SuPPLementarY InFormatIon10.1038...Supplementary information for reagent chemicals Gln analogs,...

w w w. n a t u r e . c o m / n a t u r e | 1

SuPPLementarY InFormatIondoi:10.1038/nature10656

1

Supplementary information

Supplementary information for reagent chemicals

Gln analogs, 6-diazo-5-oxonorleucine (DON) and azaserine (AZA), enter

catalytic centers of a number of glutamine-utilizing enzymes, such as

Gln:fructose-6-phosphate amidotransferase (GFAT), which is a rate-limiting

enzyme of the HBP, and Gln:phosphoribosyl amidotransferase, which is

involved in the biosynthesis of inosine monophosphate. Due to the strong

activity of the diazo group, while they are frequently used as as HBP

inhibitors, many cytotoxic side-effects are also known.

Supplementary figure legends

Supplementary figure S1. Proposed model for the function of

GlcNAcylated H2B in the O-GlcNAc signaling pathway.

Glucose is metabolized to UDP-GlcNAc through the HBP, which branches off

from the glycolytic pathway. Because OGT activity depends on the

intracellular concentration of UDP-GlcNAc, the level of GlcNAcylation of

H2B S112 therefore reflects the availability of extracellular glucose. H2B

S112 GlcNAc is targeted by the BRE1 complex on the C-terminal α-helix of

H2B, facilitating ubiquitination of H2B K120. Hence, these coordinated

SUPPLEMENTARY INFORMATION

2 | w w w. n a t u r e . c o m / n a t u r e

RESEARCH

Supplementary Figure S1

OHOH

HO

OOH

CH2OH

MLL5

GlcNAc

GlcNAc

RNAPII

MeK4

Ub

Me

K4

K120K120

RAD6

BRE1

E1

OGTs

ExtracellularGlucose

S112S112GlcNAc

UbK120K120

S112S112

HO

OOH

CH2OH

NHCOCH3

O

UDP

UDP-GlcNAc Glucose

HBP

Metabolic genes etc.

H2BH3K4HKMT

1

Supplementary information

Supplementary information for reagent chemicals

Gln analogs, 6-diazo-5-oxonorleucine (DON) and azaserine (AZA), enter

catalytic centers of a number of glutamine-utilizing enzymes, such as

Gln:fructose-6-phosphate amidotransferase (GFAT), which is a rate-limiting

enzyme of the HBP, and Gln:phosphoribosyl amidotransferase, which is

involved in the biosynthesis of inosine monophosphate. Due to the strong

activity of the diazo group, while they are frequently used as as HBP

inhibitors, many cytotoxic side-effects are also known.

Supplementary figure legends

Supplementary figure S1. Proposed model for the function of

GlcNAcylated H2B in the O-GlcNAc signaling pathway.

Glucose is metabolized to UDP-GlcNAc through the HBP, which branches off

from the glycolytic pathway. Because OGT activity depends on the

intracellular concentration of UDP-GlcNAc, the level of GlcNAcylation of

H2B S112 therefore reflects the availability of extracellular glucose. H2B

S112 GlcNAc is targeted by the BRE1 complex on the C-terminal α-helix of

H2B, facilitating ubiquitination of H2B K120. Hence, these coordinated

2

modifications likely promote transcriptional initiation of some metabolic

genes, as shown in H3K4me.

Supplementary Figure S2. Proteomic analysis of GlcNAcylated proteins in

chromatin.

a, Purification of GlcNAcylated glycoproteins from chromatin. The levels of

GlcNAc in chromatin in HeLa cells which were cultured in DMEM with 1 g/L

(Low) or 4.5 g/L (High) glucose, were analyzed by immunoblots. b, c,

Purification with α-O-GlcNAc (RL2) antibody and proteomic analysis. The

bound proteins were eluted with GlcNAc-O-serine (#1 and #2) or acidic

conditions (#3), and were analyzed by Ag staining (b) and WB (c). In, input;

E, elution from WGA beads; R, residual WGA resin. d, The O-GlcNAc

glycoproteins, which were purified from the chromatin of HeLa cells as

shown in Figure 1 and as listed in Supplementary Table S1, were

functionally categorized with the DAVID bioinformatic database. e, The

Venn diagram was obtained from a comparison of the 284 identified

O-GlcNAc glycoproteins with the 689 proteins previously reported by Wang

et al 20.

