conventional purification of histone h1.4k26 methyltransferase 
  activity  hela nuclear pellet fraction was prepared following the 
  protocol of dignam et al . 
 
approximately 10 g of 
  nuclear pellet fraction were subjected to 1000 ml of de52 resin ( whatman ) that 
  was packed into a low - pressure chromatography column ( 15 cm diameter ; 
  spectrum ) and equilibrated with bd buffer containing 100 mm 
  ammonium sulfate ( bd-100 ; 20 mm tris , ph 7.9 , 0.2 mm 
  edta , 2 mm dithiothreitol , 20% glycerol , 100 mm ammonium 
  sulfate , 0.2 mm phenylmethylsulfonyl fluoride ) .
bound proteins were 
  eluted by gravity flow using bd containing 350 mm ammonium sulfate 
  ( bd-350 ) .
the elution fraction was dialyzed against bd-100 and subjected to 
  100 ml of heparin - sepharose 6ff ( ge healthcare ) that was equilibrated with 
  bd-100 .
bound proteins were eluted with a 10-cv linear gradient from 200 to 
  700 mm ammonium sulfate in bd .
fractions containing h1.4k26 hkmt 
  activity were pooled , dialyzed , and subjected to a 54-ml deae 5pw column 
  ( tosoh ) equilibrated with bd-100 .
subsequently , fractions containing the 
  activity were pooled , dialyzed against bd-100 , and subjected to a mono s 
  hr10/10 ( ge healthcare ) column .
the elution of bound proteins was carried out 
  as described for the deae 5pw step .
fractions containing activity were pooled , 
  dialyzed against bd-100 , and subjected to a mono q hr5/5 column ( ge 
  healthcare ) .
the elution of bound proteins was carried out as described for 
  the deae 5pw step .
fractions containing activity were pooled and subjected to 
  mass spectrometric analysis as well as to a superose 6 hr10/30 gel filtration 
  column ( ge healthcare ) .
fractions from the gel filtration chromatography were 
  resolved by sds - page and visualized by silver staining or subjected to 
  immunoblotting . protein expression and purification , western blot , and silver 
  staining  recombinant histone h1.4 wild type or mutant ( lysine 26 
  changed to alanine ; h1.4 k26a ) was produced in bacteria as an ha - tagged fusion 
  protein using standard techniques .
affinity purified h1.4 protein exhibited 
  multiple bands in sds - page likely due to truncated versions of the protein . 
 
recombinant full - length g9a was obtained using a baculoviral expression system 
  according to standard techniques .
baculovirally expressed gst - jmjd2a 
  ( residues 1506 ) , gst - jmjd2b ( residues 1424 ) , gst - jmjd2c 
  ( residues 1372 ) , and flag - jmjd2d ( residues 1510 ) were purified 
  using glutathione - sepharose 4b ( ge healthcare ) or anti - flag(m2 ) affinity gel 
  ( sigma ) .
western blotting was performed using standard techniques with 
  antibodies against ha ( sigma ) , flag(m2 ) ( sigma ) , histone h1.4k26me2 ( abcam ; we 
  find that the antibody showed a certain extent of cross - reactivity to 
  h1.4k26me1 and -me3 in peptide dot blot experiments ( supplemental fig . 
 
s2a ) ) , mouse h1.4k26me1 ( jenuwein lab ; for details of antibody 
  generation strategy see ref . 
  31 ) , mouse h1.4k26me2/3 
  ( 7 ) ( jenuwein lab ) histone h1 
  ( santa cruz ) , h3k9me1 ( 32 ) 
  ( jenuwein lab ) , h3k9me2 ( millipore ) , h3k9me3 
  ( 32 ) ( jenuwein lab ) , h3k27me3 
  ( abcam ) , h3k27me2/3 ( reinberg lab ) , h4k20me1 ( millipore ) , h4k20me3 
  ( millipore ) , h3 ( cell signaling ) , gal4 ( santa cruz ) , -actin ( ambion ) , 
  gst ( ge healthcare ) , ezh2 ( abcam ) , suz12 ( abcam ) , eed ( abcam ) , and g9a 
  ( yoshihiro nakatani ) .
silver staining was performed using standard molecular 
  biology methods . in vitro hkmt assay 
briefly , the reaction was performed in a total volume of 30 l in hkmt 
  buffer ( 20 mm tris , ph 8.0 , 5 mm mgcl2 ) with 
  0.6 l of 0.2 m dithiothreitol , 1 l of 
  [ methyl - h]adomet ( trk581 - 1mci , ge healthcare ) , 110 
  l of a source of enzymatic activity ( either fractions of conventional 
  purification or 50 nm recombinant g9a ) , and 8 l of 
  substrate . the substrate consisted of 2 g of recombinant histone h1.4 
  ( wild type or k26a mutant ) or 5 g of synthetic peptides corresponding to 
  either the n terminus of histone h3 ( residues 415 ) or that of h1.4 
  ( residues 1734 ) .
hkmt assays were resolved by 15% sds - page , transferred to 
  polyvinylidene difluoride membrane , and analyzed by fluorography using 
  [ h]enhance spray ( perkinelmer life sciences ) . in vitro hkdm assay
 the demethylation reaction was carried 
  out for 38 h at 37 c in the presence of 30200 ng/l of 
  recombinant jmjd2 proteins , 2 m peptide substrate , 20 
  m fe(ii)-sulfate , 2 m zncl2 , 500 
  m 2-oxoglutarate , 500 m ascorbic acid , 20 
  mm tris - hcl , ph 8.0 , and 120150 mm kcl in a final 
  volume of 8 l .
to inhibit demethylation the iron chelating agent 
  deferoxamine ( sigma ) was added to a final concentration of 0.2 mm . 
 
