Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 on its heterochromatin assembly
Heterochromatin , a highly condensed chromatin structure , can be divided into two classes: invariably constitutive heterochromatin ( CH ) , and developmentally controlled facultative heterochromatin . CH generally consists of repetitive DNA and is transcriptionally silent , and its higher-order chromatin structure can spread , causing the heritable inactivation of genes adjacent to the CH ( Grewal and Elgin , 2002; Grewal and Moazed , 2003; Richards and Elgin , 2002 ) . The establishment of CH is tightly correlated with changes in posttranslational histone-tail modifications . The trimethylation of histone H3 lysine 9 ( H3K9me3 ) , which is a hallmark of CH , creates specific binding sites for H3K9me3 reader molecules such as HP1 proteins ( Bannister et al . , 2001; Lachner and Jenuwein , 2002; Lachner et al . , 2001; Nakayama et al . , 2001 ) , and these epigenetic states are maintained throughout replication ( Audergon et al . , 2015; Margueron and Reinberg , 2010; Ragunathan et al . , 2015 ) . CH is critical for functional chromosomal domains such as the pericentromere , and epigenetic pericentromere layers are important for accurate chromosome segregation in mitosis and for genome stability ( Allshire et al . , 1995; Peters et al . , 2001 ) . SUV39H ( 1 and 2 ) , an H3K9me3 methyltransferase in mammals ( Rea et al . , 2000 ) , targets CH domains and intact transposons ( Bulut-Karslioglu et al . , 2014 ) . H3K9me3 in the pericentric regions is mainly catalyzed by SUV39H ( Peters et al . , 2003 ) . Therefore , in Suv39h1/2 double-knockout ( Suv39h dn ) cells , the H3K9me3 levels over the pericentromere are severely diminished and HP1 proteins do not accumulate ( Lachner et al . , 2001 ) . The nature of CH and its epigenetic machineries are well conserved in various species , including fission yeast and flies ( Elgin and Reuter , 2013; Grewal and Jia , 2007 ) . In fission yeast Schizosaccharomyces pombe ( S . pombe ) , the RNA interference ( RNAi ) -dependent pathway is essential for pericentric heterochromatin formation and maintenance , and the process of pericentric transcription is coupled with the targeting of the SUV39H homologue Clr4 to chromatin ( Castel and Martienssen , 2013; Goto and Nakayama , 2012; Grewal and Jia , 2007; Moazed , 2009 ) . In the Drosophila ovary , a similar small-RNA-mediated process promotes transcriptional silencing by H3K9me3 and by HP1A , an HP1-family protein in flies ( Castel and Martienssen , 2013 ) . However , it is not clear whether the RNAi-dependent pathway ( or another RNA-based mechanism ) is involved in the process of establishing and maintaining heterochromatin in mammals , such as in creating target specificities for SUV39H and SUV39H-mediated H3K9me3 formation . Suv39h-family proteins have two functionally distinct domains , the N-terminal chromodomain ( CD ) and the C-terminal SET domain . The CD functions as a binding module that targets H3K9me3 marks , and the SET domain is responsible for SUV39H’s enzymatic activity . Since the N-terminal CD of Clr4 is required for the efficient spread and stable inheritance of H3K9me3 marks ( Al-Sady et al . , 2013; Noma et al . , 2004; Ragunathan et al . , 2015; Zhang et al . , 2008 ) , it has been suggested that Suv39/Clr4 uses a direct read-write mechanism to maintain the H3K9me3 marks on CH . However , this mechanism alone is not sufficient for converting H3K9me3-free , naïve chromosomal regions to silent heterochromatin . Here we demonstrated that Suv39h1-CD can bind nucleic acids , and that this binding is crucial for Suv39h1’s function . Suv39h-CD prefers to bind ssRNA rather than dsDNA , and Suv39h-CD’s nucleic acid binding is independent of its H3K9me3 recognition . Mutational analyses revealed that both the nucleic acid- and H3K9me3-binding activities of Suv39h1 are important for its induction of heterochromatin assembly . We also showed that Suv39h1 bound major satellite RNA in vivo in a manner dependent on its nucleic acid-binding activity and that knockdown of major satellite RNAs lowered Suv39h1 retention on pericentric heterochromatin . Our data suggest that chromatin-bound RNAs contribute to the targeting and retention of Suv39h1 at specific chromosomal regions .
We recently reported that Chp1 , a CD protein functioning in the RNAi pathway in fission yeast , binds nucleic acid via its CD , and that this activity is required for heterochromatin assembly ( Ishida et al . , 2012 ) . Experiments in the same study revealed that Clr4 , the fission yeast homologue of SUV39H , also binds nucleic acid via its CD , although the importance of Clr4-CD’s nucleic acid binding in heterochromatin assembly remains elusive . Here , to investigate whether Suv39h1 also binds nucleic acids , we produced a GST-fusion protein containing Suv39h1-CD ( residues 39–105 ) ( Figure 1A , B , GST-Suv39h1-CD ) and conducted electrophoretic mobility-shift assays ( EMSAs ) using in vitro transcribed , single-stranded 130-nt major satellite RNA ( ssRNA ) as a probe . GST-fusion proteins containing fission yeast Chp1-CD ( GST-Chp1-CD ) or GST alone were used as controls ( Ishida et al . , 2012 ) . As shown in Figure 1C , GST-Suv39h1-CD bound ssRNA robustly and far more efficiently than did GST-Chp1-CD , although how these CDs activities contribute to the overall RNA-binding efficiency of full-length proteins remains elusive . Clr4-CD’s binding of nucleic acids was detected only when K9-methylated H3 peptide was added to the EMSA ( Ishida et al . , 2012 ) ; thus , although evolutionarily conserved roles need to be considered , nature of Suv39h1-CD’s binding of nucleic acid appeared to be distinct from that of Clr4-CD . 10 . 7554/eLife . 25317 . 003Figure 1 . Suv39h1-CD can bind nucleic acids . ( A ) Schematic of full-length Suv39h1 , GST-fused Suv39h1-CD ( 39–105 ) , and S . pombe Chp1-CD ( 20–75 ) . ( B ) The recombinant GST-fused proteins used in ( C ) ; these proteins were visualized by CBB staining . ( C ) An EMSA using GST-fused proteins . 8 μM of GST or GST-fusion was used in one assay . Fluorescein-labeled 130-nt major satellite ssRNA was used as a probe . ( D and E ) Titration EMSAs using GST-Suv39h1-CD ( 0–8 μM with 0 . 5-fold dilutions ) incubated with ( D ) 130-nt ssRNA or ( E ) 130 bp dsDNA . DNA probe was detected as doublet bands in gels , because the number of the fluorescent dye , which was conjugated at the single or both 5’-ends , could slightly affect the migration . ( F ) The binding isotherm of Suv39h1-CD to ssRNA and dsDNA; plots were calculated from the unbound fractions . ( G ) The dissociation constants measured by titration ssRNA and dsDNA EMSA experiments ( D and E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 00310 . 7554/eLife . 25317 . 004Figure 1—figure supplement 1 . Characteristics of Suv39h1-full-length and -CD’s RNA binding . ( A ) , ( B ) , ( D ) , ( E ) , and G–I ) EMSAs were conducted with GST-fused Suv39h1-CD ( 39–105 ) and the following probes: ( A ) 130-nt alfa satellite ssRNA , ( B ) 130-nt ACTB mRNA , ( D ) 74-nt major satellite ssRNA , ( E ) 189-nt major satellite ssRNA , ( G ) 60-nt ( UUAGGG ) x10 ssRNA , ( H ) 74-nt major satellite DNA/RNA hybrid , or ( I ) 130-nt major satellite dsRNA . ( C and F ) The binding isotherms of Suv39h1-CD to various ssRNA probes . ( J ) MBP-fusion proteins used in ( K–N ) were resolved by SDS-PAGE and visualized by Coomassie staining . ( K–N ) EMSAs were conducted with MBP ( K ) , MBP-fused Suv39h1-CD ( 39-105 ) ( L ) , MBP-fused full-length Suv39h1-WT ( M ) or 4A ( N ) using 130-nt major satellite ssRNA as the probe . ( O ) The binding isotherms of MBP-fused Suv39h1 proteins to 130-nt major satellite ssRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 004 We previously showed that fission yeast Chp1-CD and Clr4-CD bind both ssRNA and double-stranded ( ds ) DNA ( Ishida et al . , 2012 ) . We tested whether Suv39h1-CD could also bind DNA by an EMSA using 130 bp major satellite dsDNA . Although Suv39h1-CD was able to bind dsDNA , titration analysis revealed that Suv39h1-CD had a lower binding affinity for dsDNA than for ssRNA ( Figure 1D–G; KD = 0 . 35 ± 0 . 02 μM for ssRNA and 0 . 93 ± 0 . 