Row 869 order and cluster labels are the same as Fig. 1J. G. Quantitative analysis of H3K27me3 and 870 H3K27ac ChIP signals near TSSs shown in F by box plots. H. Genome browser view of 871 H3K27me3 and H3K27ac ChIP-seq signals at the Acta1 or Myl7 loci. The regions highlighted 872 in gray show that gain of H3K27ac in EedCKO was reset to normal under EED rescue
- 30 -
873 conditions regardless of H3K27me3 status. B, C, and E, Unpaired Student’s t-test; G, 874 Wilcoxon-Mann-Whitney test. *, P<0.05; **, P<0.01; ***, P<0.001. 875
876 Figure 4. EED interacts with and co-localizes with HDAC to repress transcription 877 through enhancing its deacetylation activity. A. Co-Immnoprecipitation analysis of EED- 878 HDAC interaction in 293T cells. HA-EED pull down with HA antibody co-precipitated FLAG- 879 HDAC1/2/3 (left). Reciprocally, FLAG-HDAC1/2/3 pull down with Flag antibody co-precipitated 880 HA-EED (right). Data on HA-EED and Flag-HDAC4-9 interaction are presented in Fig. 4 -- 881 figure supplement 1C,D. B. Interaction between endogenous EED and HDAC2. EED, 882 immunoprecipitated from HL-1 cardiomyocyte-like cells, co-precipitated HDAC2. Arrowhead 883 denotes the specific band and asterisk denotes the non-specific IgG heavy chain band. C. 884 Venn diagram showing overlap of EED and HDAC2 peaks in WT. D. Heatmaps showing ChIP- 885 seq signals for EED, H3K27me3, H3K27ac and HDAC2 at ± 5kb of EED peaks. Rows were 886 sorted by ascending EED peak signal. E. Aggregate plot showing HDAC2 ChIP signals in WT 887 and EedCKO, centered on EED peak centers. F. HDAC2 occupancy of the indicated chromatin 888 regions in isolated cardiomyocytes from WT and EedCKO mice at 2 months of age. Occupancy 889 was measured by ChIP followed by quantative PCR (ChIP-qPCR). Chromatin regions are 890 named by the adjacent gene and the distance to the TSS. G. Aggregation plot showing 891 HDAC2 ChIP-seq signals in WT and EedCKO at ± 5 kb of TSS of genes that were upregulated, 892 unchanged or downregulated in EedCKO. H. Effect of EED on HDAC2 activity. In vitro 893 deacetylation assay was performed using recombinant active HDAC2 (50 ng) in the presence 894 of BSA or 5 to 100 ng of recombinant EED, purified from insect cells. Deacetylation activity 895 was measured by colorimetric assay. F, H, I, J, K, Unpaired Student’s t-test. *, P<0.05; **, 896 P<0.01; ***, P<0.001. 897 898 Figure 5. Re-introduction of HDAC1/2 restored normal heart function. A. AAV- 899 HDAC1/2 rescue of EedCKO. Schematic shows the rescue experiment design. WT and CKO 900 pups were injected with AAV-luc or AAV-HDAC1/2 at P3, and assays were done at 2 months 901 of age. B. Heart function (FS%) was measured at 2 months of age by echocardiography. C. 902 Gross morphology and heart to body weight ratio of hearts from mice at 2 months of age. Bar = 903 1 mm. D. Analysis of heart failure gene expression. Acta1, Myh7 and Nppa levels in isolated 904 cardiomyocytes were measured by qRTPCR. E, F. Heatmaps (E) and box plots (F) showing
- 31 - 905 ChIP signals for H3K27ac and H3K27me3 at ± 5kb of TSS of genes upregulated in EedCKO. 906 Row order and cluster labels are the same as Fig. 1J. Comparative analysis of ChIP-seq 907 signals was performed within each cluster as indicated. G. Aggregation plots for H3K27me3 or 908 H3K27ac ChIP-seq signals at ± 5kb of TSS of EedCKO upregulated genes in Het, CKO+luc, 909 and CKO+HDAC1/2 groups. B,C,D, Unpaired Student’s t-test. F, Wilcoxon-Mann-Whitney test. 910 *, P<0.05; **, P<0.01; ***, P<0.001. 911 912 Figure 6. HDAC inhibition antagonized rescue of EedCKO by EED re-expression. A. 913 Schematic of the experimental design. B. Heart function was measured by echocardiography 914 as fraction shortening (FS%) in 2 month old EedCKO or Het mice that received the indicated 915 treatments. Two of five EedCKO + EED + SAHA mice died prior to the study endpoint and were 916 not available for echocardiography. C. qRTPCR measurement of 5 selected genes that were 917 aberrantly expressed in EedCKO hearts. P-value by Student’s t-test. *, P<0.05; **, P<0.01; NS, 918 not significant. 