Supplementary figure S3. CBB-staining analysis for the purification of

recombinant OGT.


SUPPLEMENTARY INFORMATION RESEARCH

RNA processing

Translation

Transport

Nuclear pore

RNA binding

Chromatin assemble

Tubulin

Heat shock

Unknown

Cytoskeleton

Total identified : 284 proteinsAnalyzed : 257 proteinsUnknown : 27 proteins

19.019.0

27.627.6

18.318.34.34.3

14.414.4

3.13.15.15.1

3.93.91.61.6 2.72.7


d e

Wang et al.(689)

Fujiki et al.(284)

581 108 176

a b c

H3H2BH2AH4

In WGA

E R

IgG1

#2#1 #3

α-GlcNAc (RL2)

#2#1 #3

IgGH

IgGL

In WG

A

IgG1

α-GlcN

Ac (R

L2)

WB: α-H2A

WB: α-H4

HeLa S3 cells

Low/High glucose (24 h)

Chromatin extract (0.5 g)

WGA lectin

kDa

150

100

75

50

Low High

WB: α-GlcNAc (RL2)

α-GlcNAc (RL2) affinity

LC-MS/MS

WB: α-H3

WB: α-H2B

Glc:

kDa

250

150

50

75

37

25

100

15

20

Glc High: Glc High

2




chromatin.













et al 20.


recombinant OGT.



RESEARCH

rec. OG

T

kDa

15010075

50

37

25

20


2




chromatin.













et al 20.


recombinant OGT.

2




chromatin.













et al 20.


recombinant OGT.

3

FLAG-tagged OGT was expressed by baculovirus in Sf9 insect cells according

to the manufacturer’s instructions (Invitrogen), and was purified by

anti-FLAG immunoaffinity purification. Arrowheads point to the indicated

proteins.

Supplementary figure S4. In vitro analysis of GlcNAcylation of fly histone

octamers.

Core histone octamers (0.5 µg) derived from human HeLa cells and fly S2

cells were reacted with recombinant OGT (0.5 µg) and UDP-[3H-]GlcNAc (0.2

µCi) for 10 hrs at 37 ºC. The reactions were resolved by SDS PAGE, and

electro-transferred onto the membrane. The reacted histone octamers were

visualized by CBB staining, and subjected to autoradiography.

Supplementary figure S5. q-TOF analysis of in vitro GlcNAcylated H2B.

H2B (0.5 µg) was GlcNAcylated by OGT (0.1 µg) in vitro, and the reactants

were analyzed by q-TOF MS.

Supplementary figure S6. Mapping of O-GlcNAc sites of H2B by

ETD-MS/MS analysis.

In vitro GlcNAcylated H2B (0.5 µg) was digested with trypsin, and the

glycopeptides were partially enriched with lectin-conjugated magnetic beads.




Fly

(-)

Hum

an

1 2 3

H3H2BH2AH4

H2BAuto

CBB

kDa

9

16

9

16

3




proteins.


octamers.










ETD-MS/MS analysis.





RESEARCH

+203 Da

+203 Da

+203 Da

unmodified

monoGlcNAcylated

triGlcNAcylated

diGlcNAcylated

13000 13500 14000 1425013250 13750 14500 14750 m/z15000


3




proteins.


octamers.










ETD-MS/MS analysis.





Full scan

ETD-MS/MS

Supplementary Figure S6a

GlcNAc

GlcNAc GlcNAc

GlcNAc



RESEARCH

Full scan

ETD-MS/MS

Supplementary Figure S6b

GlcNAc

GlcNAc GlcNAc

GlcNAc



Full scan

ETD-MS/MS

Supplementary Figure S6c

GlcNAc

GlcNAc GlcNAc

GlcNAc

3




proteins.


octamers.










ETD-MS/MS analysis.