05 μM for dsDNA ) . Although nucleosomal DNA might contribute to Suv39h1’s chromatin targeting , the present study focused on Suv39h1-CD’s ssRNA-binding activity . It has been proposed that noncoding transcripts corresponding to satellite repeats are functionally linked with CH assembly and/or kinetochore formation ( Maison et al . , 2011; Rošić et al . , 2014 ) . To determine the type of sequences that are targeted by Suv39h1-CD , we prepared several different ssRNAs and subjected them to an RNA EMSA ( Figure 1—figure supplement 1 ) . Suv39h1-CD was able to bind both 130-nt alpha satellite ssRNA and 130-nt ACTB ORF ssRNA with a similar affinity as for 130-nt major satellite ssRNA ( Figure 1D , Figure 1—figure supplement 1A–C ) . Suv39h1-CD bound similarly to major satellite ssRNAs with a length of 74–189 nt ( Figure 1D , Figure 1—figure supplement 1D–F ) . Recently , Porro et al . reported that GST-SUV39H1 preferentially binds to telomeric-repeat ( ( UUAGGG ) 10 ) ssRNAs and that part of SUV39H1’s N terminus ( residues 1–106 ) is responsible for this RNA-binding activity ( Porro et al . , 2014 ) ; these findings are consistent with our results . However , we found that GST-Suv39h1-CD’s binding affinity for 60-nt ( UUAGGG ) 10 was similar to that of 74-nt major satellite ssRNAs ( Figure 1—figure supplement 1G ) . DNA–RNA hybrid formation is reported to play a role in heterochromatin assembly in fission yeast ( Nakama et al . , 2012 ) . In addition , transcripts of major satellite repeats are produced in both sense and antisense orientation ( Bulut-Karslioglu et al . , 2014 ) , implying existence of chromatin-associated double-stranded RNAs ( dsRNAs ) . Therefore , we investigated whether Suv39h1-CD has a higher binding affinity for DNA-RNA hybrid strands or dsRNA than for ssRNA . However , Suv39h1-CD bound major satellite RNA-DNA hybrid strands and dsRNA with similar affinities to that of ssRNA ( Figure 1—figure supplement 1D , H , I ) , indicating that RNA-DNA hybrid molecules or dsRNA do not have a unique role in this recognition . Taken together , these results suggested that Suv39h1-CD binds RNA without a major preference for particular structures or sequences . In the cases of fission yeast Chp1-CD and Clr4-CD , nucleic acid binding is coupled with H3K9me2/3 binding ( Ishida et al . , 2012 ) . Therefore , we next examined whether Suv39h1-CD’s RNA binding is linked with its H3K9me3 recognition . We first determined whether Suv39h1-CD shows a preference for methylated H3K9 peptide . As shown in Figure 2—figure supplement 1A , isothermal titration calorimetry ( ITC ) revealed that GST-Suv39h1-CD bound H3K9me3 peptide ( H3K9me3 ) with an affinity of KD = 26 µM , but no significant binding was observed for unmodified H3 peptide ( H3unmod ) ; this was consistent with previous observations ( Wang et al . , 2012 ) . Furthermore , a peptide pull-down assay demonstrated that GST-Suv39h1-CD had higher binding affinities for di- or trimethylated H3K9 peptide ( H3K9me0 < me1 << me2 ≦ me3 ) ( Figure 2C , WT ) . 10 . 7554/eLife . 25317 . 005Figure 2 . The residues required for Suv39h1-CD to bind RNA . ( A ) The alignment of wild-type ( WT ) and mutant Suv39h1-CD ( 39–105 ) . The upper lines indicate secondary structure . The positively charged amino acids in the C-terminal α-helix are indicated in red . The aromatic cage residues responsible for Suv39h1-CD’s recognition of H3K9me3 were indicated in green . ( B , F ) The recombinant proteins used in ( C–E and G–J ) ; the proteins were visualized by CBB staining . ( C ) An in vitro peptide-binding assay using wild-type ( WT ) and mutant ( Y67A or 4A ) Suv39h1-CDs . Biotin-tagged H3 ( 1–21 ) peptide ( 100 pmol ) was incubated with GST-Suv39h1-CD ( 1 pmol ) , and the pulled-down proteins were analyzed by western blotting with an anti-GST antibody . ( D , E , H–J ) Titration EMSAs using serially diluted GST-fused WT or mutant Suv39h1-CD . ( G ) An EMSA using GST-fused WT or mutant Suv39h1-CD . ( K ) Binding isotherms of WT and mutant Suv39h1-CD proteins for ssRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 00510 . 7554/eLife . 25317 . 006Figure 2—figure supplement 1 . Suv39h1-CD’s RNA binding is independent of its H3K9me3 recognition . ( A ) Isothermal titration calorimetry ( ITC ) showing the binding affinities of GST-Suv39h1-CD ( 39–96 ) for unmodified H3K9 , H3K9me1 , H3K9me2 , and H3K9me3 peptides . ( B ) EMSAs using GST-fused S . pombe Chp1-CD and Suv39h1-CD; GST-fused proteins were incubated with a 130-nt ssRNA probe in the presence or absence of equal molar quantities of unmodified and K9-trimethylated H3 peptides . ( C ) Binding isotherms of GST-Suv39h1-CD and ssRNA in the presence or absence of equal molar amounts of the indicated H3 peptides . ( D ) In vitro peptide-binding assay using WT Suv39h1-CD in the presence of major satellite ssRNA or dsDNA . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 006 To examine the relationship between Suv39h1-CD’s RNA binding and H3K9me3 binding , we introduced an amino acid substitution at Y67; this residue , along with F43 and W64 , is responsible for Suv39h1-CD’s recognition of H3K9me3 ( Wang et al . , 2012 ) ( Figure 2A , indicated in green ) . Although a Suv39h1-CD mutant with Y67A ( Suv39h1-CD-Y67A , Figure 2B ) retained residual binding activity for unmodified or K9-methylated H3 peptide , it lost its preference for di- and tri-methylated H3K9 ( Figure 2C , Y67A ) . An EMSA demonstrated that Suv39h1-CD-Y67A bound ssRNA with an affinity almost the same as that of wild-type Suv39h1-CD ( Figure 2D , E ) , indicating that Suv39h1-CD’s RNA binding was distinct from its H3K9me3 recognition . To confirm this possibility , we added H3K9me3 peptide to the EMSA; this addition enhanced Chp1-CD’s RNA binding ( Figure 2—figure supplement 1B ) as previously observed ( Ishida et al . , 2012 ) , but did not affect Suv39h1-CD’s RNA binding ( Figure 2—figure supplement 1B , C ) . These results suggested that Suv39h1-CD’s ability to bind RNA is an intrinsic property that is distinct from its ability to recognize H3K9me3 . Although addition of unmodified H3 peptide appeared to have a negative effect on the RNA-binding activity for both Chp1-CD and Suv39h1-CD ( Figure 2—figure supplement 1B ) , it was presumably due to an electrostatic interaction between the unmodified H3 peptide and RNA probe . We also investigated whether Suv39h1-CD’s nucleic acid binding influences its recognition of methylated H3K9 , even though its RNA binding was not affected by adding H3K9me3 peptide . We performed peptide pull-down assays in the presence or absence of major satellite ssRNA or dsDNA ( Figure 2—figure supplement 1D ) . The presence of these nucleic acids slightly repressed rather than enhanced the overall amount of co-pulled-down Suv39h1-CD , suggesting that Suv39h-CD’s recognition of methylated H3K9 is also independent of its nucleic acid binding . This slight reduction in Suv39h1-CD’s H3K9me3 binding was probably due to an electrostatic interaction between the H3 peptide and the ssRNA/dsDNA probe . Furthermore , we confirmed that Suv39h1-CD’s recognition of methylated H3K9 was not affected by the nucleic acid-binding defective mutation , 4A ( Figure 2C , see below ) . To understand the biological implications of Suv39h1-CD’s RNA binding , we next investigated the residues required for binding RNA . The CD consists of three β-sheets and a C-terminal α-helix ( Figure 2A ) ( Jacobs and Khorasanizadeh , 2002; Nielsen et al . , 2002; Wang et al . , 2012 ) . Our previous studies of fission yeast Chp1-CD and Clr4-CD demonstrated that a stretch of basic residues in the C-terminal α-helix is required for their RNA binding ( Ishida et al . , 2012 ) . Although Suv39h1-CD does not appear to have such a stretch of basic residues , it has a series of basic residues in its C-terminal α-helix ( Figure 2A , indicated in red ) . We introduced five different amino acid substitutions into Suv39h1-CD ( Figure 2A , F; mut1–4 and 4A ) and tested the ability of these mutants to bind RNA . In EMSAs , a Suv39h1-CD mutant ( mut1 ) with N-terminal distal amino acid substitutions bound RNA more weakly than wild-type Suv39h1-CD; the mutants with other substitutions almost completely lost the ability to bind RNA ( Figure 2G ) . Titration analyses confirmed that these basic residues were critical for Suv39h1-CD’s RNA binding ( Figure 2H–K ) . Our results demonstrated that the basic residues in Suv39h1-CD’s C-terminal α-helix are also involved in its RNA binding as those in Chp1-CD or Clr4-CD are . Fission yeast Chp1 binds to RNA via both its N-terminal CD and its central RNA recognition motif ( RRM ) , and the CD and RRM cooperate to repress centromeric transcripts ( Ishida et al . , 2012 ) . To determine how other Suv39h1 domains contribute to its CD’s RNA/DNA binding , we prepared Suv39h1-CD and full-length Suv39h1 as maltose binding protein ( MBP ) -fused versions ( Figure 1—figure supplement 1J ) , since we noticed that the solubility of MBP-Suv39h1 was better than that of GST-Suv39h1 ( data not shown ) . We first confirmed that MBP-Suv39h1-CD bound 130-nt ssRNA with a similar affinity to that of GST-Suv39h1-CD ( Figure 1D , Figure 1—figure supplement 1L ) . Interestingly , we found that MBP-fused full-length