919 920 Figure 7. Working model delineates a non-canonical mechanism by which EED 921 represses gene transcription. Two mechanisms for EED repression were operative in the 922 postnatal heart. One subset of repressed genes was occupied by EED and H3K27me3 in WT, 923 and EED inactivation reduced H3K27me3 in association with gain of H3K27ac. Upregulation of 924 these genes in EedCKO could be due to a combination of loss of H3K27me3 (canonical 925 mechanism). Loss of EED itself with subsequent gain in H3K27ac might also contribute to 926 regulation of these genes. A second subset of repressed genes was also occupied by EED 927 and H3K27me3 in WT, but H3K27me3 was not reduced by EED inactivation. While H3K27me3 928 may contribute to the repression of these genes in WT, their upregulation in EedCKO was not 929 attributable to H3K27me3, which was unchanged. Rather, our data suggest that upregulation 930 was directly due to loss of EED itself, with consequent reduction of HDAC activity and gain in 931 H3K27ac. A third subset of genes was occupied by EED but little H3K27me3 in WT. These 932 genes had significant H3K27ac at baseline, which was further increased by Eed inactivation. 933 Thus these genes may represent a set “poised” for activation; in WT EED occupancy 934 represses these genes by collaborating with HDAC to limit gene activity. EED inactivation 935 reduces HDAC activity, resulting in H3K27ac accumulation and gene upregulation. 936
- 32 -
937 Figure 1 -- figure supplement 1. Eed depletion in WT and EedCKO mice. Heart apexes 938 were harvested for qRT-qPCR for relative Eed mRNA expression on postnatalday 0 (P0) and 5 939 (P5). Heart apex contains both cardiomyocytes and non-cardiomyocytes. The non-myocytes 940 likely account for the detected level of Eed mRNA in EED-CKO. P-value by Student’s t-test. 941 ***, P<0.001. NS, not significant.
942
943 Figure 1 -- figure supplement 2. Characterization of EedCKO mice. A. Progressive 944 cardiac dysfunction and dilatation after cardiomyocyte-restricted ablation of Eed. w, weeks. B. 945 Nppa mRNA level in WT and CKO hearts at the indicated ages. w, weeks. C, D. Cardiac 946 fibrosis was evident by Mason Trichrome staining at 2 months of age. Fraction of myocardial 947 area occupied by fibrotic tissue (blue staining) was quantified using ImageJ. Bar = 50 µm. E, F. 948 Immunofluorescence for cardiomyocyte marker TNNI3 and cardiomyocyte membrane marker 949 WGA, and quantification of cell size from WGA-stained cardiomyocyte outlines (f). Bar= 50 µm. 950 G. Immunostaining for TNNI3 and H3K27me3. Isolated adult cardiomyocytes were > 95% pure 951 and EedCKO CMs had little H3K27me3 signal. Bar = 50 µm. H. PCR of genomic DNA from 952 purified CMs using primers that amplify unexcised floxed DNA (233 bp product) or Cre-excised 953 DNA (453 bp product). In CKO-purified CMs, unexcised floxed DNA was not detected, 954 consistent with highly efficient Cre-mediated gene inactivation, as well as high purity of 955 dissociated CMs. I. RNA-seq track view showing deletion of floxed exons 3-6 of Eed (red box).
956 J,K. Genome browser view ofH3K27me3 and H3K27ac ChIP-seq signals on Myh6 (J) and 957 Vim (K) loci in purified adult cardiomyocytes. L. EED enrichment on downstream genes was 958 measured by ChIP-qPCR in P5 heart ventricle apex. Numbers following gene names indicate 959 the number of nucleotides between the probed amplicon and the TSS. M. Box and scatter 960 plots of H3K27me3 at TSS ± 500 bp of EED target genes in four quantiles of WT H3K27me3 961 intensity. N. Aggregation plots of H3K27me3 ChIP-seq signals near the TSS of genes 962 upregulated, downregulated, or unchanged between WT and EEDCKO. O. H3K27me3 963 enrichment was measured by ChIP-qPCR on target genes using adult cardiomyocytes isolated 964 from WT and EEDCKO hearts. *, P<0.05; **, P<0.01; ***, P<0.001 by ANOVA with Dunnett’s 965 post-hoc test using Eedfl/+::Myh6-Cre– as the control group (A) , by Welch’s t-test 966 (B,D,F,N,O), or by Wilcoxon-Mann-Whitney test (M).
967
- 33 - 968 Figure 2 -- figure supplement 1. H3K27ac ChIP-qPCR validation. H3K27ac enrichment 969 was measured by ChIP-qPCR on target genes using adult cardiomyocytes isolated from WT 970 and EEDCKO hearts. *, P<0.05; **, P<0.01; ***, P<0.001 by Welch’s t-test.