1 0 | w w w. n a t u r e . c o m / n a t u r e

RESEARCH

4

The supernatants (first and third columns) and eluates (second and fourth

columns) were subject to LC-ETD-MS/MS. Note that the indicated amino

acid numbers are counted from the first methionine. a, ETD spectrum of

m/z 731.6080 (3+) identified the peptide, which was derived from 78 to 94

amino acids of H2B with a single GlcNAc moiety attached to serine 91. b,

ETD spectrum of m/z 588.0620 (4+) identified the peptide, which was derived

from 95 to 114 amino acids of H2B with a single GlcNAc moiety attached to

serine 112. c, ETD spectrum of m/z 491.9090 (3+) identified the peptide,

which was derived from 115 to 126 amino acids of H2B with a single GlcNAc

moiety attached to serine 123.

Supplementary figure S7. Scanning for GlcNAc sites with the H2B

peptide library.

a, The sequences of H2B peptide library are listed. b, Positions of H2B

peptides are illustrated (upper). The values were normalized against cognate

alanine-mutated control peptides.

Supplementary figure S8. MALDI-TOF MS analysis of the number of

O-GlcNAc moieties attached to the H2B peptide.

The H2B peptide 101-115 (a) or the 111-125 peptide (b) was used as a

substrate for the in vitro OGT assay. The reacted peptides (0.5 µg) were

w w w. n a t u r e . c o m / n a t u r e | 1 1



Covering H2B peptide control peptide(a.a.) (Original) (Ser/Thr to Ala)

1 -15 PDPAKSAPAPKKGSK PDPAKAAPAPKKGAK6 -20 SAPAPKKGSKKAVTK AAPAPKKGAKKAVAK11 -25 KKGSKKAVTKAQKKD KKGAKKAVAKAQKKD16 -30 KAVTKAQKKDGKERK KAVAKAQKKDGKERK21 -35 AQKKDGKERKRSRKE AQKKDGKERKRARKE26 -40 GKERKRSRKESYSIY GKERKRARKEAYAIY31 -45 RSRKESYSIYVYKVL RARKEAYAIYVYKVL36 -50 SYSIYVYKVLKQVHP AYAIYVYKVLKQVHP41 -55 VYKVLKQVHPDTGIS VYKVLKQVHPDAGIA46 -60 KQVHPDTGISSKAMG KQVHPDAGIAAKAMG51 -66 DTGISSKAMGIMNSF DAGIAAKAMGIMNAF56 -70 SKAMGIMNSFVNDIF AKAMGIMNAFVNDIF61 -75 IMNSFVNDIFERIAG IMNAFVNDIFERIAG66 -80 VNDIFERIAGEASRL VNDIFERIAGEAARL71 -85 ERIAGEASRLAHYNK ERIAGEAARLAHYNK76 -90 EASRLAHYNKRSTIT EAARLAHYNKRAAIA81 -95 AHYNKRSTITSREIQ AHYNKRAAIAAREIQ86 -100 RSTITSREIQTAVRL RAAIAAREIQAAVRL91 -105 SREIQTAVRLLLPGE AREIQAAVRLLLPGE96 -110 TAVRLLLPGELAKHA AAVRLLLPGELAKHA101-115 LLPGELAKHAVSEGT LLPGELAKHAVAEGA106-120 LAKHAVSEGTKAVTK LAKHAVAEGAKAVAK111-125 VSEGTKAVTKYTSSK VAEGAKAVAKYAAAK

ba

10 20 30 40 50 60 70 80 90 100 110 1203

2

1

0

H2B

a.a

α1 α2 α3 αCL1 L2

Fold

O-G

lcN

Acyl

atio

n(W

T/m

utan

t)

N-terminal tail

4












peptide library.










RESEARCH


a

H2B peptide (101-115)




+ GlcNAc- H2O

(203 Da)

+ GlcNAc- H2O

(203 Da)

bUDP-GlcNAc (-)

UDP-GlcNAc (+)

UDP-GlcNAc (-)

UDP-GlcNAc (+)

4












peptide library.








5

deionized with a reverse-phase micro column, and subjected to MALDI-TOF

MS analysis.

Supplementary figure S9. In vitro OGT assay with point mutants in the

N-terminal tail of H2B.

a, Illustration of the recombinant H2B mutants used for the in vitro OGT

assay. b, c, In vitro OGT assay using recombinant Xenopus H2B lacking its

N-terminal tail (H2BΔN, b) or substituted with the indicated Ser/Thr to Ala

(c). Recombinant H2B mutants (0.5 µg) were reacted with UDP-[3H-]GlcNAc

(0.2 µCi) by recombinant OGT (0.5 µg), and subjected to autoradiography

(upper) and CBB staining (bottom).