Suv39h1 bound ssRNA with a relatively higher affinity compared with MBP-Suv39h1-CD , and the 4A mutation partially impaired this binding ( Figure 1—figure supplement 1L–O ) . These results suggest that other Suv39h1 domain ( s ) contribute to Suv39h1-CD’s RNA-binding activity , which was reminiscent of fission yeast Chp1 ( Ishida et al . , 2012 ) . To elucidate how Suv39h-CD’s nucleic acid–binding activity is integrated into the Suv39h1-mediated establishment and maintenance of pericentric heterochromatin , we prepared retroviral expression vectors expressing FLAG-tagged Suv39h1 mutants defective in nucleic acid binding ( 4A ) , methylated H3K9 binding ( Y67A ) , or both ( Y67A-4A ) , and FLAG-tagged wild-type Suv39h1 ( Figure 3A ) . We transduced these constructs into Suv39h dn immortalized mouse embryonic fibroblasts ( iMEFs ) ( Lachner et al . , 2001 ) . On day 2 after infection with these viral particles , the GFP-positive cells were sorted out to collect transgene-expressing Suv39h dn iMEFs . The sorted cells were analyzed for pericentric heterochromatin assembly on day 3 and day 6 ( Figure 3C–F and Figure 3—figure supplement 1A , B ) . We monitored these cells’ exogenously expressed wild-type and mutant Suv39h1 on day 8 , and confirmed that their expression levels were comparable to or slightly higher than that of endogenous Suv39h1 ( Figure 3B ) . 10 . 7554/eLife . 25317 . 007Figure 3 . Suv39h1-CD’s nucleic acid binding and H3K9me binding are both crucial for pericentric heterochromatin assembly . ( A ) Alignment of wild-type ( WT ) and mutant full-length Suv39h1 . Boxes outline the chromodomain ( shaded black ) , and the SET domain ( shaded gray ) . ( B ) Suv39h1 protein expression level . *Endogenous Suv39h1; arrowhead: FLAG-Suv39h1 . ( C–D ) Kinetic comparison of the appearance ratio of the cells containing H3K9me3+ DAPI-dense foci after induction of WT and mutant Suv39h1 in Suv39h dn iMEFs . Immunohistochemical staining for H3K9me3 in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged 4A , Y67A , Y67A-4A , or WT Suv39h1 at ( C ) 3 days or ( D ) 6 days post-infection . The H3K9me3-positive cells within a population of 100 or more cells were counted ( n = 3 , mean ± SD , *p<0 . 05 , **p<0 . 001 ) . ( E ) The rate of HP1β heterochromatin accumulation in WT and mutant Suv39h1 during heterochromatin establishment . Immunohistochemical staining for HP1β in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged 4A , Y67A , Y67A-4A , or WT Suv39h1 at 6 days post-infection . ( F ) HP1β accumulated at heterochromatin in cells where H3K9me3 was restored . The radii of the circles indicate the number of cells in each category . Immunohistochemical staining for HP1β in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged 4A , Y67A , Y67A-4A , or WT Suv39h1 at 6 days post-infection . The cells with HP1β heterochromatin accumulation within a population of 100 cells or more were counted ( n = 3 , mean ± SD , *p<0 . 05 , **p<0 . 005 ) . The radii of the circles indicate the % ratio of cells in each category . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 00710 . 7554/eLife . 25317 . 008Figure 3—figure supplement 1 . H3K9me3 or HP1β in wild-type and mutant Suv39h1 during heterochromatin assembly . ( A ) Immunohistochemical staining for H3K9me3 in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged WT or mutant ( 4A , Y67A , or Y67A-4A ) Suv39h1 at 3 and 6 days post-infection . Arrowheads indicate H3K9me3 fluorescence signals . ( B ) HP1β and H3K9me3 in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged WT or mutant ( 4A , Y67A , or Y67A-4A ) Suv39h1 during heterochromatin assembly , at 6 days post-infection . An anti-HP1β antibody ( BMP002: MBL ) and anti-H3K9Me3 antibody ( clone 2F3 ) were used for co-immunofluorescence . Arrowheads indicate HP1β heterochromatin accumulation . ( C ) Native chromatin immunoprecipitation ( ChIP ) assays showing H3K9me3 enrichment at major satellite repeats in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged WT or mutant ( 4A , Y67A , or Y67A-4A ) Suv39h1 at 6 days post-infection ( n = 2 , mean ± SD , ) . Anti-H3K9me3 antibody ( clone 2F3 ) was used . ( D ) qRT-PCR assays for major satellite RNA transcripts in Suv39h dn iMEFs expressing FLAG-tagged WT or mutant ( 4A , Y67A , or Y67A-4A ) Suv39h1 at 14 days post-infection . Representative results of two independent biological replicates are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 00810 . 7554/eLife . 25317 . 009Figure 3—figure supplement 2 . Comparable histone H3 methylation by wild-type and mutant Suv39h1 in vitro . ( A ) Western blots of purified FLAG-tagged wild-type ( WT ) or mutant ( mut ) Suv39h1 . ( B ) FLAG-tagged WT or mutant Suv39h1 was incubated with 14C-labeled SAM . The proteins were separated by SDS-PAGE and stained with Coomassie Blue ( bottom ) . Autoradiography was detected with the BAS-5000 Image Analyzer ( top ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 00910 . 7554/eLife . 25317 . 010Figure 3—figure supplement 3 . Cell proliferation rates of Suv39h dn iMEFs were not changed by expressing FLAG-tagged WT or mutant Suv39h1 . Suv39h dn iMEFs stained by CellTrace Far Red Cell Proliferation Kit were infected with retroviruses for FLAG-Suv39h1 ( wt or mutant ) -IRES-GFP . Cell proliferation rates were analyzed by FACS . This panel shows representative results of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 01010 . 7554/eLife . 25317 . 011Figure 3—figure supplement 4 . Suv39h1’s N-terminal region also contributes to Suv39h1-mediated pericentric H3K9me3 formation . ( A ) Alignment of wild-type ( WT ) and mutant full-length Suv39h1 . Boxes outline the chromodomain ( shaded black ) , and the SET domain ( shaded gray ) . ( B ) Suv39h1 protein expression level . *Endogenous Suv39h1; black arrowhead: FLAG-Suv39h1 WT or R24 , K27A; gray arrowhead: FLAG-Suv39h1-DN . ( C ) Comparison of the rate of increase of H3K9me3 in WT and mutant Suv39h1 during the establishment of heterochromatin . Immunohistochemical staining for H3K9me3 in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged R24 , K27A , DN , or WT Suv39h1 at 3 days ( white bar ) or 6 days ( gray bar ) post-infection . The H3K9me3-positive cells within a population of 100 or more cells were counted . Right and left panel show representative results of two independent experiments . ( D ) Immunohistochemical staining for H3K9me3 in WT iMEFs , Suv39h dn iMEFs , or Suv39h dn iMEFs expressing FLAG-tagged WT or mutant ( R24 , K27A or DN ) Suv39h1 at 3 or 6 days post-infection . Arrowheads indicate H3K9me3 fluorescence signals . Right and left panel show representative results of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 011 The induction of wild-type Suv39h1 ( Suv39h1-WT ) expression in Suv39h dn iMEFs rescued pericentric H3K9me3 signals in the DAPI-dense pericentric regions ( H3K9me3 foci+ ) , 64 . 0 ± 7 . 9% of the cells on day 3 and 97 . 0 ± 1 . 3% on day 6 were H3K9me3-positive ( Figure 3C , D ) . Inducing the H3K9me3-binding-deficient Suv39h1-Y67A mutant also rescued the H3K9me3 signals on most of the DAPI-dense regions on day 6 ( 90 . 1 ± 3 . 2% ) . The formation of H3K9me3 foci on DAPI-dense regions was also induced by the RNA-binding-deficient Suv39h1-4A mutant , but this induction was significantly less efficient than that observed with Suv39h1 ( 40 . 3 ± 1 . 9% on day 3 and 87 . 3 ± 0 . 6% on day 6 ) . The Suv39h1-Y67A-4A double mutant had an additive effect , and H3K9me3 foci+ cells were further decreased on day 6 ( 71 . 9 ± 1 . 9% ) . We further examined H3K9me3 status on pericentromere ( major satellite repeat regions ) by chromatin immunoprecipitation ( ChIP ) analysis . H3K9me3 level at major satellite regions was severely diminished in Suv39h dn iMEFs and recovered by FLAG-tagged Suv39h1-WT expression on day 6 after transduction ( Figure 3—figure supplement 1C ) . Suv39h1 mutants ( Suv39h1-Y67A , −4A and -Y67A-4A ) also increased H3K9me3 levels , but Suv39h1-Y67A-4A had a minimal effect . An in vitro HMTase assay showed that all of the Suv39h1 mutants had histone methyltransferase ( HMTase ) activity for free H3 , comparable to Suv39h1-WT ( Figure 3—figure supplement 2 ) . Cell proliferation analysis demonstrated that the number of cell divisions during the first 3 days after viral infection were not much different among the Suv39h1 dn iMEFs infected with an empty or expression vector for Suv39h1 proteins ( Figure 3—figure supplement 3 ) . Thus , it is unlikely that the different rescue efficiencies for H3K9me3 resulted from altered enzymatic activity or by cell cycle delay . In the case of pericentric HP1β accumulation , Suv39h1-WT expression induced >80% of cells to become HP1β foci-positive ( HP1β foci+ ) on day 6 ( 83 . 