Methylation of histone H3 lysine9
occurs during translation
Speaker: Kuan-Yu Huang
Adviser: Kung-Yao Chang
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The chromatin package
Histone
Chromatin
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Methyl-histone H3 lysine 9
 A key mark in the estabilishment of functional chromatin
(Biochim Biophys. Acta. 2014)
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SetDB1/HP1α/CAF-1 complex establishes the H3
methylation and chromatin fromation
(in nuclear)
(EMBO Rep. 2009)
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(in cytoplasm)
Histone maturation cascade
Complex I
Complex II
Complex III
Complex IV
(Nat. Struct. Mol. Biol. 2010)
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The western-blot analysis of affinity-purified
cytoplasmic H3 protein complexes
(Nat. Struct. Mol. Biol.6 2010)
Motivation
1. When does H3K9me1 occur in the first time?
2. What is the complex responsible for its catalysis?
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Histone H3K9 mono- and dimethylation occurs at
the ribosome
HeLa cells were treated by cycloheximide
0.5%
Cell lysed
Sucrose cushion(elongating ribosomes purify)
Western blot analysis
Cycloheximide
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The associated H3 molecules most likely represent
its recent translation
complex I:Hsc70
complex II:Hsp90 ,tNASP
complex III:sNASP ,HAT1
complex IV:ASF-1B Imp4
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The preparation for MS
ribosome bands were separated by SDS-PAGE
(stained with Coomassie)
KSTGGKAPR
Protected
17KDa band recovered
Chemical modified by propionic anhydride
(protect lysine to tryptic cleavage)
Digest with trypsin
LC-MS/MS
propionic anhydride
MW=130.14
propionyl lysine
MW=193.19
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The LC-MS/MS analysis of mono- and
dimethylated H3(9-17) peptide
9 KSTGGKAPR 17
Protected
MW=901.03
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Quantification of the modifications on H3
peptides(3-8) and(18-26)
3 TKQTAR 8
18 KQLATKVAR 26
H3K9 methylation occurs while H3 is associated to ribosomes
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MOLDI-TOF mass spectrometry detects the
methylated products in histone methyltransferase
assay
The purified ribosome
complexes incubated with
H3 peptide substrates(mixed
with SAM)
MALDI-TOF
14 Da
14 Da
1 RTKQTARKSTGGKAPRKQLA 20
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Quantitation analysis of the methylated histone
peptides
53%
30%
The ribosome complexes
feature a selective H3K9
mono- and
dimethyltransferase activity
Equation: (AUC H3K9me1+AUC H3K9me2+ AUC H3K9me3)/(AUC
H3+ AUC H3K9me1+AUC H3K9me2+AUC H3K9me3)
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AUC: area under the curve
The procedure employed to in vitro puromycilate
nascent polypeptides
RNase A or EDTA
3’
puromycin
 Puromycin is an aminonucleoside antibiotic, similar to
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the 3’ end of Tyr-tRNATyr .
The active ribosomes are required to label nascent
polypeptides
RNase A or EDTA
Horseradish peroxidase(HRP):
It is used to detect specific
protein in western blot.
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The methylation of H3K9 can occur cotranslationally
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SetDB1 associates to ribosome complex
HeLa cells treated by cycloheximide
Cell lysed
Sucrose gradient
Fractioned
Western blot analysis Co-immunoprecipitation
Western blot analysis
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The ribosome complex has SetDB1 catalytic
activity
HeLa cells were transfected with siSetDB1
HeLa cells treated by cycloheximide
Cell lysed
Western blot analysis Sucrose gradient
Fractioned
Western blot analysis
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Radioactive methyltransferase assay
HeLa cells were transfected with siSetDB1
cpm(an unit): count per minute
Ribosome extraction
Histone H3 peptide incubate(3H-SAM contained)
Spot on filter paper
Scintillation counter measures the cpm
H3(1-20) peptide H3K9 methylation value=cpmH3-cpmH3K9me3
SetDB1 associates to ribosomes and methylates H3K9
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The ribosome associated SetDB1 has a nonprocessive methylation mechanism
Purified the
recombinant SetDB1
and ribosome
Incubated with H3 peptide
substrates
MALDI-TOF
Relative abundance(mMass
v5.5.0)
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The addition of excess full-length H3 acts as a
competitive inhibitor
H3 full length
SetDB1 associates to ribosomes to establish the mono- and dimethylation
mark on H3K9 during translation in non-processive manner
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Conclusion
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Thanks for your attention
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The chromatin package
Database (2016)
 The main function of chromatin are 1) to package DNA into a more
compact, denser shape ,2) to control gene expression and DNA
replication, etc.
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Histone H3K9 mono- and dimethylation occurs at
the ribosome
HeLa cells were treated by cycloheximide
Cell lysed
35% Sucrose cushion
Western blot analysis
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Histone H3 exists in ribosome complex
HeLa cells were treated by cycloheximide
Cell lysed
15-55% Sucrose gradient
Fraction
Western blot analysis
& agarose gel electrophorisis
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The isotopic distribution of mono- and
dimethylated H3(9-17) peptide
(MS/MS analysis)
propionic anhydride
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Histone post-translational modification
Histone proteins are heavily modified post-translationally, and
patterns of these modifications mark specific functional nuclear
domains.
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Neurotherapeutic (2013)
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(Nat. Struct. Mol. Biol. 2010)
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Identification of the methylated H3K9 residue in
ribosomal bound histone H3
The ribosome bound histone H3 features H3K9me1 and H3K9me2
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Histone bands were separated by SDS-PAGE
Digest with trypsin
Run HPLC
Fraction
Western blot analysis
& agarose gel electrophorisis
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Recombinant SetDB1
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