Supplementary figure S10. Generation and validation of the α-H2B S112

GlcNAc monoclonal antibody.

a, The peptide used for immunization. b, c, Validation of α-H2B S112 GlcNAc

antibody with ELISA (b), and WB analysis with in vitro GlcNAcylated H2B

and H2A (0.5 µg each, c). d, Validation of α-H2B S112 GlcNAc antibody.

Chromatin lysates, in which H2B was twice depleted (upper), were

individually subjected to WB (middle), and ChIP on peak 4888 (bottom).

Error bars, means and s.d. (n = 3). Sequences of the primer sets used for

ChIP assay were as follows: 5’- AGA CAA CGG CAA CCG AAA AG -3’, and 5’-




kDa

Auto

CBB

9

16

9

16

kDa

Auto

CBB16

16

10 20 30 40 50 60 70 80 90 100 110 120

H2B 4 125

H2BΔN 12527

a.a

1C

ont.

H2B

H2B

ΔN

H2B

H2B

ΔN

2 3 4 5 6

hH2B

OGT + - +- + +1 2 3 4

H2B

H2B

S6A

H2B

S14A

H2B

T21A

a

b c

H2B S6A 4 125S

H2B S14A 4 125S

H2B T21A 4 125T

5


MS analysis.




















RESEARCH


C-KHAVSEGTK-CONH2

CH2

GlcNAc

O

a d

c

α-H2B-S112GlcNAc

α-H2B

- + - +OGT

kDa

16

16

16

16

H2B H2A

α-H2A

α-GlcNAc (RL2)

b

e

10002000

40008000

1600032000

64000128000

3.5

3

2.5

2

1.5

1

0.5

0

Opt

ic d

ensi

ty

CKHAVS(GlcNAc)EGTK-NH2

CKHAVSEGTK-NH2

Dilution factor

0.1 μg

0.05 μg

0.025 μg

0.0125 μg

7 8

α-H2B S112 GlcNAc

H2B-C (S112 GlcNAc)

H2B-NH2B-N (S36 GlcNAc)

H2A (T101 GlcNAc)

H2AH2B-C

GlcNAc Ser

Ser

1 2 3 4 5 6

α-H2B

α-H2B

IP: IgGIP: H2B

Input

1st depletion

2nd depletion

α-H2B S112 GlcNAc

Input1st depletion

2nd depletion

Input1st depletion

2nd depletion

% in

put

0.0

0.2

0.4

0.6

0.8

1.0

peak 4888negative control

5


MS analysis.





















1.28 5.24

88.27 5.21

6.69 0.04

93.23 0.04

Glc depletion 0 h

Glc depletion 24 h

PIPI

Annexin V

Annexin V

6

CAT AGC CAT CCA ATC GAA CG -3’ (peak 4888); 5’- CCA CCC CTT CCA

AAA TGA TG -3’, and 5’- TAG AGA TGG GTG GCT TCC TTT G -3’ (80 kbp

upstream from peak 4888 as a negative control). e, Dot blot analysis of the

cross-reactivity of α-H2B S112 GlcNAc antibody. The indicated amounts of

the indicated peptides and amino acids were spotted on a PVDF membrane,

and blotted with α-H2B S112 GlcNAc antibody. The sequences of the used

peptides were as follows: KHAV-S/S(GlcNAc)-EGTK (H2B-C

unmodified/S112 GlcNAc), SRKE-S/S(GlcNAc)-YSVY (H2B-N

unmodified/S36 GlcNAc), LGKV-S/S(GlcNAc)-IAQG (H2A unmodified/T101

GlcNAc).

Supplementary figure S11. Glucose depletion of HeLa cells: the effect of

cell death.

The cells were cultured in glucose-free DMEM for 24 hrs, and then were

subjected to flow cytometry with annexin V and PI staining. The value in

each partition shows the percentage of the cell populations.

Supplementary figure S12. Glucose depletion of HeLa cells: the effect on

histone modifications.