0 ± 4 . 9% ) , but the phenotype was rescued only poorly by the Suv39h1 mutants ( Figure 3E; 63 . 0 ± 3 . 7% , 44 . 3 ± 19 . 8% , and 33 . 2 ± 9 . 4% for Suv39h1-Y67A , −4A , and -Y67A-4A on day 6 , respectively ) . Dual immunostaining of the Suv39h dn iMEFs on day 6 further demonstrated that compared to iMEFs rescued with Suv39h1-WT , iMEFs rescued with the Suv39h1 mutants contained larger cell populations that were negative for both H3K9me3 and HP1β ( Figure 3F , bottom left of each panel ) or were positive for only H3K9me3 ( Figure 3F , top left of each panel ) , suggesting that SUV39H-mediated heterochromatin assembly was less efficient in those mutant cells . To examine whether introduced Suv39h1 actually restored the repressive heterochromatin structure , we analyzed the expression levels of major satellite RNA . As reported previously ( Bulut-Karslioglu et al . , 2012 ) , major satellite repeats were derepressed in Suv39h dn iMEFs ( ~10 times higher than that of WT iMEFs ) and introduction of Suv39h1-WT suppressed this up-regulation of major satellite repeats ( Figure 3—figure supplement 1D ) . Mutant Suv39h1 with Y67A , 4A , or Y67A-4A also decreased major satellite repeat transcripts , but their repression levels were milder than that of Suv39h1-WT . Furthermore , among these mutants , Suv39h1-Y67A-4A’s effect was the weakest . These results are well consistent with the restored levels of H3K9me3 and HP1β . Collectively , these results indicate that Suv39h1-CD’s nucleic acid–binding activity integrates and cooperates with its recognition and binding of methylated H3K9 to establish/maintain Suv39h1-mediated pericentric heterochromatin such as H3K9me3 formation , HP1 accumulation , and transcriptionally repressive status . Despite having impaired binding of both RNA and methylated H3K9 , the Suv39h1 mutant Y67A-4A was able to assemble CH on pericentric regions , although less efficiently and completely than wild-type Suv39h1 ( Figure 3 ) . These data indicate that the Y67A-4A mutant retains other critical factors or functions that promote Suv39h1’s association with CH sufficiently for Y67A-4A to induce pericentric heterochromatin assembly . As to which factor or function is involved in this process , HP1 or an HP1-associated function is a reasonable candidate , since Suv39h1’s N terminus interacts with HP1 ( Melcher et al . , 2000; Yamamoto and Sonoda , 2003 ) . Indeed , we recently found that the induction of pericentric H3K9me3 is extremely poor in Suv39h dn iMEFs complemented with an N-terminal deletion ( Δ1–41 ) mutant of Suv39h1 ( ΔN , Figure 3—figure supplement 4A , C , D ) expressed at an endogenous level ( Muramatsu et al . , 2016 ) ( Figure 3—figure supplement 4B ) . Although ΔN does not bind HP1 , it was recently reported that the N-terminal region of Suv39h1 ( 1-41 ) also possesses chromatin-binding activity independent of its HP1 interaction ( Müller et al . , 2016 ) . Therefore , we validated the role of this chromatin-binding activity in Suv39h1-mediated H3K9me3 formation . We used the Suv39h1-R24 , K27A mutant , which is reported to be defective for chromatin binding but to still interact with HP1 in pericentric H3K9me3 formation in Suv39h dn iMEFs ( Müller et al . , 2016 ) ( Figure 3—figure supplement 4B–D ) . We found that the pericentric H3K9me3 formation was clearly diminished in Suv39h dn cells complemented with Suv39h1-R24 , K27A , but the impact of ΔN was much stronger than that of the R24 , K27A mutation . Thus , we concluded that both the HP1-binding and chromatin-binding activities of the N-terminal region also contribute to Suv39h1-mediated heterochromatin assembly . Since Suv39h1-CD had a higher binding affinity for RNA than for DNA in EMSAs ( Figure 1D–G ) , and this binding activity appeared to be crucial for Suv39h1-mediated pericentric heterochromatin formation , we examined whether Suv39h1 binds RNA transcribed from pericentric major satellite repeats inside the cell . In experiments with Suv39h dn iMEFs expressing FLAG-tagged wild-type or 4A Suv39h1 , we immunoprecipitated cellular RNAs associated with the wild-type or mutant Suv39h1 and assessed the levels of the major satellite repeats by dot-blot hybridization , which allowed us to evaluate the levels of repetitive DNA without amplification . As shown in Figure 4 , the signal for the major satellite repeats was clearly detected in precipitates from iMEFs expressing wild-type Suv39h1 but was severely diminished in precipitates from iMEFs expressing mutant Suv39h1-4A ( Figure 4B ) , even though similar amounts of FLAG-Suv39h1-WT or −4A were immunoprecipitated ( Figure 4A ) . These major satellite signals were totally lost by pretreating the samples with RNase , indicating that the detected signals were derived from RNA , not from contaminating genomic DNA . Although our EMSA using full-length Suv39h1 suggested the involvement of Suv39h1 domains other than CD in RNA binding ( Figure 2—figure supplement 1J–O ) , these data show that Suv39h1 binds major satellite RNA primarily via its CD . The major satellite repeats are reported to be expressed during G1/S transition and in M phase ( Lu and Gilbert , 2007 ) . Therefore , this expression during G1/S transition may have a role in H3K9me3 inheritance after replication . 10 . 7554/eLife . 25317 . 012Figure 4 . Suv39h1-CD’s nucleic acid binding is required for its interaction with major satellite RNAs in vivo . ( A ) Whole cell lysates ( input ) of Suv39h dn iMEFs expressing FLAG-tagged wild-type ( WT ) or mutant ( 4A ) Suv39h1 and FLAG immunoprecipitates ( IP ) were subjected to immunoblotting using an anti-FLAG M2 antibody . Parental Suv39h dn iMEFs ( none ) and Suv39h dn iMEFs with an empty vector ( vector ) were used as controls . ( B ) Dot-blot analysis of immunoprecipitated RNAs . RNAs associated with WT or mutant ( 4A ) Suv39h1 in Suv39h dn iMEFs were precipitated with the anti-FLAG M2 antibody and subjected to dot-blot analysis using a labeled probe for major satellite repeats ( left ) and 18S rRNA ( right ) . ( C , D ) Chromatin fractionation assays were performed using control iMEFs ( C ) and Suv39h dn iMEFs expressing FLAG-tagged WT or mutant Suv39h1 ( D ) . Soluble ( Sup ) and insoluble chromatin-enriched ( Ppt ) fractions were resolved by SDS-PAGE and analyzed by immunoblotting . For comparison , anti-α-tubulin and anti-histone H3 antibodies were used to detect soluble- and chromatin-associated proteins , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 01210 . 7554/eLife . 25317 . 013Figure 4—figure supplement 1 . Both Suv39h1-CD’s nucleic acid- and H3K9me3 binding activities are required for its interaction with major satellite RNAs in vivo . RNA immunoprecipitation ( RNA-IP ) experiments using parental Suv39h dn iMEFs and Suv39h dn iMEFs expressing FLAG-tagged WT or mutant ( 4A , Y67A , or Y67A-4A ) Suv39h1 ( n = 3 , mean ± SD ) . An anti-FLAG M2 antibody was used to precipitate Suv39h1-interacting RNAs . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 013 The interaction between Suv39h1 and major satellite RNAs was also examined by RT-qPCR analysis ( Figure 4—figure supplement 1 ) . Suv39h1-WT stably bound major satellite RNAs , and this interaction was impaired for Suv39h1-4A . The interaction was also lost for Suv39h1-Y67A mutant , suggesting that H3K9me3 recognition by Suv39h1-CD is also required for the Suv39h1’s stable interaction with the major satellite RNAs . Intriguingly , Suv39h1-Y67A-4A interacted partially with the major satellite RNAs . This result does not seemingly fit with our model that Suv39h1-CD’s RNA- and H3K9me3-binding activities cooperatively function in Suv39h1’s chromatin targeting . However , we also found that Suv39h1-Y67A-4A loosely bound chromatin ( see below ) . Considering this observation , Suv39h1-Y67A-4A may interact cellular RNAs without stable binding to chromatin . To determine whether the interaction between Suv39h1 and major satellite RNA occurs in the nucleus , we conducted chromatin fractionation assays . Cells were harvested and separated into soluble ( Sup ) and insoluble , chromatin-enriched ( Ppt ) fractions , and endogenous Suv39h1’s chromatin association was evaluated by immunoblotting . We found that most of Suv39h1 was present in the chromatin-enriched fraction ( Figure 4C ) . We confirmed that FLAG-tagged wild-type Suv39h1 expressed in Suv39h dn iMEFs was also associated with chromatin ( Figure 4D ) . Interestingly , although Suv39h1 mutants ( 4A and Y67A ) failed to interact with major satellite RNA in the RNA-IP experiments ( Figure 4B and Figure 4—figure supplement 1 ) , most of these mutants were also detected in the chromatin-enriched fractions ( Figure 4D ) , suggesting that Suv39h1 mutants are still associated with chromatin