971
972 Figure 3 -- figure supplement 1. AAV9-EED rescue of EEDCKO mice. A. Schematic of 973 AAV9 constructs expressing Flag-tagged EED (Flag-EED), GFP or luciferase. ITR, Inverted 974 Terminal Repeart. PTnnt2, cardiac specific troponin T promoter. B. AAV9-EED expression of 975 Flag-EED. Mice were treated with AAV9-EED at P5 or P25. Heart extracts were prepared at 2 976 months of age. WT, untreated wild-type mice. C. Brightfield and GFP fluorescent signals in 977 hearts of mice injected with AAV9-GFP at P5 or P25 and harvested at 2 months of age. Bar = 978 500 µm. D. qRTPCR was performed to validate the expression of the indicated genes in 979 cardiomyocytes from 2 month-old mice treated with AAV9-luc or AAV9-EED at P14 and P25.
980
981 Figure 4 -- figure supplement 1. HDAC-EED interaction. A. RNA-seq data showing that 982 HDAC transcript levels did not change in 2 month old EedCKO cardiomyocytes. DE, 983 Differential Expression; NS, not significant. B. Immunoblotting for HDAC1, 2, and 5 proteins in 984 adult cardiomyocytes isolated from hearts of control (WT) and EedCKO (CKO). C. HA-EED, 985 pulled down with HA antibody, co-immunoprecipitated HDAC4/5/6/7/9. HA-EED and HDAC4-9 986 were co-transfected into 293T cells and harvested for immunoprecipitation assay 48 hours 987 after transfection. Arrowheads indicate full-length HDAC proteins. D. Flag-HDAC4/5/6/7/9, 988 pulled down with Flag antibody, co-immunoprecipitated HA-EED. Arrowheads indicate full- 989 length HDAC proteins. E. HDAC5 occupancy measurement by ChIP-qPCR. HDAC5 990 occupancy of the indicated chromatin regions in isolated adult CMs from WT and EedCKO 991 mice at 2 months of age. Occupancy was measured by ChIP followed by quantative PCR 992 (ChIP-qPCR). Chromatin regions are named by the adjacent gene and the distance to the 993 transcriptional start site (TSS).
994
995 Figure 4 -- figure supplement 2. Validation of HDAC2 and EED proteins purity and 996 dCas9-EED interaction with EZH2. Coommassie brilliant blue staining of recombinant 997 purified HDAC2 and EED proteins. The lot # specific staining gel image was proided by the 998 manufacturer, Cayman Chemical.
- 34 -
999
1000 Figure 5 -- figure supplement 1. Effect of over-expression of HDAC1/2 on genome- 1001 wide H3K27ac and H3K27me3 accumulation at H3K27ac peaks with increased signal in 1002 EedCKO. A. Schematic of AAV9 construct expressing Flag-tagged HDAC1/2 (Flag-HDAC1/2). 1003 ITR, Inverted Terminal Repeart. B. Validation of Flag tagged HDAC1 and HDAC2 protein 1004 expression in AAV-treated hearts. Mice were treated with AAV9-Flag-HDAC1/Flag-HDAC2 or 1005 AAV9-Flag-EED at P3. Hearts were analyzed at 2 months of age. C. Validation of ectopic and 1006 endogenous expression of HDAC1 and HDAC2 proteins in AAV-treated hearts of mice at 2 1007 months of age. D-F. Analysis of H3K27ac and H3K27me3 ChIP signals around all H3K27ac 1008 peaks gained in CKO, in het CKO_luc, and CKO_HDAC1/2. Heatmap (D) was sorted by 1009 descending value of the ratio of H3K27ac signal in CKO_luc to het. E, aggregate plots of 1010 H3K27ac and H3K27me3 ChIP signals. F, scatterplot of signal ratios comparing change in 1011 EED loss of function to change in HDAC1/2 rescue. Signals are from the peak center ± 0.5 kb. 1012 G. Immunoblotting and quantification of H3K27ac and H3K27me3 levels in adult 1013 cardiomyocytes isolated from hearts of three groups. H3K27ac or H3K27me3 immunoblot 1014 signals were normalized to total H3. NS, not significantly different.
1015
1016 LEGENDS FOR THE SUPPLEMENTARY FILES
1017 Supplementary file 1. Tables for differential peaks for H3K27me3 and H3K27ac, and 1018 differentially expressed genes in WT and CKO.
1019 Supplementary file 2. The list of siRNAs, antibodies information and PCR primers. A. 1020 The sequence of the TriFECTa Dicer-Substrate siRNAs (DsiRNAs). B. Antibodies information. 1021 C. The list of PCR primers.
1022 Supplementary file 3. The list of Next-generation sequencing data.
1023
- 35 - Ai_Fig. 1.