HeLa cells were cultured in glucose-free DMEM for the indicated time. The

histones, prepared from the cells, were analyzed by WB with antibodies

6










GlcNAc).


cell death.










RESEARCH

Glc depletion (h)

α-H3 K4me3

α-H3 K14Ac

α-H2B K120ub

α-H2B S112GlcNAc

36120 24 48

α-H2B

α-H3 K56Ac

α-H4 K16Ac


6










GlcNAc).


cell death.








7

against the indicated histone modifications.

Supplementary figure S13. The effect of retreatment with the physiologic

concentrations of glucose.

HeLa cells were cultured in glucose-free DMEM for 24 hrs. After glucose

depletion, the cells were further cultured in the media, which were

supplemented with the indicated concentrations of glucose, or 150 µM

PUGNAc for 24 hrs.

Supplementary figure S14. Generation and validation of retroviruses

delivering shRNA against OGT.

a, The sequence of shRNA against OGT (shOGT) and the structure of

recombinant retrovirus is shown (upper). The experimental procedure is

shown (middle). The shOGT was retrovirally transferred into HeLa cells,

and the infected cells were sorted as ZsGreen -positive cells. The sorting of

the infected cells was validated by flow cytometry (lower). b, The efficiency of

the OGT knockdown was confirmed by WB. The intensities of the bands were

quantified with the ImageJ program. c, The GlcNAc level in chromatin

derived from the cells knocked down with OGT.

Supplementary figure S15. Analysis of nucleosome incorporation of



α-H2B S112GlcNAc

α-H2B

1.00.5 PUG0 4.5Glc (g/L)


7







PUGNAc for 24 hrs.














RESEARCH


a b

Infection Sortingfor ZsGreen+ cells

shOGT/pSIREN-ZsGreen ZsGreenLTR shOGT LTR

shLuc :5'-TGC GTT GCT AGT ACC AAC-3'

shOGT :5'-GCA CAT AGC AAT CTG GCT TCC-3'

shLuc shOGT

kDa150

100

75

c

α-GlcNAc (RL2)kDa

GlcCont. GlcCont.

shLuc shOGT (1291)

26

16

38

α-H2B

α-OGT

α-βActin

1.0 0.15

ZsGreen

89.5 %

97.8 %

ZsGreen

10.1 %

97.8 %

7







PUGNAc for 24 hrs.














Flag: Cont.

S P S P S P S P

H2B(WT)

H2B(AA)

H2B(K120R)


α-FLAG

α-H2B

EtBr200 b

p lad

der

7







PUGNAc for 24 hrs.












8

exogenously expressed FLAG-tagged H2B mutants.

Non-chromatin proteins were extracted by 0.1% NP40 treatment of the HeLa

cells which were stably expressing FLAG-tagged H2B mutants (S;

supernatant). The histones in chromatin were solubilized by micrococcal

nuclease digestion of the residual pellet fractions (P; pellet).

Supplementary figure S16. Reagents affecting cellular glycolysis and

protein GlcNAcylation.

a, HeLa cells were cultured in glucose-free DMEM for 24 hrs, and then were

treated with 1 g/L D-glucose, 1 g/L L-glucose (not transported into cells), 1

g/L 3-O-methyl-D-glucopyranose (transported, but not phosphorylated at the

C-6 position), or 1 g/L 2-deoxyglucose (transported, phosphorylated at the

C-6 position, but not at the C-1 position) for an additional 24 hrs. The

chromatins derived from the treated cells were subject to WB with the

indicated antibodies. b, HeLa cells were cultured in DMEM in the presence

or the absence of PUGNAc (an inhibitor of OGA) and 0.1 mM iodoacetate (an

inhibitor of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) for 24 hrs.

Of note, while iodoacetate is frequently used for inhibiting GAPDH in cells or

tissues, it often provokes a number of side effects that are caused by the

nonspecific reaction of iodoacetate with thiol reagents.



RESEARCH


IAA

PUG

α-H2B K120ub

α-H2B S112GlcNAc

α-H2B

α-H2B K120ub

α-H2B S112GlcNAc

α-H2B

L-glucose

3-O-methyl-D-glucopyranose

2-deoxyglucose

ContD-glucose

a b

-

- -

+ +

+

8

exogenously expressed FLAG-tagged H2B mutants.