seventy l of matrix solution ( 5 mg / ml -cyano-4-hydroxycinnamic 
  acid , 37% ( v / v ) acetonitrile , 0.12% trifluoroacetic acid ) were added to the 
  reaction products , 1 l of the sample was spotted on a maldi sample plate , 
  and spectra were acquired as described 
  ( 34 ) .
the following peptides 
  were used as substrates : h3k4me3 , art[kme3]qtarkstggkaprkql - cys ; h3k9me3 , 
  artkqtar[kme3]stggkaprkql - cys ; h3k27me3 , kaar[kme3 ] sapatggvkkphryrp - cys ; 
  h4k20me3 , sgr gkgg[kme3]glgkggakrhrk - cys ; h1.4k26me3 , pvkkkar[kme3]saggakrk or 
  pvkkkar[kme3]aaggakrk ; h1.4k26me2 , tpvkkkar[kme2]aaggakr - cys ; and h1.4k26me1 , 
  pvkkkar[kme1]saggakrk .
histone samples were excised 
  from sds - pa gels , in - gel digested with trypsin ( at a w / w ratio of 1:200 , 
  trypsin : protein , estimated by the staining intensity of coomassie blue - stained 
  histones ) for 2 h before the reaction was stopped by addition of 5% formic 
  acid and 60% acetonitrile .
dried digests were solubilized in 20 
  l of 50 mm ammonium hydrogen carbonate followed by addition of 
  50 l of a propionic anhydride solution ( at a v / v ratio of 30:70 , propionic 
  anhydride : methanol ) and adjustment of ph 7 with nh4oh .
the samples 
  were incubated at 50 c for 20 min and dried under vacuum before 
  lc - ms / ms .
all lc - ms / ms experiments were performed using a dionex ultimate nanoflow 
  hplc system ( sunnyvale ca ) and a thermo ltq mass spectrometer ( san jose , ca ) . 
 
histone samples were first solubilized in 0.1% trifluoroacetic acid and loaded 
  onto a trap column ( dionex acclaim pepmap 100 c18 , 5 m , 100  , 
  300-m inner diameter  5 mm ) . after washing with 0.1% 
  trifluoroacetic acid at 20 l / min for 2 min ,
the peptides were back - flashed 
  onto a 75 m  12-cm emitter column packed with magic c18aq , 3-m 
  200  ( michrom bioresources inc .
, aubum , ca ) , and eluted with a linear 
  gradient of 2 to 45% acetonitrile containing 0.1% formic acid in 30 min at a 
  flow rate of 250 nl / min .
mass spectrometry data were acquired using a 
  data - dependent acquisition procedure with each full ms followed by analysis of 
  the five most intense ions by a zoom scan and a ms / ms scan .
data dependent 
  analysis used a repeat count of two and a dynamic exclusion duration of 60 
  s. transfections , stable cell line3
 10 
  293 t cells were transfected with 10 g of plasmid dna using fugene hd 
  ( roche ) according to the manufacturer 's instructions .
cells were incubated for 
  48 h post - infection , harvested , and whole cell extract prepared using ripa 
  buffer ( 10 mm tris , ph 7.4 , 150 mm nacl , 1% nonidet 
  p-40 , 0.5% deoxycholic acid , 0.1% sds , 1 mm edta ) .
the generation of the 
  nih3t3-tet off cells ( clontech ) with a stably integrated , inducible 
  jmjd2b(1424)-green fluorescent protein expression construct was 
  described previously ( 35 ) . 
 
induction was performed for 12 h and green fluorescent protein - positive cells 
  were fluorescence - activated cell sorter - sorted and histones extracted .
histone extraction  core and linker histones were extracted 
  from 293 , 293 ft , hela , 293trex - luciferase ( see below ) , nih 3t3 , and embryonic 
  stem cells using 0.2 m sulfuric acid according to standard 
  protocols .
the acidic supernatant ( containing histones ) was neutralized by the 
  addition of 0.75 volume of 1.5 m tris , ph 8.8 .
cells were either 
  untreated or treated with the g9a small molecule inhibitor bix-01294 ( kindly 
  provided by boehringer ingelheim pharmaceuticals , inc . ) or the histone 
  deacetylase inhibitor trichostatin a ( sigma ) .
immunoprecipitation  one mg of solubilized nuclear pellet was 
  mixed with 1 volume of bd-200 plus 0.05% nonidet p-40 .
five g of antibody 
  were added and the reaction mixture ( 200 l ) incubated overnight at 4 
  c on a rotating wheel .
twenty l of pre - equilibrated protein g - agarose 
  ( roche ) were added and incubation continued for 1 h at 4 c on a rotating 
  wheel .
reactions were spun in a microcentrifuge at 400  g for 
  3 min , the supernatant was discarded , and beads washed two times with 400 
  l of bc-200 containing 0.05% nonidet p-40 , one time with 400 l of 
  bc-500 , 0.05% nonidet p-40 , followed by one time with 400 l of bc-100 . 
 
reverse transcriptase - pcr  hela cells were grown in the 
  presence of 1% dmso or 2.5 m decitabine 
  ( 5-aza-2-deoxycytidine ) in dmso for 72 h. total rna was 
  isolated using trizol ( invitrogen ) and cdna was produced using the 
  superscript iii cdna synthesis kit according to the manufacturer 's 
  instructions .
real time pcr was performed using the brilliant ii 
  sybr green qpcr master mix ( stratagene ) .
we previously generated a 293 trex cell line 
  ( invitrogen ) that is stably transfected with a constitutively expressing 
  luciferase transgene containing gal4 dna binding sites upstream of the 
  transcriptional start site ( 293trex - luciferase 
  ( 8) ) .
this cell line was stably 
  transfected with pcdna4to ( invitrogen ) containing the gal4-dbd and full - length 
  cdna of human g9a ( inserted between the hindiii and ecori sites ) .
gal4-tagged 
  g9a was expressed upon addition of 2 g / ml tetracycline / doxycycline for 24 
  or 48 h. chip was performed in duplicate according to standard techniques and 
  as previously described ( 36 ) 
  using the following primer set : upper , 
  5-caccgagcgaccctgcataagc-3 and lower , 
  5-gcttctgccaaccgaacggac-3. briefly , cells were treated with 1% 
  formaldehyde in the growth medium for 10 min at room temperature . 
 