WT CKO A 1.5 C J RNA TSS H3K27me3 TSS EED WT CKO P0 WT CKO WT CKO WT 1.0 NS EED WT CKO Tpm2
P0 60 Myl4 0.5 Nppb C1 Ankrd1 GAPDH 4 4 *** 0 40 Myl9 1.5 Mybpc1 WT CKO Nppa C2
P5 FS (%) 20 Tnni1 * Rel EED Protein 1.0 Myh7 *** P5 8 9 Mybpc2 EED 0 0.5 Acta1 C3 4 4 Myl7 GAPDH Tnnt1 0 Tbx15 n=50 WT CKO B 100 D WT 15 80 CKO ** 60 10 40 n=46 -5k 5k
20 5 -3 +3 0 3 0 3 Percent survival 0 8 9 K organ morphogenesis 4 6 8 10 12 HW/BW Ratio (mg/g) 0 Age (weeks) E F skeletal system development extracellular matrix organization WT CKO WT CKO regulation of anatomical structure morphogenesis
central nervous system development
muscle organ development
0 5 10 15 20 25 30 35 -log10 (P-value) 1 kb genomic G H intervals I L 3 *** WT CKO WT CKO H3K27me3 Peaks *** 2 WT CKO 20 NS H3K27me3 15 1 44982 5009 2086 10 2 7 m e 3 signals NS 3 K 5 GAPDH H 0 0 WT CKO H3K27me3 ChIP signals C1 C2 C3 P0 1.2 NS 1.0 0.8 0.6 0.4
mRNA 0.2 0 WT P5 CKO 1.2 1.0 0.8 Relative Eed 0.6 *** 0.4 0.2 0
Figure 1 -- figure supplement 1. Eed depletion in WT and EedCKO mice. Heart apexes were harvested for qRT-qPCR for relative Eed mRNA expression on postnatalday 0 (P0) and 5 (P5). Heart apex contains both cardiomyocytes and non-cardiomyocytes. The non-myocytes likely account for the detected level of Eed mRNA in EED-CKO. P-value by Student’s t-test. ***, P<0.001. NS, not significant. A B C D WT CKO WT CKO 5.0 0.60 12 0.20 *** 4.0 * *** 0.54 9 *** 0.15 3.0 0.30 ** *** 0.10 2.0 Expression 6 ** *** 0.15 0.05 1.0 3 Fibrosis (%) Nppa WT CKO 0.00 0 0 0
Rel 2w 4w TNNI3 WGA DAPI 4.0 0.09 Age E F 0.08 500 *** *** 0.07 )
3.0 2 ** 0.06 400 * 0.05 fl/+ _ 2.0 0.04 * Eed ::Myh6-Cre 0.03 Eedfl/+::Myh6-Cre+ 300 1.0 0.02 fl/fl _ Eed ::Myh6-Cre 200 0.01 fl/fl + 0 Eed ::Myh6-Cre 0 CM area (µm 2w 4w 8w 2w 4w 8w 100 WT CKO 0
G WT CKO H I fl/fl
Marker Eed CKO
WT TNNI3 H3K27me3 453 bp Excised CKO 233 bp Floxed Eed exon 6 5 4 3 DAPI TNNI3 H3K27me3 J K 10 10 K27me3 K27me3 K27ac K27ac Myh6 Vim L M O 20 50 WT P=5.25e-30 40 15 CKO 30 10 WT CKO 20 50 n.s. n.s. 5 10 n.s. 40 of EED target genes EED Fold Enrichment 0 0 H3K27me3 at TSS ± 500 bp H3K27me3 at weak strong 30 apd h 800 0 WT H3K37me3 at TSS G n2a -ts s Six1-ts s Eya 1-tss 15 + 20 Acta1-tss x b Tbx15+30 0 Cd k T Intergenic ctr l 10 H3K27me3 Fold Enrichment N H3K27me3; WT H3K27me3; CKO Peak threshold 0 Upregulated Unchanged Downregulated (863 Genes) (20081 Genes) (392 Genes) 2.0 2.0 2.0 n2a -ts s
p=0.0794 p=2.7E-77 p=9.6E-121 Acta1-tss
1.5 1.5 1.5 Cd k Tbx15+30 0 Intergenic ctr l 1.0 1.0 1.0
0.5 0.5 0.5 ChIP signals TSS regioins
0.0 0.0 0.0 -5 -2.5 TSS 2.5 5 -5 -2.5 TSS 2.5 5 -5 -2.5 TSS 2.5 5 Figure 1 -- figure supplement 2. Characterization of EedCKO mice. A. Progressive cardiac dysfunction and dilatation after cardiomyocyte-restricted ablation of Eed. w, weeks. B. Nppa mRNA level in WT and CKO hearts at the indicated ages. w, weeks. C, D. Cardiac fibrosis was evident by Mason Trichrome staining at 2 months of age. Fraction of myocardial area occupied by fibrotic tissue (blue staining) was quantified using ImageJ. Bar = 50 µm. E, F. Immunofluorescence for cardiomyocyte marker TNNI3 and