even without being stably bound to major satellite RNA . Even more interestingly , a relatively higher level of Suv39h1-Y67A-4A was detected in the soluble fractions ( Figure 4D ) . Together , these results confirmed that Suv39h1 interacts with major satellite RNA within the nucleus , but not in the cytoplasm , and that both RNA- and H3K9me3-binding activities contribute to Suv39h1’s chromatin association . Our rescue experiments of Suv39h dn iMEFs clearly indicated that Suv39h1-CD’s H3K9me3- and RNA-binding functions are both critical for establishing H3K9me3 marks in pericentric regions without preexisting H3K9me3 marks . On the other hand , we were still able to induce pericentric H3K9me3 and HP1 accumulation in Suv39h dn iMEFs complemented with 4A , Y67A , or Y67A-4A Suv39h1 mutants ( Figure 3 ) , albeit less efficiently than in iMEFs expressing wild-type Suv39h1 . Thus , Suv39h1-CD’s function might be dispensable for maintaining H3K9me3 marks once established . However , Suv39h1-4A’s association with major satellite RNA was severely impaired in cells with rescued H3K9me3 and HP1 ( Figure 4 ) , suggesting that defective RNA binding may affect Suv39h1 dynamics in the pericentromere , where major satellite repeat elements are transcribed . Consistent with this possibility , a single K87A amino acid substitution in SUV39H1 ( included in mut1 and 4A in our study ) was reported to enhance the SUV39H1 exchange rate in CH regions ( Bosch-Presegué et al . , 2011 ) , although the biological implication of this observation remains unclear . It is quite possible that Suv39h1 dynamics in the pericentromere were also affected in the mutant with defective H3K9me3 binding . To test the possibility that Suv39h1’s RNA- and H3K9me3-binding contribute to its dynamics on CH , we measured these dynamics in Suv39h dn iMEFs with stable expression of GFP-tagged wild-type or mutant Suv39h1 ( Figure 5—figure supplement 1A ) . As expected , GFP-Suv39h1-WT localized to the nucleus and formed foci on Hoechst-dense pericentric regions ( Figure 5A ) . GFP-Suv39h1-4A , -Y67A , and -Y67A-4A also formed foci on Hoechst-dense regions ( Figure 5A ) . However , semi-quantitative analysis to measure their heterochromatin enrichment ( represented as the intensity ratio of euchromatin over heterochromatin; Figure 5B ) revealed that GFP-Suv39h1-Y67A-4A fluorescence was significantly increased in euchromatin compared to the wt ( Figure 5B ) , suggesting that Suv39h1-Y67A-4A’s affinity to heterochromatic regions was decreased . GFP-Suv39h1-Y67A was also slightly less enriched on heterochromatin , and Y67A and 4A appeared to have an additive effect . We next assayed these molecules’ binding time to heterochromatin by fluorescence recovery after photobleaching ( FRAP ) . As clearly shown in Figure 5C and D , GFP-Suv39h1-4A and -Y67A exchanged more rapidly than Suv39h1-WT ( GFP-Suv39h1-4A and -Y67A had recovery half-times of ~6 s , versus ~20 s for Suv39h1-WT ) , and their mobile fraction was increased ( 88–89% for GFP-Suv39h1-4A and -Y67A , versus ~73% for GFP-Suv39h1-WT ) . GFP-Suv39h1-Y67A-4A was much more dynamic , with an ~3 s recovery half-time and ~91% mobile fraction . GFP-Suv39h1-mut1 and -mut3 ( Figure 5—figure supplement 1 ) also exchanged more rapidly than GFP-Suv39h1-WT ( Figure 5—figure supplement 1C , D ) . These data suggest that Suv39h1-CD’s binding of nucleic acid and of methylated H3K9 are both crucial for Suv39h1’s retention of heterochromatin in cells . 10 . 7554/eLife . 25317 . 014Figure 5 . Both Suv39h1-CD’s nucleic acid binding and H3K9me binding contribute to Suv39h1’s retention on heterochromatin . ( A ) Subcellular localization of Hoechst staining and wild-type ( WT ) or mutant GFP-Suv39h1 in Suv39h dn iMEF cells . ( B ) Box plot showing the signal-intensity ratio between euchromatin and heterochromatin . Center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by R software , whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles , and outliers are represented by dots; n = 20 sample points . ( *p<0 . 005 , **p<0 . 00005 ) ( C ) FRAP analysis of WT or mutant GFP-Suv39h1 in Suv39h dn iMEFs . The means of the relative intensity in the bleached area are indicated with the SD ( n ≥ 29 ) . ( D ) Quantification and statistical analysis of the FRAP analysis in C; and the mobile population fraction ( mobile ) and half-time of fluorescence recovery ( t1/2 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 01410 . 7554/eLife . 25317 . 015Figure 5—figure supplement 1 . Wild-type and mutant GFP-Suv39h1 analysis . ( A ) Protein levels of wild-type ( WT ) or mutant GFP-Suv39h1 . *Endogenous Suv39h1; **FLAG-Suv39h1 . Arrowhead: GFP-Suv39h1 . ( B ) The subcellular localization of Hoechst staining and WT , mut1 , and mut3 GFP-Suv39h1 in Suv39h dn iMEF cells . ( C ) FRAP analysis of WT , mut1 , and mut3 GFP-Suv39h1 in Suv39h dn iMEF cells . The means of the relative intensity in the bleached area are indicated with the SD ( n ≥ 11 ) . ( D ) Quantification and statistical analysis of the FRAP analysis in C , showing the mobile population fraction ( mobile ) and fluorescence recovery half-time ( t1/2 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 015 Since the Suv39h1-4A mutant is defective in major satellite RNA binding and more dynamic on CH in vivo , it is highly possible that major satellite repeat transcripts contribute to the retention of Suv39h1 on heterochromatin . However , the above experiments could not rule out the possibility that Suv39h1-CD targets chromatin via its DNA-binding activity ( Figure 1E ) . We therefore employed knock-down of major satellite repeat transcripts using locked nucleic acid ( LNA ) -DNA gapmers ( Probst et al . , 2010 ) . As shown in Figure 6A , introduction of major satellite repeat-specific LNA-DNA gapmers into Suv39h dn iMEFs that express GFP-Suv39h1 significantly and specifically decreased major satellite repeat transcripts . In major satellite knock-down cells , GFP-Suv39h1 on pericentric heterochromatin became more mobile than the control ( Figure 6B ) . This data further supports the notion that major satellite RNAs contribute to Suv39h1 stability on pericentric regions . 10 . 7554/eLife . 25317 . 016Figure 6 . Major satellite RNAs contribute to Suv39h1’s retention on heterochromatin . ( A ) qRT-PCR for major satellite RNAs or nuclear non-cording RNA NEAT1 transcripts in Suv39h dn iMEFs expressing GFP-suv39h1 transfected without or with a set of two LNA-DNA GapmeRs directed against the Forward and Reverse major satellite transcripts . ( B ) FRAP analysis of GFP-Suv39h1 in the described cells ( A ) . The means of the relative intensity in the bleached area are indicated with the SD ( n ≥ 13 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25317 . 016
The CD is a highly conserved protein module most recognized for targeting methylated histone tails ( Blus et al . , 2011; Fischle et al . , 2003 ) . Although several lines of evidence suggest that the CD binds DNA and RNA in addition to targeting methylated histones , its biological relevance and physiological roles are poorly understood ( Hiragami-Hamada and Fischle , 2014 ) . In this study , we demonstrated that Suv39h1-CD not only recognizes H3K9me3 , but also binds RNA and DNA ( with a higher affinity to ssRNA than to dsDNA ) , indicating that RNA may also be crucial for Suv39h1 recruitment/function . Indeed , our current data suggest that these two activities contribute to Suv39h1-mediated CH assembly , including the accumulation of H3K9me3 and HP1 ( Figure 3 ) and the retention of Suv39h1 itself on the pericentric regions ( Figure 5 ) . Furthermore , we found that the HP1-binding and chromatin-binding activities of Suv39h1’s N-terminal region contributed to the Suv39h1-mediated pericentric H3K9me3 formation ( Figure 3—figure supplement 4C and [Muramatsu et al . , 2016] ) . Collectively , our results demonstrated that the four different activities of Suv39h1 other than its lysine methyltransferase activity cooperatively regulate Suv39h1-mediated pericentric heterochromatin assembly . Independent work by another group shows that in vitro RNA binding activity is also associated with the CD of human SUV39H1 ( Johnson et al . , 2017 ) . We found that basic residues in Suv39h1-CD’s C-terminal α-helix are involved in binding RNA , and that Suv39h1-CD’s RNA binding is distinct and independent of its recognition and binding of H3K9me3 . Considering the slower recovery of H3K9me3 in Suv39h dn iMEFs expressing Suv39h1-4A ( which is deficient in RNA binding ) than in those expressing Suv39h1-Y67A ( which is deficient in H3K9me3 binding ) , RNA-binding activity is particularly involved in recruiting Suv39h1 to H3K9me3-free chromosomal regions . Since pericentric regions