cross - linking was quenched by the addition of glycine to a final concentration 
  of 125 mm in growth medium .
cells were washed twice with 
  phosphate - buffered saline and lysed in buffer a ( 25 mm tris , ph 
  8.0 , 1% sds , 1 mm edta , 25 mm nacl ) .
cell lysate was 
  sonicated using a bioruptor 200 ( diagenode ) with 200 watts ultrasonic 
  wave output power .
sonication was carried out for 15 min with alternating 30-s 
  on / off pulses .
sheared chromatin was used for immunoprecipitation using 
  antibodies described above and antibodies against gal4 ( millipore ) and random 
  igg ( jackson ) .
h1.4k26me2 antibody competition with histone peptides was 
  carried out as follows : 5 g of antibody was mixed in a tube with 1 
  m h3k9me2 , h1k26me0 , and h1k26me2 peptides in a final volume of 
  40 l for 30 min on ice .
chip experiments on the endogenous mage - a1 promoter were 
  carried out in triplicate as described above using hela cells grown in the 
  presence or absence of 2.5 m decitabine for 72 h. the following 
  primer set was used : oligo 1 , 5-gtccaggctctgccagacatc-3 and 
  oligo 2 , 5-cgtccctcagaatggaaacctc-3. the gapdh chip 
  primer set is located around the transcriptional start site and the sequence 
  is available upon request .
a , hkmt assays carried out with increasing amounts of nuclear extract 
  ( left ) or nuclear pellet ( right ) using recombinant wild type 
  histone h1.4 or a version with a lys to ala point mutation as 
  substrate .
c , mono q peak fraction of h1.4k26 hkmt activity was 
  analyzed by sds - page and silver staining ( left ) .
specificity is 
  illustrated by hkmt assay using wild type or k26a mutant h1.4 as substrate 
  ( top ) .
the fraction was subjected to mass spectrometric analysis and 
  identified hkmts were confirmed by immunoblotting ( right ) . 
 
d , h1.4k26 hkmt activity peak from the mono q step was pooled and 
  subjected to a superose 6 gel filtration column .
fractions were either 
  analyzed by hkmt assay or resolved by sds - page and analyzed by silver staining 
  or immunoblotting .
e , immunoprecipitation experiment from nuclear 
  pellet fraction using igg , anti - g9a , and anti - ezh2 antibodies .
a , hkmt assays carried out with increasing amounts of nuclear extract 
  ( left ) or nuclear pellet ( right ) using recombinant wild type 
  histone h1.4 or a version with a lys to ala point mutation as 
  substrate .
c , mono q peak fraction of h1.4k26 hkmt activity was 
  analyzed by sds - page and silver staining ( left ) .
specificity is 
  illustrated by hkmt assay using wild type or k26a mutant h1.4 as substrate 
  ( top ) .
the fraction was subjected to mass spectrometric analysis and 
  identified hkmts were confirmed by immunoblotting ( right ) . 
 
d , h1.4k26 hkmt activity peak from the mono q step was pooled and 
  subjected to a superose 6 gel filtration column .
fractions were either 
  analyzed by hkmt assay or resolved by sds - page and analyzed by silver staining 
  or immunoblotting .
e , immunoprecipitation experiment from nuclear 
  pellet fraction using igg , anti - g9a , and anti - ezh2 antibodies . precipitated 
  material was analyzed by hkmt assays or immunoblotting .
purification of h1.4k26 methyltransferase activities  we , and 
  others previously reported that h1.4k26 is methylated in vivo 
  ( 2 , 
  5 , 
  7 ) . in an unbiased attempt to 
  identify h1.4k26-specific methyltransferases we used recombinant bacterially 
  expressed histone h1.4 as a substrate for in vitro hkmt assays and 
  readily detected the h1-specific hkmt activity in both nuclear extract and 
  pellet fractions .
the target specificity of the hkmt activity assays was 
  verified by comparing methylation of wild type h1.4 to that of a version in 
  which the lys residue was mutated to alanine ( h1.4k26a ) 
  ( fig .
notably , 
  methylation of the k26a mutant was strongly reduced but not completely 
  abolished , suggesting that other residue(s ) on histone h1.4 in addition to 
  lys are methylated .
the endogenous core histones h3 and h4 were 
  methylated when the nuclear pellet fraction was used in hkmt assays . 
  surprisingly , the addition of recombinant h1.4k26a but not wild type h1.4 
  abolished the activity toward h4 . at present
we do not know if this finding is 
  relevant and did not pursue it further in this study .
we next performed a conventional purification from the nuclear pellet 
  following h1.4 hkmt activity ( fig . 
 
1b ) and found that h1.4k26-specific and 
  non - lys - specific activities co - eluted throughout the entire 
  purification ( supplemental fig .
1c ) was subjected to mass spectrometric analysis . among 
  other polypeptides we identified three hkmts :
g9a / kmt1c , glp / kmt1d , and 
  ezh2/kmt6 and the presence of each was verified by western blot 
  ( fig .
analysis of the h1.4k26 hkmt activity by gel 
  filtration chromatography revealed that the activity peak migrated at high 
  molecular mass ( 400800 kda ) and correlated well with both the g9a and 
  ezh2 elution profiles as visualized by western blot 
  ( fig .
interestingly , a larger migrating h1.4 protein species was mainly methylated 
  in fraction b14 and its radiolabel intensity did not correlate with the g9a 
  and ezh2 elution profiles .
although fraction b14 contained g9a and ezh2 , it is 
  unclear why its substrate preference was different from that of fractions 
  b8b12 .
the methylation on this h1.4 protein was also lys 
  specific because the radiolabel was lost when the fractions were assayed with 
  an h1.4k26a mutant protein ( fig . 
 
therefore , 
  it was possible that ezh2 is solely responsible for the h1.4 methylation 
  observed . to clarify this we performed immunoprecipitations from nuclear 
  pellet fractions and demonstrated
that : 1 ) g9a and ezh2 do not interact ; 2 ) 
  both g9a and ezh2 immunoprecipitates contain h1.4 hkmt activity compared with 
  an igg control ( fig . 
 