cardiomyocyte membrane marker WGA, and quantifi- cation of cell size from WGA-stained cardiomyocyte outlines (f). Bar= 50 µm. G. Immunostaining for TNNI3 and H3K27me3. Isolated adult cardiomyocytes were > 95% pure and EedCKO CMs had little H3K27me3 signal. Bar = 50 µm. H. PCR of genomic DNA from purified CMs using primers that amplify unexcised floxed DNA (233 bp product) or Cre-excised DNA (453 bp product). In CKO-purified CMs, unexcised floxed DNA was not detected, consistent with highly efficient Cre-mediated gene inactivation, as well as high purity of dissociated CMs. I. RNA-seq track view showing deletion of floxed exons 3-6 of Eed (red box). J,K. Genome browser view ofH3K27me3 and H3K27ac ChIP-seq signals on Myh6 (J) and Vim (K) loci in purified adult cardiomyocytes. L. EED enrichment on downstream genes was measured by ChIP-qPCR in P5 heart ventricle apex. Numbers following gene names indicate the number of nucleotides between the probed amplicon and the TSS. M. Box and scatter plots of H3K27me3 at TSS ± 500 bp of EED target genes in four quantiles of WT H3K27me3 intensity. N. Aggregation plots of H3K27me3 ChIP-seq signals near the TSS of genes upregulated, downregulated, or unchanged between WT and EEDCKO. O. H3K27me3 enrichment was measured by ChIP-qPCR on target genes using adult cardiomyocytes isolated from WT and EEDCKO hearts. *, P<0.05; **, P<0.01; ***, P<0.001 by ANOVA with Dunnett’s post-hoc test using Eedfl/+::Myh6-Cre– as the control group (A) , by Welch’s t-test (B,D,F,N,O), or by Wilcoxon-Mann-Whitney test (M). Ai_Fig. 2 A E CKO WT EED WT EEDCKO TSS regions Upregulated Unchanged Downregulated 5 H3K9ac (863 Genes) (20081 Genes) (392 Genes) 5 H3 ** p=6.9E-26 p=0.8804 p=1.3E-13 4 4 3 H3K14ac 2 H3 3 1 ** ** ChIP signals p=0.074 0 H4ac -5 -2.5TSS 2.5 5 -5 -2.5TSS 2.5 5 -5 -2.5 TSS2.5 5 2 distance from TSS (kb) H3 Enhancer regions Upregulated Unchanged Downregulated
Relative proteins level 1 H3K27ac (863 Genes) (20081 Genes) (392 Genes) 5 H3 p=4.5E-21 p=0.0775 p=3.7E-102 4 0 H3K14acH3K27ac H3K9ac H4ac 3 2 B 1.2 6 1 si-NC si-Eed ChIP signals 0 1.0 5 *** EED -5 -2.5 0 2.5 5 -5 -2.5 0 2.5 5 -5 -2.5 0 2.5 5 * distance from peak center (kb) 0.8 4 GAPDH H3K27ac; WT 0.6 3 F H3K27ac ** H3K27ac; CKO 0.4 TSS H3K27ac H3K27me3 Relative EED 2
Relative H3K27ac WT CKO GAPDH 0.2 1 G 4 4 4 4 C1 0 0 C D si-NC si-Eed si-NCsi-Eed WT CKO *** genomic 1 kb intervals C2 *** *** ***
H3K27ac Peaks UP 5 5
1.5 *** genes WT CKO 4 4 C3 3 3 1.0 2 2 1 1
5950 20799 8161 0.5 H3K27ac ChIP signals 0 0 H3K27ac Signal genes
DOWN C1 C2 C3 UP genes DOWN 0 -5k 5k genes WT CKO 0 3 WT CKO 30 ** 25
20
15
10 * * ** ** ** ** ** ** ** ** ** * 5 H3K27ac Fold Enrichment 0 Six1-tss Myl7-tss Myh7-tss Tnni1-tss Acta1-tss Nppa-100 Nppa-500 Myh7-668 Nppa+300 Myh7+200 Myh7+408 Tnnt1+200 Nppa-1000 Myh7-9374 Myh7-9974 Tbx15+300 Myh7+3377 Myh7-10027 Intergenic ctrl
Figure 2 -- figure supplement 1. H3K27ac ChIP-qPCR validation. H3K27ac enrichment was measured by ChIP-qPCR on target genes using adult cardiomyocytes isolated from WT and EEDCKO hearts. *, P<0.05; **, P<0.01; ***, P<0.001 by Welch’s t-test. Ai_Fig. 3.