consist of major satellite repeats , it is possible that nascent transcripts derived from such regions possess specialized properties that are recognized by Suv39h1 . Although SUV39H1 is reported to preferentially bind TERRA transcripts derived from telomeric repeats ( Porro et al . , 2014 ) , our study demonstrates that Suv39h1-CD also efficiently binds ACTB mRNA and does not show any particular sequence specificity ( Figure 1—figure supplement 1 ) . Thus , it is unlikely that Suv39h1 targets specific chromosomal regions by recognizing a specific RNA sequence or structure . However , it is possible that transcripts derived from repetitive chromosomal regions are not efficiently processed , leading to their stable association with transcribed regions . Pericentric transcripts with this kind of feature might become a target of Suv39h1 . Since another independent work now demonstrates that the N-terminal basic domain of mouse Suv39h2 also shows RNA binding activity and this activity is crucial for the recruitment and stabilization of Suv39h2 to heterochromatin . ( Velazquez Camacho et al . , 2017 ) , Suv39h-family proteins may use a similar RNA binding activity for their targeting . In addition to its role in establishing H3K9me3 , Suv39h1-CD’s RNA-binding activity also contributes to Suv39h1’s stable association with H3K9me3-rich pericentromeric regions ( Figure 5 ) . Knockdown of major satellite repeat transcripts ( Figure 6 ) further supports the notion that RNA ( major satellite RNA ) binding of Suv39h1 contributes to its stability on chromatin . While we showed that Suv39h1-CD binds H3K9me3 peptide , its binding affinity is weaker than those of the CDs of HP1 proteins ( Figure 2—figure supplement 1A and [Jacobs and Khorasanizadeh , 2002; Wang et al . , 2012] ) . Suv39h1-CD’s RNA-binding activity might increase its affinity for H3K9me3-rich pericentric regions; alternatively , RNA binding might modulate Suv39h1-CD’s enzymatic activity . A recent report showed that Suv39h1’s CD inhibits its methyltransferase activity , and the CD’s binding of H3K9me3 is suggested to allosterically regulate Suv39h1’s enzymatic activity ( Müller et al . , 2016 ) . It is not clear how Suv39h1’s methyltransferase activity is controlled by the CD; however , it is possible that Suv39h1-CD’s RNA binding is also coupled with its allosteric regulation . Physical interaction between the CD and SET domains appeared to affect the CD’s RNA-binding activity: we demonstrated that full-length Suv39h1 bound RNA with a relatively higher affinity compared with Suv39h1-CD ( Figure 1—figure supplement 1M ) . Considering the CD’s role in targeting Suv39h1 to H3K9me3-free chromatin regions , Suv39h1’s physical association with other factors such as chromatin-unbound non-coding RNAs or HP1 proteins may modulate the CD’s C-terminal α-helix to bind RNAs . In general , the assembly of higher-order chromatin structure has two steps , establishment and maintenance ( Hall et al . , 2002; Sadaie et al . , 2004 ) . H3K9me3 marks on CH have to be maintained during DNA replication , and given that Suv39h1 possesses a CD that recognizes H3K9me3 , a direct read-write mechanism involving Suv39h1 is proposed to maintain H3K9me3 ( Audergon et al . , 2015; Ragunathan et al . , 2015 ) . However , this mechanism does not fully explain the restoration of H3K9me3 in Suv39h dn iMEFs . Thus , Suv39h1-CD’s RNA-binding activity appears to contribute to its ability to target pericentric regions without preexisting H3K9me3 and to establish H3K9me3 marks; this function is consistent with the role of nucleic acid binding in Chp1 and Clr4 in fission yeast ( Ishida et al . , 2012 ) . Moreover , HP1 can also bind RNA and methylated H3K9 ( Keller et al . , 2012; Muchardt et al . , 2002 ) . HP1’s ability to bind RNA is SUMO-dependent , and an HP1-Ubc9 fusion molecule with enhanced sumoylation on the HP1 part can be targeted to the pericentric regions , even in Suv39h dn iMEFs ( Maison et al . , 2011 ) . Thus , HP1 can recruit mutant Suv39h1 to pericentric regions via binding between sumoylated HP1 and chromatin-bound major satellite RNA . Once Suv39h1-Y67A-4A is targeted to pericentric regions and deposits H3K9me3 , HP1 further increases its affinity for pericentric regions , and this positive feedback loop between HP1 and Suv39h1-Y67A-4A may be sufficient for this mutant Suv39h1 to mediate pericentric heterochromatin assembly . The ΔN phenotype supports this possibility . In this study we demonstrated that Suv39h1-CD’s RNA-binding activity is required for its targeting to and assembly of heterochromatin , highlighting the functional role of pericentromeric transcripts in introducing H3K9me3 marks . Recent studies using fission yeast demonstrated that H3K9me3 can be maintained during DNA replication as a self-perpetuating epigenetic mark ( Audergon et al . , 2015; Ragunathan et al . , 2015 ) . Interestingly , the maintenance of H3K9me3 requires intact Clr4-CD . Epe1 , a protein containing a jmjC-domain , counteracts this process by directly removing the H3K9me3 mark . Such counteracting enzymatic activities appear to be important for preventing the inappropriate inactivation of euchromatic genes by stochastically introduced H3K9me3 marks . Although it is not clear whether similar counteracting demethylase activities play a role in maintaining genome integrity in mammalian cells , pericentromeric transcripts may modulate such counteracting enzymatic activity to directly regulate chromatin dynamics .
EMSAs were performed as described previously ( Ishida et al . , 2012 ) using a PCR-amplified major satellite sequence as a dsDNA probe . For RNA probes , DNA fragments ( major satellite , ACTB ORF , and alfa satellite ) were PCR-amplified with a T7 promoter sequence , and the resultant PCR fragments were used for in vitro transcription by Thermo T7 RNA polymerases ( Toyobo ) . Transcribed RNAs were purified on denaturing 7 M Urea Polyacrylamide Gel and extracted with Biomasher Ι ( Assist ) . Purified ssRNAs and dsDNAs were labeled with the 5’ EndTag Nucleic Acid Labeling System ( Vector Laboratories , Cat# MB-9001 , RRID:AB_2336073 ) , and the labeled probes were incubated with GST- or MBP-fused proteins in 10 μl of binding buffer ( 20 mM HEPES-KOH pH 7 . 6 , 100 mM KCl , 0 . 01% NP-40 , and 1 mM dithiothreitol [DTT] ) containing 4 U RNaseOUT ( Invitrogen ) . The fluorescent dye can be conjugated with dsDNA at the single or both 5’-ends , and the number of conjugated dye could affect the probe migration in gels , resulting in appearance of doublet bands . Binding reactions were carried out on ice for 30 min . For titration EMSAs , serially diluted proteins ( diluted 3:5 or 1:2 ) were used . To examine the relationship between RNA binding and H3K9me3 recognition , an equimolar amount of H3K9me3 peptide was added to the binding reactions . The reaction samples were loaded onto a 5% polyacrylamide gel with 0 . 5 × Tris-borate EDTA buffer ( TBE ) , and the labeled probes were detected with a Typhoon 9400 ( GE Healthcare ) . The unbound fractions were measured with ImageMaster 1D software ( GE Healthcare ) . Igor Pro software ( WaveMetrics , RRID:SCR_000325 ) was used for curve fitting . Eco-Pack 2–293 cells ( Clontech ) and 293T cells ( gifted from Dr . Yao [Ito et al . , 2001] ) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum . Wild-type and Suv39h dn iMEFs ( Lachner et al . , 2001 ) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum , 0 . 1 mM beta-mercaptoethanol , and 1x non-essential amino acids . The following antibodies were used in this study: anti-FLAG-M2 ( F3165: Sigma-Aldrich , RRID:AB_259529 ) , anti-FLAG-M2-HRP ( A8592: Sigma-Aldrich , RRID: AB_439702 ) , anti-α-tubulin ( T5168: Sigma , RRID: AB_477579 ) , anti-Suv39h1 ( 8729: Cell Signaling , RRID: AB_10829612 ) , anti-H3 ( ab21054: Abcam , RRID: AB_880437 ) , anti-H3K9me3 ( ab8898: Abcam , RRID: AB_306848 and 2F3 ( RRID: AB_2616099 ) ( Chandra et al . , 2012 ) , anti-HP1β ( BMP002: MBL , RRID: AB_843158 ) , and anti-GST ( 27-4577-01: GE Healthcare , RRID: AB_771432 ) . DNA fragments encoding Suv39h1-CD ( residues 39–105 or 39–96 ) were PCR-amplified from pCAG-IRES-Puro-FLAG-Suv39h1 ( Muramatsu et al . , 2016 ) . The amplified Suv39h1-full length or -CD was cloned into the TOPO pCRII vector ( Clontech ) and subcloned into the bacterial expression vector pGEX-6P-3 ( GE Healthcare ) to make the GST-fusion molecule . The GST-Suv39h1-CD ( 39–96 ) was only used for ITC . To express FLAG-tagged wild-type or mutant Suv39h1 , DNA fragments encoding FLAG-Suv39h1 were generated by PCR using pCAG-IRES-Puro-FLAG-Suv39h1 as the template , and the amplified FLAG-Suv39h1 was cloned into the TOPO pCRII vector . Suv39h1 mutagenesis was performed with the TOPO pCRII vector according to the manufacturer’s instructions ( Stratagene ) . Next , the wild-type or mutant FLAG-Suv39h1 was subcloned into the pMCs-IRES-GFP retrovirus expression vector ( Cell Biolabs ) . To express GFP-Suv39h1 fusion molecules , the PCR-amplified DNA of wild-type or mutant Suv39h1 was subcloned into the pEGFP-C1 vector ( Addgene ) , and the GFP-Suv39h1 fusion part was amplified again by PCR and subcloned into the BamH1 and SalI sites of pMCs-IRES-GFP from which IRES and the vector’s own GFP sequences were deleted . For retrovirus production , Eco-Pack 2–293 cells ( Clontech ) were transfected with pMCs-FLAG-Suv39h1WT/mut-IRES-GFP or pMCs-GFP-Suv39h1WT/mut . The resultant retrovirus supernatants were used to infect control or suv39h dn iMEF cells ( Peters et al . , 2001 ) . To obtain populations expressing similar levels of wild-type or mutant FLAG-Suv39h1 or GFP-Suv39h1 , infected cells expressing the same levels of GFP were sorted by fluorescence-activated cell sorting ( FACS ) . Cells cultured on chamber slides ( Matsunami Glass , SCS-008 ) were fixed in 4% paraformaldehyde for 8 min at room temperature . The fixed cells were permeabilized with 0 . 