1e ) ; and 3 ) the presence of histone octamers in the hkmt 
  assay does not abolish g9a activity toward h1.4 ( supplemental fig . 
 
significantly less activity is precipitated with anti - ezh2 but 
  this might be explained by the fairly stringent wash conditions that partially 
  disrupted the ezh2 containing prc2 complex .
g9a methylates h1.4 at lysine 26given that g9a was shown 
  previously to methylate native histone h1 in vitro 
  ( 37 ) we used recombinant 
  full - length g9a to perform in vitro hkmt assays with either wild type 
  h1.4 or its k26a mutant as substrate .
we detected significant 
  [ h]ch3 incorporation on wild type h1.4 as evidenced by 
  fluorography ( fig .
2a ) 
  but a signal was not detected on h1.4k26a . however , longer exposure times gave 
  evidence of minor [ h]ch3 label ( data not shown ) 
  suggesting that other lysines might be methylated with low efficiency .
the 
  reaction was carried out in substrate excess to attain the maximum amount of 
  methylation under the given assay conditions to facilitate mass spectrometric 
  analysis ( see fig . 
 
we monitored the increase in lys 
  methylation upon incubation of ha - tagged h1.4 with g9a and adomet using 
  anti - h1.4k26me2-specific antibody ( fig . 
 
importantly , the antibody was methyl - specific 
  because recombinant un - methylated h1.4 was not recognized despite being 
  recognized by an anti - ha antibody ( fig . 
 
the multiple bands are recognized by anti - ha and 
  anti - h1.4k26me2 antibodies likely resulted from h1.4 truncations ( see 
   experimental procedures  ) .
next , we used synthetic peptides 
  corresponding to residues 1734 of h1.4 that were either unmethylated or 
  harbored one , two , or three methyl groups at lys as substrates 
  for g9a - dependent hkmt assays .
g9a only utilized unmethylated and 
  monomethylated peptides in a time - limiting reaction , similar to the result 
  obtained when h3k9-peptides were used as substrate 
  ( fig .
therefore , 
  g9a predominantly functions as an h3k9 and h1.4k26 mono- and dimethylase 
  in vitro , although extended incubation times allowed g9a to perform 
  trimethylation of the substrate ( data not shown ; previously discussed in ref . 
 
g9a - methylated h1.4 was subjected to mass spectrometry to unambiguously 
  identify target sites . due to the high lysine content of h1.4
the sample was 
  propionylated before digestion to better distinguish mass differences in 
  lc - ms / ms .
the mass spectra of recombinant h1.4 incubated with g9a in the 
  presence of adomet resulted in the detection of peptides with mono- and 
  dimethylated h1.4k26 ( fig . 
 
2d , middle and bottom panels , spectra 
  shown in black ) . in the absence of g9a or adomet ,
2d , top 
  panel , spectra shown in black and red , middle and 
  bottom panels , spectra shown in red ) .
in addition to h1k26 
  we found minor amounts of methyl groups incorporated on several other lysine 
  residues ( fig .
g9a methylates h1.4 at lysine 26 in vivo  we next tested if 
  g9a impacted h1.4k26 methylation in cell - based assays . to this end
overproduction of g9a significantly increased global h1.4k26 dimethylation and 
  also moderately increased the level of h3k9me2 .
gal4-dbd alone or fused to the 
  h4k20-specific hkmt pr - set7/kmt5a did not alter h1.4k26me2 or h3k9me2 levels 
  ( fig .
previous 
  reports indicated that the lack of g9a substantially reduced h3k9me2 and -me1 
  in mouse embryonic stem cells 
  ( 32 , 
  39 , 
  40 ) .
we extracted core and 
  linker histones from these wild type and g9a stem 
  cell lines ( tt2 and 2210 , respectively ) , and observed a global reduction in 
  h1.4k26me2 in the case of g9a cells as visualized by 
  western blot ( fig . 
 
to demonstrate by alternative means that g9a is partially responsible for 
  h1.4k26 methylation in vivo we took advantage of a recently 
  identified , small molecule inhibitor of g9a .
the compound termed bix-01294 is 
  specific for g9a as even the highly homologous glp protein is far less 
  effectively inhibited ( 41 ) . 
 
hela cells were treated for 48 h with bix-01294 at a final concentration of 9 
  m , histones were extracted and analyzed by western blot . 
 
inhibition of g9a led to significant decreases in h1.4k26me2 and h3k9me2 
  levels ( fig .
3c ) , 
  whereas other histone lysine methylation marks were unaffected 
  ( fig . 3c and data not 
  shown ) .
interestingly , trichostatin a - treated cells also showed a considerable 
  reduction in global h1.4k26me2 levels .
previously , we demonstrated that 
  h1.4k26 is acetylated in vivo and that the class iii histone 
  deacetylase sirt1 targeted h1.4k26 
  ( 8) . now , our data suggest that 
  lys is also targeted by class i and ii histone deacetylases and 
  that acetylation of h1.4k26 might protect it from being methylated .
g9a methylates h1.4k26 on chromatin in a gene - specific 
  manner  generally , histone h1 deposition condenses chromatin 
  structure and negatively regulates gene expression , although various histone 
  h1 isotypes seem to affect transcription differently ( recently reviewed in 
  ref .
as histone h1.4 
  repressed transcription in reporter gene assays 
  ( 7 ) , we tested if g9a and h1.4 
  can co - occupy repressed genes . to this end a 293 cell line was stably 
  transfected with a construct encoding gal4-dbd fused in - frame to g9a 
  ( gal4-g9a ) under the control of a promoter that contains a tet - operator 
  sequence .
upon addition of doxycycline ( + tet ) gal4-g9a protein binds to gal4-specific 
  dna binding sites integrated in the promoter of the luciferase reporter gene 
  ( fig .
4b ) consistent 
  with earlier reports that described g9a as a transcriptional co - repressor 
  ( 3942 ) . 
 
induction of gal4-g9a expression for 48 h was monitored by a significant 
  increase in gal4-g9a protein as well as by a global increase in h1.4k26me2 and 
  h3k9me2 levels ( fig . 
 