A B C D
Myh7 Acta1 60 30 16 *** *** *** *** 50 *** 14 *** Het CKO-luc CKO-EED 25 *** 40 12 CKO-EED-P14 CKO-ED-P25 Eed-Het CKO-luc 20 10 30 15 8 P14 AAV P5 FS % 20 6
Rel mRNA Exp 10 6 4 6 6 6 4 6 6 P14 4 10 6 4 6 6 5 2
P25 P25 AAV 0 0 0 Het Het Het 2 mo Echo CKO-luc-P14 CKO-luc-P14 CKO-luc-P14 qRTPCR CKO-EED-P1CKO-EED-P24 5 CKO-EED-P1CKO-EED-P24 5 CKO-EED-P1CKO-EED-P24 5 Molecular studies E P14 treatment P25 treatment Flag (EED) H3K27me3 H3K27ac Flag (EED) H3K27me3 H3K27ac 1.5 4 1.5 8 NS NS * 3 H3K27me3 ** 6 H3K27me3 1.0 1.0 2 4 H3K27ac 0.5 H3K27ac 0.5 3 3 3 Rel Level 1 Rel Level 2 3 3 3 3 3 3 3 3 3 0 0 0 0 H3 H3 Het Het Het Het
Het Het CKO-luc-P14 CKO-luc-P14 CKO-luc-P25 CKO-luc-P25 CKO-EED-P14 CKO-EED-P14 CKO-EED-P25 CKO-EED-P25
CKO-luc-P14 CKO-luc-P25 CKO-EED-P14 CKO-EED-P25 F G TSS ± 500 bp of 863 UP genes TSS ± 500 bp of 863 UP genes
EED-P14CKO-luc EED-P14EED-25 CKO-lucEED-P14EED-25 CKO-luc 10.0 20 RNA TSS H3K27me3 TSS H3K27ac *** 7.5 *** 15 NS C1 NS 10 5.0 C2 5 2.5
863 UP genes in CKO 0 C3 H3K27ac ChIP signal (ChIP-Input) 0.0 H3K27me3 ChIP signal (ChIP-Input)
-5k 5k
-3 +3 0 3 0 3 CKO-luc EED-P14 EED-P25 CKO-luc EED-P14 EED-P25 H
15 kb 15 kb 30 20 CKO-luc CKO-luc 30 20 EED-P14 EED-P14 EED-P25 30 EED-P25 20 H3K27me3 H3K27me3 CKO-luc 10 CKO-luc 10
EED-P14 10 EED-P14 10 10 10
H3K27ac EED-P25 H3K27ac EED-P25
Myl7 Acta1 P FLAG-EED A Tnnt2 B P5 2 mon P25 2 mon AAV9-EED AAV-EED WT AAV-EED ITR ITR P GFP or luciferase Flag Tnnt2 AAV9-GFP AAV9-luciferase GAPDH ITR ITR C P5 2 mon P25 2 mon
D Slc9a3 Tnnt1 Tbx15 Nppa Tnni1
*** 30 * 100 ** 40 15 *** 15 ** *** ** 25 ** 80 *** *** 20 30 10 10 60 15 20 40 10 5 5 10 Rel mRNA Exp 20 5 0 0 0 0 0
Het Het Het Het Het
CKO-lucEED-P1EED-P24 5 C`KO-lucEED-P1EED-P24 5 CKO-lucEED-P1EED-P24 5 CKO-lucEED-P1EED-P24 5 CKO-lucEED-P1EED-P24 5
Figure 3 -- figure supplement 1. AAV9-EED rescue of EEDCKO mice. A. Schematic of AAV9 constructs expressing Flag-tagged EED (Flag-EED), GFP or luciferase. ITR, Inverted Termi-
nal Repeart. PTnnt2, cardiac specific troponin T promoter. B. AAV9-EED expression of Flag-EED. Mice were treated with AAV9-EED at P5 or P25. Heart extracts were prepared at 2 months of age. WT, untreated wild-type mice. C. Brightfield and GFP fluorescent signals in hearts of mice injected with AAV9-GFP at P5 or P25 and harvested at 2 months of age. Bar = 500 µm. D. qRTPCR was performed to validate the expression of the indicated genes in cardiomyocytes from 2 month-old mice treated with AAV9-luc or AAV9-EED at P14 and P25. G F C A HDAC2 ChIP signals 0 1 2 3 4 5 Intergenic ctrl
-5 HDAC1 HDAC2 Enrichment IP: HA-EED 0 2 4 6 8 + HA-EED Chromatin Occupancy 4583 4042 EED HDAC2 CKO HDAC2 WT HDAC2 -2.5 Cdkn2a-tss Upregulated (863 Genes)
NS HDAC3 WT TSS Acta1-tss in WT pcDNA3.1 NS HDAC