1% Triton X-PBS for 15 min at room temperature and blocked with 3% BSA in 0 . 1% Tween-20 TBS ( 3% BSA-TTBS ) for 15 min at room temperature . Next , the cells were incubated with primary antibodies diluted in 3% BSA-TTBS for 1 hr at room temperature , washed twice with PBS , and incubated with secondary antibodies conjugated with Alexa568 , 647 Fluor ( Invitrogen ) diluted in 3% BSA-TTBS for 1 hr at room temperature . The nuclei were counterstained with DAPI and observed under a confocal microscope ( Olympus , FV1000 ) at the Support Unit for Bio-Material Analysis in the RIKEN BSI Research Center ( RRC ) . iMEFs in eight 150 mm dishes ( ~80% confluent ) were fixed with 1% formaldehyde for 10 min at room temperature . The harvested cells were lysed in SDS lysis buffer ( 50 mM HEPES-KOH pH 7 . 6 , 1% SDS , and 10 mM EDTA ) , and the cleared cell lysates were sonicated with a Bioruptor ( Cosmo Bio ) . The samples were diluted with RIP dilution buffer ( 50 mM HEPES-KOH pH 7 . 6 , 167 mM NaCl , 0 . 01% SDS , 1 . 1% Triton X-100 , and 5 mM EDTA ) supplemented with 40 U/ml RNase inhibitor ( RNaseOUT , Invitrogen ) and were immunoprecipitated with anti-FLAG M2 antibody–conjugated Dynabeads ( Invitrogen ) at 4°C overnight . The beads were washed with ice-cold low-salt buffer ( 20 mM HEPES-KOH pH 7 . 6 , 150 mM NaCl , 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , and 20 U/ml RNaseOUT ) , high-salt buffer ( 20 mM HEPES-KOH pH 7 . 6 , 500 mM NaCl , 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , and 20 U/ml RNaseOUT ) , LiCl buffer ( 10 mM HEPES-KOH pH 7 . 6 , 0 . 25 M LiCl , 1% Nonidet P-40 , 1% sodium deoxycholate , 1 mM EDTA , and 20 U/ml RNaseOUT ) , and diethylpyrocarbonate ( DEPC ) -treated water . The protein–RNA complexes were eluted with RIP elution buffer ( 10 mM HEPES-KOH pH 7 . 6 , 300 mM NaCl , 0 . 5% SDS , 5 mM EDTA , and 40 U/ml RNaseOUT ) and treated with 0 . 8 µg/ml Proteinase K for 1 hr at 42°C , followed by de-crosslinking at 65°C for at least 4 hr . Immunoprecipitated RNAs were further purified with TRIzol LS Reagent ( Invitrogen ) and treated with 2 U of DNase Turbo ( Ambion ) in 50 µl reaction buffer for 20 min at 37°C . The immunoprecipitated RNAs were extracted with phenol/chloroform ( pH 5 . 2 ) and recovered by adding an equal volume of 2-propanol and 1/10 vol of 3 M sodium acetate ( pH 5 . 2 ) using 1 µl of ethachinmate ( Nippon Gene ) as a carrier . The precipitated RNAs were dissolved in 50 μl of DEPC-treated water . Samples were denatured for 15 min at 65°C in loading buffer ( 6 . 6% formaldehyde , 50% formamide , 20 mM MOPS , 1 mM EDTA , 5 mM sodium acetate , and 0 . 1 U/µl RNaseOUT ) before dot-blot analysis . For the quantitative RIP experiment , the dissolved RNAs were further treated with 2 U of DNase Turbo in 50 µl reaction buffer for 20 min at 37°C , extracted with phenol/chloroform ( pH 5 . 2 ) , and recovered by adding an equal volume of 2-propanol and 1/10 vol of 3 M sodium acetate ( pH 5 . 2 ) . cDNA was synthesized by SuperScript III reverse transcriptase ( Invitrogen ) with random hexamers . Quantitative real-time PCR was performed by the Fast SYBR Green Master Mix ( Applied Biosystems ) and the StepOnePlus Real Time PCR system ( Applied Biosystems ) . Cells were harvested by trypsin treatment , washed twice with ice-cold PBS , and resuspended in lysis buffer ( 50 mM Tris-HCl pH 7 . 5 , 5 mM MgCl2 , 100 mM NaCl , 1% Triton-X100 , 10% glycerol , and 1 mM sodium fluoride ) . After 10 min incubation on ice , the lysates were centrifuged at 14 , 010 ×g for 10 min at 4°C to separate supernatant and chromatin-enriched pellet fractions . Whole cell lysates and supernatant and pellet fractions were resuspended in SDS-sample buffer and sonicated using a Bioruptor ( Diagenode ) to reduce viscosity before SDS-PAGE analysis . Proteins in each fraction were analyzed by western blotting . FRAP was performed as described previously ( Hayashi-Takanaka et al . , 2009 ) using a confocal microscope ( FV-1000; Olympus; operated by the built-in software FV10-ASW ver . 4 . 2 ) , with a PlanApoN 60× OSC NA = 1 . 4 oil-immersion objective lens . iMEFs were grown on a glass-bottom dish ( Mat-Tek ) set on a heated stage at 37°C ( Tokai Hit ) in a 5% CO2 atmosphere control system ( Tokken ) . Two images were collected at a 5 s interval ( 0 . 8% 488 nm laser transmission; 2 . 0 μs/pixel; 512 × 512 pixels; pinhole 800 μm;×8 . 0 zoom; line averaging 2 ) , a circle ( 2 . 55 μm diameter ) covering a heterochromatin region was bleached ( 100% 488 nm laser transmission; 4 . 0 μs/pixel; 1 iteration ) , and 25 images were further acquired every 5 s using the original settings . Fluorescence intensities were measured using the ImageJ 1 . 47v program ( http://rsb . info . nih . gov/ij/ ) . The net intensities of the bleached and unbleached heterochromatin regions were obtained by background subtraction in each time frame . The intensity of the bleached heterochromatin was normalized to that of the unbleached heterochromatin , and the intensity relative to the original intensity before bleaching was plotted . The recovery curves from single cells were individually fitted to single exponential kinetics to obtain the half-time ( t1/2 ) and mobile fraction ( Kimura and Cook , 2001 ) , using ImageJ 1 . 47v . The recovery curve was governed by R = 0 . 2 + P ( 1-exp-kt ) , where R = relative intensity , P = plateau value , k = association constant , and t = time . The images obtained before bleaching were also used to measure the heterochromatin:euchromatin ratio . To confirm the enrichment of GFP-Suv39h and its mutants in Hoechst-dense heterochromatin , iMEFs were fixed with 4% paraformaldehyde ( Electron Microscopy Sciences ) for 10 min and stained with 100 ng/ml Hoechst33342 ( Nacalai Tesque ) for 1 hr before collecting images with a confocal microscope ( FV-1000; Olympus; operated by the built-in software FV10-ASW ver . 4 . 2 ) , with a PlanApoN 60× OSC NA = 1 . 4 oil-immersion objective lens ( sequential scanning using 0 . 1% 405 nm and 4% 488 nm lasers; 2 . 0 μs/pixel; 800 × 800 pixels; pinhole 800 μm;×3 . 0 zoom; line averaging 6 ) . We obtained locked nucleic acid ( LNA ) -DNA GapmeRs from Exiqon . An LNA-DNA GapmeR for major satellite RNA was previously reported ( Probst et al . , 2010 ) . The sequence of the Neat1 LNA-DNA GapmeR was ccagTCCACCCGTCtcca ( LNA nucleotides in lower case letters ) . For knockdown experiments with locked nucleic acid ( LNA ) -GapmeRs , trypsinized Suv39h dn iMEFs ( 1 × 106 ~cells ) were suspended in 100 μl MEF II nucleofector solution ( Lonza ) and then mixed with LNA-DNA GapmeRs ( 250 nM final concentration ) ) . Transfection was carried out in an electroporation cuvette using the Nucleofector instrument ( Lonza ) . The transfected cells were transferred to fresh medium and incubated at 37°C for 24 hr . Then these cells were changed into fresh medium and incubated at 37°C for 24 hr , followed by harvesting cells for RNA preparation or FRAP analysis . Immunoprecipitated RNA or DNA samples were blotted onto a Hybond N+ nylon membrane ( GE Healthcare ) by Bio-Dot ( Biorad ) according to the manufacturer’s instructions . The membrane was rinsed with 10X SSC ( 1 . 5 M NaCl and 150 mM sodium citrate ) and crosslinked with a UV Stratalinker ( Stratagene ) . The membrane was pre-hybridized at 55°C for at least 15 min and hybridized with a probe at 55°C overnight . PCR-amplified major satellite or 18S rRNA probes were labeled and hybridized using the Alkphos Direct Labeling and Detection System ( GE Healthcare ) according to the manufacturer’s instructions . The membrane was washed three times with a first-wash buffer ( 2 M Urea , 0 . 