a , recombinant full - length g9a derived from a 
  baculoviral expression system was used to methylate increasing amounts of 
  recombinant wild type or k26a mutant ha - tagged h1.4 using 
  h - labeled adomet as methyl donor .
shown is the fluorography after 
  an exposure time of 12 h ( top panel ) and the coomassie blue - stained 
  membrane to visualize the amount of h1.4 and h1.4k26a proteins ( bottom 
  panel ) .
b , as in a but using unlabeled adomet as methyl 
  donor . detection of methyl groups incorporated at h1.4k26 was carried out by 
  western blot using a specific anti - h1.4k26me2 antibody .
c , as in a but 
  using synthetic peptides corresponding to the sequence shown for h1.4 or h3 
  ( top ) as substrate .
d , recombinant wild type h1.4 was incubated with g9a in the presence 
  or absence of unlabeled adomet as methyl donor .
the reactions were resolved by 
  sds - page , stained with coomassie brillant blue , and the h1.4 bands then cut 
  from the gel and subjected to mass spectrometry .
ion chromatograms of 
  kksagaar peptides are shown with different modifications 
  generated from lc - ms / ms data of propionylated partial tryptic digest of 
  histone h1.4 .
only the sample incubated with g9a and adomet led to the 
  detection of monomethylated ( [ m + 2h ] = 578.1 ) and dimethylated 
  lys ( [ m + 2h ] = 557.2 ) ( spectra shown in 
  black ) .
only unmodified h1.4k26 peptides were detected in the absence 
  of adomet ( spectra shown in red ) . in the absence of g9a
only 
  unmodified ( [ m + 2h ] = 570.8 ) lys peptides were 
  detected .
e , the table summarizes other potential h1.4 target sites 
  and the percentage of methylation compared with an unmethylated h1.4 
  control .
a , recombinant full - length g9a derived from a 
  baculoviral expression system was used to methylate increasing amounts of 
  recombinant wild type or k26a mutant ha - tagged h1.4 using 
  h - labeled adomet as methyl donor .
shown is the fluorography after 
  an exposure time of 12 h ( top panel ) and the coomassie blue - stained 
  membrane to visualize the amount of h1.4 and h1.4k26a proteins ( bottom 
  panel ) .
detection of methyl groups incorporated at h1.4k26 was carried out by 
  western blot using a specific anti - h1.4k26me2 antibody .
c , as in a but 
  using synthetic peptides corresponding to the sequence shown for h1.4 or h3 
  ( top ) as substrate .
d , recombinant wild type h1.4 was incubated with g9a in the presence 
  or absence of unlabeled adomet as methyl donor .
the reactions were resolved by 
  sds - page , stained with coomassie brillant blue , and the h1.4 bands then cut 
  from the gel and subjected to mass spectrometry .
ion chromatograms of 
  kksagaar peptides are shown with different modifications 
  generated from lc - ms / ms data of propionylated partial tryptic digest of 
  histone h1.4 . only the sample incubated with g9a and adomet led to the 
  detection of monomethylated ( [ m + 2h ] = 578.1 ) and dimethylated 
  lys ( [ m + 2h ] = 557.2 ) ( spectra shown in 
  black ) .
only unmodified h1.4k26 peptides were detected in the absence 
  of adomet ( spectra shown in red ) . in the absence of g9a
only 
  unmodified ( [ m + 2h ] = 570.8 ) lys peptides were 
  detected .
e , the table summarizes other potential h1.4 target sites 
  and the percentage of methylation compared with an unmethylated h1.4 
  control .
a , 293 t cells were 
  transiently transfected with gal4 , gal4-pr - set7 , or gal4-g9a .
whole cell 
  extract was prepared from transfected cells or acid extraction was performed 
  to isolate core and linker histones and both fractions were analyzed by 
  western blot using antibodies indicated on the right .
b , acid extraction of histones was performed 
  from wild type ( tt2 ) or g9a ( 2210 ) 
  embryonic stem cells and purified histones were analyzed by western blot using 
  antibodies indicated on the right .
c , 293 t cells were treated for 48 
  h with dmso , compound bix-01294 ( a specific g9a inhibitor ; final concentration 
  9 m ) , or trichostatin a ( final concentration 300 
  nm ) .
high salt nuclear extracts were prepared from treated cells 
  and analyzed by western blot using the antibodies indicated on the 
  right .
a , 293 t cells were 
  transiently transfected with gal4 , gal4-pr - set7 , or gal4-g9a .
whole cell 
  extract was prepared from transfected cells or acid extraction was performed 
  to isolate core and linker histones and both fractions were analyzed by 
  western blot using antibodies indicated on the right .
b , acid extraction of histones was performed 
  from wild type ( tt2 ) or g9a ( 2210 ) 
  embryonic stem cells and purified histones were analyzed by western blot using 
  antibodies indicated on the right .
c , 293 t cells were treated for 48 
  h with dmso , compound bix-01294 ( a specific g9a inhibitor ; final concentration 
  9 m ) , or trichostatin a ( final concentration 300 
  nm ) .
high salt nuclear extracts were prepared from treated cells 
  and analyzed by western blot using the antibodies indicated on the 
  right . to determine whether g9a - mediated luciferase repression correlates with h1 
  deposition and h1.4k26 methylation we performed chip experiments . as expected , 
 
gal4-g9a was recruited to the promoter region of the luciferase gene in the 
  presence of doxycycline .
interestingly , histone h1 occupancy was low on the 
  luciferase gene but increased upon luciferase repression by gal4-g9a
. 
  moreover , the levels of h3k9me2 and h1.4k26me2 increased in the presence of 
  doxycycline , whereas those of h3k27me3 remained unchanged 
  ( fig .
4d ) . to rule out 
  that the h1.4k26me2-specific antibody used for chip cross - reacted with h3k9me2 
  we performed peptide competition experiments .
dot blot analysis confirmed that 
  the anti - h1.4k26me2 antibody specifically recognized h1.4 peptides but not 
  h3k9 , h3k27 , or h4k20 peptides .
moreover , the recognition of h1.4k26me2 could 
  be abolished only upon preincubating anti - h1.4k26me2 antibodies with 
  h1.4k26me2 peptides , but not with h3k9me2 or h3k27me2 peptides ( supplemental 
  fig .
we repeated the chip with h1.4k26me2 antibodies that were competed 
  with various histone peptides prior to the immunoprecipitation step .
analysis 
  of precipitated material by real time - quantitative pcr showed that h3k9me2 and 
  h1.4k26me0 peptides did not significantly reduce the amount of precipitated 
  material ( p > 0.05 ) .
of note , an h3k9me2-specific antibody was 
  completely neutralized by the h3k9me2 peptide under identical conditions . 
 
competition with h1.4k26me2 peptides reduced significantly the signal 
  intensity as determined by paired t - tests ( p < 0.05 ; 
  fig .
the fact 
  that the h1.4k26me2 peptide did not fully abolish the signal indicates that 
  the antibody shows a low level of cross - reactivity .
results from the chip experiments on the luciferase transgene suggest that 
  g9a alters the chromatin structure by promoting the recruitment of h1 . to test 
  this hypothesis on an endogenous gene we focused on mage - a genes , 
  previously shown as being occupied by g9a and with its presence
mage - a gene silencing is also mediated by dna methylation in a number 
  of tumor cell lines .
it is well established that global and gene - specific dna 
  methylation decreases upon addition of the cytosine analog decitabine . 
 