2.5 Six1-tss 5827 Tbx15+300 NS
2 HDAC1 5% Input + HA-EED
T NS
5 b x CKO 15+ HDAC2
8000 NS 0 1 2 3 4 5
-5 HDAC3 D Kb fromTSSofgenes pcDNA3.1 -2.5 (20081 Genes) HA (EED) Flag (HDAC) Unchanged EED peaks (8625) TSS -5k 5k EED 2.5 H3K27 me3 IP: Flag-HDAC 5 HDAC3 + HA-EED H3K27 ac 0 1 2 3 4 5 HDAC2 -5 HDAC2
WT HDAC1 -2.5 Downregulated (392 Genes) HDAC2 CKO + HA-EED
HDAC3 5% Input TSS
HDAC2 1722 E3 1830 E2 5073 E1 2.5 HDAC1 0 123 GAPDH Flag (HDAC) HA (EED) 5 E
H HDAC2 ChIP signals 0 1 2 3 4
HDAC2 activity (a.u.) distance fromEEDpeakcenter(kb) -5 B 0 0 0 0 0 0 HDAC2 CKO HDAC2 WT ...... 0 1 2 3 4 5 6 * * EED peaks(8625) -2.5 EED only IP: IgG BSA HDAC2 0 IP: EED * EED (ng) 2.5
* 5% Input * EED HDAC2 5 Ai_Fig. 4. A B WT CKO DE WT CKO Hdac9 NS Hdac8 NS HDAC5 Hdac7 NS Hdac6 NS HDAC2 Hdac5 NS Hdac4 NS HDAC1 Hdac3 NS GAPDH Hdac2 NS Hdac1 NS 2 6 log2 (TPM)
C IP: HA-EED 5% Input + HA-EED + HA-EED pcDNA3.1 HDAC9 HDAC8 HDAC7 HDAC6 HDAC5 HDAC4 pcDNA3.1 HDAC9 HDAC8 HDAC7 HDAC6 HDAC5 HDAC4
Flag-HDACs
HA
D IP: Flag-HDACs 5% Input + HA-EED + HA-EED pcDNA3.1 HDAC9 HDAC8 HDAC7 HDAC6 HDAC5 HDAC4 pcDNA3.1 HDAC9 HDAC8 HDAC7 HDAC6 HDAC5 HDAC4
HA-EED
Flag-HDACs
GAPDH
E WT CKO NS NS 40
30 NS nrichmen t
E 20 NS NS
10 HDAC5 0 Six1-ts s Acta1-ts s Tbx15+30 0 Cdkn2a-tss Tbx1 5+8000 Intergenic ctr l Figure 4 -- figure supplement 1. HDAC-EED interaction. A. RNA-seq data showing that HDAC transcript levels did not change in 2 month old EedCKO cardiomyocytes. DE, Differential Expression; NS, not significant. B. Immunoblotting for HDAC1, 2, and 5 proteins in adult cardiomyocytes isolated from hearts of control (WT) and EedCKO (CKO). C. HA-EED, pulled down with HA antibody, co-immunoprecipitated HDAC4/5/6/7/9. HA-EED and HDAC4-9 were co-transfected into 293T cells and harvested for immunoprecipitation assay 48 hours after transfection. Arrow- heads indicate full-length HDAC proteins. D. Flag-HDAC4/5/6/7/9, pulled down with Flag antibody, co-immunoprecipitated HA-EED. Arrowheads indicate full-length HDAC proteins. E. HDAC5 occupancy measurement by ChIP-qPCR. HDAC5 occupancy of the indicated chromatin regions in isolated adult CMs from WT and EedCKO mice at 2 months of age. Occupancy was measured by ChIP followed by quantative PCR (ChIP-qPCR). Chromatin regions are named by the adjacent gene and the distance to the transcriptional start site (TSS). HDAC2 protein EED protein kD kD 2µg 5µg 10µg 200ng 250 250 150 150 100 75 EED 100 75 50
HDAC2 39 50 25 39
25 Provided by Cayman Chemical
Figure 4 -- figure supplement 2. Validation of HDAC2 and EED proteins purity and dCas9-EED interaction with EZH2. Coommassie brilliant blue staining of recombinant purified HDAC2 and EED proteins. The lot # specific staining gel image was provided by the manufacturer, Cayman Chemical. Ai_Fig. 5.