1% SDS , 50 mM NaHPO4 pH 7 . 0 , 150 mM NaCl , 1 mM MgCl2 , and 0 . 1% blocking reagent ) at 55°C and twice with a second-wash buffer ( 50 mM Tris , 100 mM NaCl , and 1 mM MgCl2 ) at room temperature . Images were captured with a LAS-3000 Mini ( Fujifilm ) . A recombinant GST-fusion protein was expressed in E . coli BL21 ( DE3 ) cells . Cells were lysed by sonication ( Branson , Sonifier 450D ) , and proteins in the lysates were purified using Glutathione Sepharose ( GE Healthcare ) according to the manufacturer’s instructions . To produce maltose binding protein ( MBP ) -fused recombinant Suv39h1 ( MBP-Suv39h1 ) proteins , the corresponding Suv39h1 cDNAs were introduced into the pMAL-c2X vector ( NEB ) . Proteins expressed in the E . coli strain BL21 ( DE3 ) were purified using Amylose Resin ( NEB: E8021S ) according to the manufacturer’s instructions . The GST- or MBP-fused recombinant proteins were further purified by anion-exchange chromatography ( SOURCE 15Q; GE Healthcare ) . The eluted material was dialyzed against phosphate-buffered saline ( PBS ) with 10% glycerol , divided into aliquots , and stored at −80°C until use . ITC was performed on a MicroCal VP-ITC calorimeter ( GE Healthcare ) at 20°C . The proteins were dissolved in phosphate buffer ( 20 mM KPO4 pH 6 . 8 and 10 mM NaCl ) . A typical titration consisted of injecting 10 μl aliquots of the ligand ( H3K9me0-3; ARTKQTAR ( Lys[Me]0–3 ) STGGKAPRKQLAY ) into the protein sample at 3 min intervals to ensure that the titration peak returned to the baseline . The ITC data were analyzed using the Origin program . Peptide pull-down assays were performed as described previously ( Ishida et al . , 2012 ) . GST-CD fusion molecules ( 1 pmol ) were incubated with 10 μl of Dynabeads M-280 Streptavidin-conjugated with biotinylated unmodified , K9-monomethylated , K9-dimethylated , or K9-trimethylated H3 peptide ( 100 pmol ) in 150 μl of binding buffer ( 20 mM Tris-HCl pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , and 0 . 1% Triton X-100 ) for 1 hr at 4°C . To examine the relationship between nucleic acid–binding and H3K9me3 recognition , 1 pmol of major satellite RNA or dsDNA probe was added to the binding reaction . The beads were washed twice with binding buffer , and the bound proteins were eluted in SDS sample buffer and analyzed by immunoblotting with an anti-GST antibody . A small scale ChIP analysis was performed as described previously ( Brind'Amour et al . , 2015 ) . iMEF cells ( 1 × 105 cells ) were harvested and re-suspended in nuclear isolation buffer ( Sigma ) . The chromatin was fragmented for 7 min at 37°C , and diluted in ChIP immunoprecipitation buffer ( 20 mM Tris-HCl pH 8 . 0 , 2 mM EDTA , 150 mM NaCl , 0 . 1% Triton X-100 , 1 × EDTA-free protease inhibitor cocktail and 1 mM PMSF ) . Lysate mixture was incubated with anti-H3K9me3 ( 2F3 ) coupled with 10 μl of mouse IgG Dynabeads for over night at 4°C . The immune complexes were sequentially washed in low salt wash buffer ( 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , 20 mM Tris-HCl pH 8 . 0 , 150 mM NaCl ) , and high salt wash buffer ( 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , 20 mM Tris-HCl pH 8 . 0 , 500 mM NaCl ) . The immune complex was purified with 1 . 8×volume of Ampure XP DNA purification beads ( Agencourt ) , then analyzed by real-time PCR using primers reported as specific for major satellite DNA ( Matsui et al . , 2010 ) . PCR primers used for RT-qPCR were as follows: Major Satellite ChIP F ( 5’- GACGACTTGAAAAATGACGAAATC-3’ ) , Major Satellite ChIP R ( 5’- CATATTCCAGGTCCTTCAGTGTGC-3’ ) , Gapdh ChIP F ( 5’- ATCCTGTAGGCCAGGTGATG-3’ ) , Gapdh ChIP R ( 5’- AGGCTCAAGGGCTTTTAAGG-3’ ) , Gapdh RT-PCR F ( 5’- ATGAATACGGCTACAGCAACAGG-3’ ) , Gapdh RT-PCR R ( 5’- CTCTTGCTCAGTGTCCTTGCTG-3’ ) , NEAT1-6 F ( 5’- TGCTTACACGGCTTGTTCAG-3’ ) , NEAT1-6 R ( 5’- AACTCCAAGGTCCCTGTCCT-3’ ) . Suv39h dn iMEFs were seeded onto a 10 cm dish the day before the experiment . Cells were labeled with 10 µM CellTrace Far Red ( Thermo Fisher ) on the dish for 20 min at 37°C , then washed twice in medium and returned to the incubator for 10 min at 37°C . Then , these labeled Suv39h dn iMEFs were infected with a retrovirus containing pMCs-FLAG-Suv39h1WT/mut-IRES-GFP , and incubated for 3 days at 37°C . Analysis was performed on 10 , 000 cells/ GFPdull+ sample using FACS . 48 hr after transfection , 293T cells expressing FLAG-tagged Suv39h1wt/mut were lysed in RIPA buffer ( 50 mM Tris-HCl pH 7 . 5 , 420 mM NaCl , 1 mM EDTA , 1% NP40 , and 0 . 25% sodium deoxycholate ) containing 1 mM PMSF , and were subjected to sonication on ice . FLAG-tagged Suv39h1wt/mut proteins were purified with anti-FLAG M2 affinity gel . An in vitro histone methyltransferase assay was performed as described previously ( Shimazu et al . , 2014 ) . FLAG-tagged wild-type or mutant Suv39h1 was incubated in 1x Collin’s buffer ( 25 mM Tris-HCl pH 8 . 5 , 5 mM DTT ) with Histone H3 ( 0 . 5 μg ) and 14C-labeled SAM ( 0 . 01 μCi , Perkin Elmer ) for 15 min at 25°C . The reaction was stopped by adding Laemmli SDS sample buffer . Proteins were resolved on a 15% acrylamide SDS-PAGE gel , and the dry gel was exposed to an imaging plate ( Fujifilm ) for 1 day . Autoradiography was detected using a BAS-5000 Image Analyzer ( Fujifilm ) .
SUV39H is the major histone H3 lysine 9 ( H3K9 ) -specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin . SUV39H recognizes trimethylated H3K9 ( H3K9me3 ) via its chromodomain ( CD ) , and enriched H3K9me3 allows SUV39H to target specific chromosomal regions . However , the detailed targeting mechanisms , especially for naïve chromatin without preexisting H3K9me3 , are poorly understood . Here we show that Suv39h1’s CD ( Suv39h1-CD ) binds nucleic acids , and this binding is important for its function in heterochromatin assembly . Suv39h1-CD had higher binding affinity for RNA than DNA , and its ability to bind nucleic acids was independent of its H3K9me3 recognition . Suv39h1 bound major satellite RNAs in vivo , and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere . Suv39h1 mutational studies indicated that both the nucleic acid–binding and H3K9me–binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly . These results suggest that chromatin-bound RNAs contribute to creating SUV39H’s target specificity .
Plants , animals and fungi store much of their DNA tightly packed with proteins in a form named heterochromatin . This arrangement helps to inactivate genes that are not needed in specific cells or at specific times , and provides a way to protect the genetic material from damage . Heterochromatin tends to form when an enzyme called a lysine methyltransferase chemically modifies some of the proteins associated with the DNA , which are known as histones . This enzyme modifies only some of the histones to get the process started , while a second protein then binds to the modified histones and causes more of the DNA to become packaged up as heterochromatin . In 2012 , researchers reported that the version of the lysine methyltransferase enzyme from yeast binds to RNA molecules via a portion known as its chromodomain . Moreover , the enzyme needed to bind to RNA to help heterochromatin to form . A similar mechanism also occurs in fruit flies , another organism that is commonly studied in the laboratory . However , it was not clear if it happened in mammals like mice and humans . Now , Shirai , Kawaguchi et al . – who include many of the researchers involved in the 2012 study – report that the corresponding enzyme from mice can also bind to RNA molecules via its chromodomain . Further experiments showed that this activity was closely linked with the enzyme’s ability to target the correct histones and efficiently form heterochromatin . The first experiments were conducted using purified enzymes in the laboratory , while follow-up experiments looked at the enzyme’s activity within mouse cells . Other studies have previously reported that mutant mice lacking the lysine methyltransferase enzyme have defective heterochromatin , tend to die young and have genetic instabilities that are associated with an increased risk of tumors and male infertility . The new findings of Shirai , Kawaguchi et al . reveal that the mechanism behind the establishment of heterochromatin has mostly likely been conserved over a billion years of evolution , which is when yeast and mammals last shared a common ancestor . By revealing more about how mammalian cells can protect their DNA , these new findings could also mark an important step toward understanding and preventing birth defects that are caused when an embryo’s genetic material becomes damaged .