treatment with decitabine was previously shown to reactivate tumor suppressor 
  genes , with g9a occupancy at these genes being substantially reduced 
  ( 44 ) .
we selected 
  mage - a1 for a comparative chip analysis before and after addition of 
  decitabine . in the absence of decitabine , g9a and histone h1 were present on 
  the mage - a1 gene and h1.4 was methylated at lys 
  ( fig .
that mage - a transcripts are detectable in hela cells 
  ( 43 ) we found mage - a1 
  and -a3 gene expression to be induced upon treatment with decitabine 
  ( fig .
, 
  g9a was lost from the mage - a1 promoter region and importantly , the 
  levels of h1k26 methylation were also reduced , presumably due to the decrease 
  in total h1 protein .
these results are in accord with those obtained using the 
  luciferase reporter gene above , suggesting that g9a promotes spatially 
  restricted h1 residency on chromatin .
figure 4.g9a methylates h1.4k26 on a specific genomic location and promotes h1 
  recruitment . a , illustration of the transgene system that allows 
  tetracycline - induced expression of gal4-g9a and targeting to the luciferase 
  promoter containing gal4 dna binding sites .
b , 
  induction of gal4-g9a effectively represses luciferase expression as 
  demonstrated by luciferase assays of two independent clones .
the experiments 
  were carried out in duplicate and the result of one representative experiment 
  is shown .
c , whole cell extracts were prepared from two independent 
  clones that were grown in the absence or presence of tetracycline for 24 h. 
  extracts were analyzed by western blot using the antibodies indicated on the 
  right .
d , chip experiments from cells grown in the absence or 
  presence of tetracycline for 24 h. antibodies used for the chips are indicated 
  on the right .
e , chips with anti - h1.4k26me2 antibodies either untreated or 
  preincubated with synthetic peptides corresponding to various histone 
  sequences and modification states . quantitative real time pcr analysis for 
  each sample was carried out in triplicate .
competition with h3k9me2 and 
  h1.4k26me2 peptides did not result in statistically relevant ( p > 
  0.05 ) reduction of pcr product .
f , quantitative real time rt - pcr 
  measuring mage - a1 and -a3 transcript levels from hela cells 
  treated for 72 h with dmso or decitabine .
g , chip experiments carried out from 
  293 cells analyzing the mage - a1 promoter in the absence or presence 
  of decitabine .
g9a methylates h1.4k26 on a specific genomic location and promotes h1 
  recruitment . a , illustration of the transgene system that allows 
  tetracycline - induced expression of gal4-g9a and targeting to the luciferase 
  promoter containing gal4 dna binding sites .
b , 
  induction of gal4-g9a effectively represses luciferase expression as 
  demonstrated by luciferase assays of two independent clones .
the experiments 
  were carried out in duplicate and the result of one representative experiment 
  is shown .
c , whole cell extracts were prepared from two independent 
  clones that were grown in the absence or presence of tetracycline for 24 h. 
  extracts were analyzed by western blot using the antibodies indicated on the 
  right .
d , chip experiments from cells grown in the absence or 
  presence of tetracycline for 24 h. antibodies used for the chips are indicated 
  on the right .
e , chips with anti - h1.4k26me2 antibodies either untreated or 
  preincubated with synthetic peptides corresponding to various histone 
  sequences and modification states .
competition with h3k9me2 and 
  h1.4k26me2 peptides did not result in statistically relevant ( p > 
  0.05 ) reduction of pcr product .
f , quantitative real time rt - pcr 
  measuring mage - a1 and -a3 transcript levels from hela cells 
  treated for 72 h with dmso or decitabine .
g , chip experiments carried out from 
  293 cells analyzing the mage - a1 promoter in the absence or presence 
  of decitabine .
figure 5.g9a - methylated h1.4 is recognized by l3mbtl1 and hp1 in 
  vitro . a , gst pull - down experiment with gst or gst fused to 
  the three mbt domains of l3mbtl1 ( gst-3mbt ) and recombinant ha - tagged h1.4 
  either unmethylated or methylated by g9a .
notably , a point mutation in the 
  second mbt domain ( p2a ) abolished binding but mutations in the first 
  and third mbt domains ( p1a and p3a ) retained binding to 
  g9a - methylated h1.4 .
b , gst pull - down experiment with gst or 
  gst - hp1 and recombinant ha - tagged h1.4 either unmethylated or 
  methylated by g9a . methylated h1.4 was visualized by western blot using 
  anti - h1.4k26me2 antibodies .
g9a - methylated h1.4 is recognized by l3mbtl1 and hp1 in 
  vitro . a , gst pull - down experiment with gst or gst fused to 
  the three mbt domains of l3mbtl1 ( gst-3mbt ) and recombinant ha - tagged h1.4 
  either unmethylated or methylated by g9a .
notably , a point mutation in the 
  second mbt domain ( p2a ) abolished binding but mutations in the first 
  and third mbt domains ( p1a and p3a ) retained binding to 
  g9a - methylated h1.4 .
b , gst pull - down experiment with gst or 
  gst - hp1 and recombinant ha - tagged h1.4 either unmethylated or 
  methylated by g9a . methylated h1.4 was visualized by western blot using 
  anti - h1.4k26me2 antibodies .
methylated at lys by g9a provides a binding 
  surface for l3mbtl1 and hp1chromodomain - containing hp1 and mbt 
  domain - containing l3mbtl1 proteins were previously shown to specifically 
  recognize methylated h1.4 ( 36 , 
  45 ) . to examine
if g9a is able 
  to mediate this binding event we performed pull - down assays using recombinant 
  h1.4 that was pre - methylated by g9a . a gst fusion protein comprising the three 
  mbt domains of l3mbtl1 co - precipitated with methylated , but not unmethylated 
  h1.4 .
we previously showed that the second mbt domain was responsible for 
  methyllysine binding ( 36 ) and 
  indeed a point mutation in the second ( p2a ) but not in the first or third 
  ( p1a , p3a ) mbt domains abolished the interaction with g9a - methylated h1.4 
  ( fig .
5a ) . similarly , 
  gst - hp1 but not gst alone interacted with g9a - methylated h1.4 
  ( fig .
5b ) . 
  importantly , unmethylated h1.4 was not precipitated when g9a was added to the 
  pull - down assay , ruling out the possibility that g9a bridges the interaction 
  between h1.4 and hp1. figure 6.members of the jmjd2 subfamily of the jmjc domain - containing proteins 
  demethylate h1.4k26 in vitro and in vivo .
a , 
  identification of mouse jmjd2d h1.4k26 demethylase activity by maldi - tof mass 
  spectrometry .
each panel contains a spectrum for h3k4me3 , h3k9me3 , h3k27me3 , 
  h4k20me3 , h1.4k26me3 , h1.4k26me2 , and h1.4k26me1 peptides incubated with or 
  without mouse jmjd2d protein .
the masses corresponding to unmodified 
  ( 0 ) , mono- ( 1 ) , di- ( 2 ) , or trimethylated 
  ( 3 ) peptides are indicated in dotted lines .
the appearance 
  of a peak corresponding to the demethylated peptide is marked with an 
  arrowhead .
the shift corresponds to a loss of 14 , 28 , or 42 daltons 
  due to the removal of methyl group(s ) .
b , jmjd2a , jmjd2b , and jmjd2c 
  were also identified as h1k26me3 demethylases .
addition of the iron chelator 
  deferoxamine at the beginning of the reaction suppressed the formation of a 
  demethylated peptide peak .
c , nih3t3 cells stably expressing mouse 
  jmjd2b display a reduction in h1.4k26me2/3 and h3k9me3
acid - extracted histones ( 1 or 2 g ) were 
  analyzed as indicated by triangles on the top .
d , ectopic 
  expression of human jmjd2d wild type but not its catalytic point mutant 
  ( h192a ) in 293 t cells causes global reduction in h1.4k26me2/3 and h3k9me2 . 
 