CKO + luc CKO + HDAC1/2 Het
A C 10 D P1 P3 P6 2 mon Acta1 Myh7 Nppa ** 25 Assay 8 ** EED 20 Het 6 ** 15 ** CKO 4 10 3 3 4 AAV-HDAC1/2 2 Rel mRNA level HW / BW (mg/g) 5 3 3 4 3 3 4 3 3 4 B 0 0 CKO+luc 3 *** CKO+HDAC1/2 3 Het 4
0 10 20 30 40 50 60 FS %
E F Het CKO + HDAC1/2 CKO + luc RNA TSS H3K27me3 TSS H3K27ac CKO CKO CKO CKO CKO CKO * * * Het Het Het 20 HDAC1/2 luc HDAC1/2 luc HDAC1/2 luc 15
C1 10
5 H3K27ac ChIP signals 0 C2 NS NS 8 *
6
4 863 genes upregulated in CKO C3 2
0 H3K27me3 ChIP signals -5k TSS 5k C1 C2 C3 -1.0 0 1.0 0 1 2 3 0 1 2 3 G 20 5
15 4 CKO + luc 3 10 CKO + HDAC1/2 2 Het 5 1 0 0
H3K27ac ChIP signals -5 -2.5 TSS 2.5 5 H3K27me3 ChIP signals -5 -2.5 TSS 2.5 5 distance from TSS (kb) distance from TSS (kb) P Flag-HDAC1 Flag-HDAC2 A Tnnt2 B Flag-HDAC1/2 WT AAV9-HDAC1/2 Flag-HDAC1 ITR P2A ITR Flag Flag-HDAC2 β-tublin C Flag-HDAC1/2 WT Flag-HDAC1/2 WT
HDAC1 Flag-HDAC1 HDAC2 Flag-HDAC2 β-tublin HDAC1 β-tublin HDAC2
H3K27ac H3K27me3 all H3K27ac peaks all H3K27ac peaks D E increased in CKO F increased in CKO CKO CKO CKO CKO Het Het 20 2 HDAC1/2 luc HDAC1/2 luc CKO + luc CKO + HDAC1/2 15 Het 1
10 0
-1
5 (CKO_luc/Het) log2 H3K27ac ratio H3K27ac ChIP signals 0 -2 -5 -2.5 0 2.5 5 -2 -1 0 1 2 log2 H3K27ac ratio (CKO_luc/CKO_HDAC1/2)
5 2 4 1 3 0 2 all H3K27ac peaks increased in CKO
(CKO_luc/Het) -1 1 log2 H3K27me3 ratio H3K27me3 ChIP signals 0 -2 -5 -2.5 0 2.5 5 -2 -1 0 1 2 -5k TSS 5k distance from peak center (kb) log2 H3K27me3 ratio 0 1 2 3 0 1 2 3 (CKO_luc/CKO_HDAC1/2)
CKO_luc CKO_HDAC1/2 Het G NS Flag-HDAC1/2 1.5
GAPDH
1.0 Het H3K27me3 CKO + HDAC1/2 NS CKO + luc H3 0.5
H3K27ac Relative Level, Normalized to H3 0 H3 H3K27ac H3K27me3
Figure 5 -- figure supplement 1. Effect of over-expression of HDAC1/2 on genome-wide H3K27ac and H3K27me3 accumulation at H3K27ac peaks with increased signal in EedCKO. A. Schematic of AAV9 construct expressing Flag-tagged HDAC1/2 (Flag-HDAC1/2). ITR, Inverted Terminal Repeart. B. Validation of Flag tagged HDAC1 and HDAC2 protein expression in AAV-treated hearts. Mice were treated with AAV9-Flag-HDAC1/Flag-HDAC2 or AAV9-Flag-EED at P3. Hearts were analyzed at 2 months of age. C. Validation of ectopic and endogenous expression of HDAC1 and HDAC2 proteins in AAV-treated hearts of mice at 2 months of age. D-F. Analysis of H3K27ac and H3K27me3 ChIP signals around all H3K27ac peaks gained in CKO, in het CKO_luc, and CKO_HDAC1/2. Heatmap (D) was sorted by descending value of the ratio of H3K27ac signal in CKO_luc to het. E, aggregate plots of H3K27ac and H3K27me3 ChIP signals. F, scatterplot of signal ratios comparing change in EED loss of function to change in HDAC1/2 rescue. Signals are from the peak center ± 0.5 kb. G. Immunoblotting and quantification of H3K27ac and H3K27me3 levels in adult cardiomyocytes isolated from hearts of three groups. H3K27ac or H3K27me3 immunoblot signals were normalized to total H3. NS, not significantly different. Ai_Fig. 6.
A B AAV9-EED or AAV9-Luc 70 60 P=0.07 P=1 P1 P6 SAHA 2 mon 50 or DMSO 40 Assay EED FS % 30 Het 20 CKO 10 3 3 3 3 0 C 5 5 ** Rx * geno AAV9 4 4 CKO EED DMSO 3 3 CKO EED SAHA NS 2 2 NS Het luc DMSO Myl9 mRNA
Acta1 mRNA Het luc SAHA 1 1
0 0 1.5 * NS 30 150 ** 1.2 NS 20 100 0.9 0.6 Tnni1 mRNA 10 Tbx15 mRNA NS 50 Mybpc2 mRNA 0.3 NS 0 0 0 Ai_Fig. 7.
WT EEDCKO
HDAC EED HDAC X Cluster 3 H3K27me3HIGH:H3K27acNO HDAC
Cluster 1
HDAC HDAC EED X H3K27me3NO:H3K27acLOW Cluster 2
H3K27ac HDAC with high activity H3K27me3 HDAC with low activity