members of the jmjd2 subfamily of the jmjc domain - containing proteins 
  demethylate h1.4k26 in vitro and in vivo . a , 
  identification of mouse jmjd2d h1.4k26 demethylase activity by maldi - tof mass 
  spectrometry .
each panel contains a spectrum for h3k4me3 , h3k9me3 , h3k27me3 , 
  h4k20me3 , h1.4k26me3 , h1.4k26me2 , and h1.4k26me1 peptides incubated with or 
  without mouse jmjd2d protein .
the masses corresponding to unmodified 
  ( 0 ) , mono- ( 1 ) , di- ( 2 ) , or trimethylated 
  ( 3 ) peptides are indicated in dotted lines .
the appearance 
  of a peak corresponding to the demethylated peptide is marked with an 
  arrowhead .
the shift corresponds to a loss of 14 , 28 , or 42 daltons 
  due to the removal of methyl group(s ) .
b , jmjd2a , jmjd2b , and jmjd2c 
  were also identified as h1k26me3 demethylases .
addition of the iron chelator 
  deferoxamine at the beginning of the reaction suppressed the formation of a 
  demethylated peptide peak .
c , nih3t3 cells stably expressing mouse 
  jmjd2b display a reduction in h1.4k26me2/3 and h3k9me3 .
acid - extracted histones ( 1 or 2 g ) were 
  analyzed as indicated by triangles on the top .
d , ectopic 
  expression of human jmjd2d wild type but not its catalytic point mutant 
  ( h192a ) in 293 t cells causes global reduction in h1.4k26me2/3 and h3k9me2 . 
 
identification of h1.4k26 demethylases  candidate approaches 
  have been useful to identify the targets of the jmjc domain - containing histone 
  lysine demethylases .
we , and others previously identified jmjd2 proteins to 
  specifically demethylate h3k9 and h3k36 
  ( 34 , 
  35 , 
  46 , 
  47 ) . here , we tested a large 
  panel of jmjc proteins among which jmjd2 proteins exhibited significant 
  activity toward methylated h1.4k26 .
we purified recombinant mouse jmjd2 
  proteins from baculovirus - infected sf9 cells ( supplemental fig .
s3 ) and 
  examined demethylase activity toward methylated h1.4k26 peptides along with 
  trimethylated h3k4 , h3k9 , h3k27 , and h4k20 peptides . recombinant jmjd2d 
  efficiently converted h1.4k26me3 and h1.4k26me2 to the monomethyl state as 
  detected by maldi - tof mass spectrometry , whereas conversion of an h1.4k26me1 
  peptide substrate to the unmethylated state was inefficient 
  ( fig .
conversion 
  of h1.4k26me3 to the monomethylated state is consistent with the previous 
  observation that human jmjd2d removes two methyl moieties from trimethylated 
  h3k9 ( 35 , 
  47 ) . under the assay 
  conditions used in fig . 
 
6a complete h3k9 demethylation was observed due to the 
  relatively high enzyme concentration , whereas the use of lower amounts of 
  enzyme or shorter incubation times predominantly resulted in accumulation of 
  h3k9me1 ( data not shown ) .
there was no detectable activity toward h3k4me3 and 
  h4k20me3 , and only weak activity toward h3k27me3 . under similar assay 
  conditions , recombinant jmjd2a ,
jmjd2b , and jmjd2c removed only one methyl 
  group from h1.4k26me3 peptides ( fig . 
 
6b ) , again consistent with their ability to convert 
  h3k9me3 to the dimethyl state in vitro 
  ( 47 ) .
the addition of the iron 
  chelating agent deferoxamine abolished the production of h1.4k26me2 
  ( fig .
we also 
  examined the enzymatic activity of recombinant mouse lsd1 and 
  drosophila lid proteins , which were shown to demethylate h3k4me1 , or 
  -me2 and me3 , respectively 
  ( 34 , 
  46 ) .
we did not detect h1k26 
  demethylation by these enzymes under conditions wherein h3k4 peptide 
  demethylation was observed ( data not shown ) .
these results indicate that in 
  addition to being h3k9me3-specific jmjd2 subfamily members are also 
  h1.4k26-specific demethylases . to test if jmjd2 proteins demethylate h1.4k26 in a cellular context we took 
  advantage of a previously generated nih3t3 cell line with stably integrated 
  inducible jmjd2b ( 35 ) . in 
  contrast to the host cell line , induction of jmjd2b exhibited a 4-fold 
  reduction in h3k9me3 levels concomitant with increased h3k9me1 levels 
  ( fig .
importantly , we also detected a reduction in h1.4k26me2/3 levels , but no 
  significant increase in global h1.4k26me1 levels .
this could be due to the 
  high basal levels of h1.4k26me1 relative to those of h1.4k26me2/3 . given that 
  mouse and human jmjd2 members are highly conserved we next assessed the 
  ability of ectopic jmjd2 proteins to target h1.4k26 in a human cell system .
we 
  transiently transfected 293 t cells with human jmjd2d , either the wild type or 
  point mutant in its catalytic domain ( h192a ; described in ref . 
 
48 ) . only functional jmjd2d 
  reduced global h1.4k26me2/3 levels and , consistent with earlier studies 
  ( 49 , 
  50 ) , those of h3k9me2 
  ( fig .
, we conclude that the mammalian family of jmjd2 proteins is 
  responsible for the enzymatic turnover of h1.4k26 methylation in 
  vivo .
our data show that h1.4k26 is subject to dynamic methylation and that g9a 
  is partially responsible for this process .
g9a is highly homologous to another 
  hkmt termed glp / euhmtase1 ( 51 ) 
  both of which were shown to exhibit specificity for h3k9 . for the purpose of 
  this study we focused on g9a but
indeed , we identified glp in our purification of h1.4k26 hkmt activity 
  ( supplemental table s1 ) and g9a and glp were shown previously to function in a 
  heteromeric complex with both enzymes contributing to global h3k9me1 and -me2 
  ( 40 ) . a recent report utilized 
  spot array technology ( described in refs . 
  52 and 
  53 ) to identify g9a substrates 
  on the basis of a g9a - target consensus sequence .
h1.4k26 was identified among 
  potential g9a substrates in the context of synthetic peptides 
  ( 54 ) .
we clarified that g9a 
  specifically methylates h1.4k26 using peptides and full - length recombinant 
  proteins with or without the presence of core histones .
importantly , we 
  demonstrated methylation of h1.4k26 by g9a in vivo using cell 
  biological , genetic , and pharmacological approaches .
recently , it was shown that the g9a and glp ankyrin repeats recognize 
  h3k9me1/2 .
it was speculated that one enzyme of the g9a - glp complex recognizes 
  the modification , whereas the other places the mark 
  ( 55 ) .
given the similarity of 
  the residues surrounding h3k9 and h1.4k26 we suspect that the ankyrin repeats 
  also recognize h1.4k26me2 .
should this be the case , it is possible that the 
  g9a - glp complex might recognize h1.4k26me2 and subsequently methylate h3k9 
  within the same chromatosome or recognize h3k9me1/2 to methylate h1.4k26 . 
  because g9a also methylates free h1.4 it is possible that binding of 
  h1.4k26me2 to the ankyrin repeats provides a means by which g9a promotes its 
  recruitment to chromatin .
4 , d and 
  e ) and it will be interesting to investigate in future 
  studies if a mutation in the ankyrin repeats that is deficient for histone 
  methyl - lysine binding affects h1-recruitment to chromatin .
the linker histone h1 was believed to constitute chromatin in a 1:1 ratio 
  with core histone octamers .
however , recent data demonstrated that h1 is not 
  omnipresent but in certain instances subject to localized recruitment to 
  establish facultative heterochromatin ( recently discussed by ref . 
 
moreover , genome - wide 
  binding data illustrated that histone h1 is present predominantly over 
  repressed genes and intergenic regions , whereas it is absent and replaced by 
  parp-1 along active genes 
  ( 56 ) .
therefore , it is likely 
  that mechanisms are in place to deposit h1 or to exchange other chromatin 
  binding factors with h1 .
covalent modifications on core histones and the 
  linker histone might affect such processes as recently demonstrated by studies 
  showing that the presence or absence of h2a monoubiquitination affects h1 
  deposition ( 57 , 
  58 ) .
5 , a and b ) that might assist in its recruitment 
  or increase its residence time on chromatin , thereby facilitating alterations 
  of the chromatin structure .
members of the jmjd2 
  subfamily of h3k9/h3k36-specific demethylases have now been shown to target 
  h1.4k26 as well .
it was proposed that jmjd2 proteins regulate gene expression 
  by locally altering histone methylation states .
demethylation of h1.4k26 would 
  diminish the ability of repressive effector proteins like hp1 and l3mbtl1 to 
  remain on chromatin and might also increase h1.4 mobility .
we previously found that h1.4k26 is methylated by the h3k27-specific hkmt 
  ezh2/prc2 and here we have shown that the h3k9-specific hkmt g9a methylates 
  h1.4k26 in vitro and in vivo .
several h3k9-hkmts in addition 
  to g9a are known in mammalian organisms and given the amino acid sequence 
  similarity around h3k9 , h3k27 , and h1.4k26 it will be interesting to examine 
  which of these histone methyltransferases , if any , have the potential to 
  methylate h1.4k26 .
previous reports and this study revealed that jmjd2 
  proteins target multiple sites including h1.4k26 but the h3k9-specific jmjd1 
  proteins and h3k27-specific demethylases like utx did not demethylate h1.4k26 
  in our assays .
this suggests that simple sequence similarity is not sufficient 
  to define a recognition site .
the crystal structural analysis of jmjd2a with 
  methylated h3k9 and h3k36 peptides nicely illustrated distinct recognition 
  mechanisms ( 59 ) and it would 
  be interesting to see if a similar or distinct mechanism applies to h1.4k26 
  binding .
 the linker histone h1 generally participates in the establishment of 
  chromatin structure . 
 however , of the seven somatic h1 isotypes in humans some 
  are also implicated in the regulation of local gene expression . 
 histone h1 
  isotype 4 ( h1.4 ) represses transcription , and its lysine residue 26 
  ( lys26 ) was found to be important in this aspect . 
 h1.4k26 is known 
  to be methylated and acetylated in vivo , but the enzymes responsible 
  for these post - translational modifications and the regulatory cues that 
  promote h1.4 residence on chromatin are poorly characterized . here 
 we report 
  that the euchromatic histone lysine methyltransferase g9a / kmt1c mediates 
  h1.4k26 mono- and dimethylation in vitro and in vivo and 
  thereby provides a recognition surface for the chromatin - binding proteins hp1 
  and l3mbtl1 . 
 moreover , we show evidence that g9a promotes h1 deposition and is 
  required for retention of h1 on chromatin 
 . we also identify members of the 
  jmjd2/kdm4 subfamily of jumonji - c type histone demethylases as being 
  responsible for the removal of h1.4k26 methylation . 