Non-chromatin proteins were extracted by 0.1% NP40 treatment of the HeLa

cells which were stably expressing FLAG-tagged H2B mutants (S;

supernatant). The histones in chromatin were solubilized by micrococcal

nuclease digestion of the residual pellet fractions (P; pellet).

Supplementary figure S16. Reagents affecting cellular glycolysis and

protein GlcNAcylation.

a, HeLa cells were cultured in glucose-free DMEM for 24 hrs, and then were

treated with 1 g/L D-glucose, 1 g/L L-glucose (not transported into cells), 1

g/L 3-O-methyl-D-glucopyranose (transported, but not phosphorylated at the

C-6 position), or 1 g/L 2-deoxyglucose (transported, phosphorylated at the

C-6 position, but not at the C-1 position) for an additional 24 hrs. The

chromatins derived from the treated cells were subject to WB with the

indicated antibodies. b, HeLa cells were cultured in DMEM in the presence

or the absence of PUGNAc (an inhibitor of OGA) and 0.1 mM iodoacetate (an

inhibitor of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) for 24 hrs.

Of note, while iodoacetate is frequently used for inhibiting GAPDH in cells or

tissues, it often provokes a number of side effects that are caused by the

nonspecific reaction of iodoacetate with thiol reagents.




α-BRE1

α-H2B S112 GlcNAc

IP: IgGIP: H2B S112 GlcNAc

a bα-BRE1

α-Flag (H2B)

IP:Flag-: Cont. H2B (WT)

Cont. Glc Cont. Glc

9

Supplementary figure S17. WB analysis of the glucose-dependent

interaction between H2B S112 GlcNAc and BRE1A.

a, FLAG-tagged H2B was expressed in HeLa cells which were cultured in

media with or without glucose. H2B was immunoprecipitated with

anti-FLAG antibody, and then subjected to WB analysis with α-BRE1A

antibody. b, H2B S112 GlcNAc was immunoprecipitated from HeLa cells

cultured under normal condition (i.e. glucose (+)-DMEM supplemented with

10 % FBS), and subjected to WB analysis with α-BRE1A antibody.

Supplementary figure S18. CBB staining analysis for the purification of

recombinant ubiquitin ligases of H2B.

a, CBB staining analysis of the purification of recombinant ubiquitin ligase

of H2B. Arrowheads point to the indicated proteins. b, In vitro

ubiquitination assay with H2B and recombinant E1-RAD6A-BRE1A/1B

ubiquitin ligases. H2B was ubiquitinated by the E1, RAD6A, and BRE1A/B

complex, and the reaction was subjected to WB analysis with α-H2B K120ub

antibody. The bands were quantified with ImageJ program (upper). Error

bars, means and s.d. (n = 3).

Supplementary figure S19. Analysis of sxc/ogt-mediated GlcNAcylation of

H2B in Drosophila polytene chromosomes.



RESEARCH

rec. E1

rec. RA

D6A

rec. BR

E1 A

/1B com

plex

kDa

150

250

10075

50

37

2520

1015

150

250

100

75

50

37

2520

kDa

a b


1 2 3 4- + + +E1+RAD6A+ - + ++BRE1A/1B

1 2 3 4Fold

(mon

o-ub

H2B

/H2B

)

0

1

2

3

n.d.

mono-ubH2B

H2B

di-ubH2Btri-ubH2B

**

α-H2B-K120ub

α-H2B

BRE1BBRE1A

9






















+/+ Sxc1/Sxc7


DAPI

H2B S112GlcNAc Merge

H3 K27 me3 DAPI

H2B S112GlcNAc Merge

H3 K27 me3

9




















10

The spread polytene chromosomes were prepared from wild-type flies (+/+)

and OGT-null flies (sxc1/ sxc7), and were stained with α-H2B S112 GlcNAc

(green), α-H3K27me3 (red) and DAPI (blue).

Supplementary figure S20. Analysis of the cellular distribution of

GlcNAcylated H2B in HeLa cells.

HeLa cells were stained with α-H2B S112 GlcNAc fluorescent antibody

(green) and DAPI (white).

Supplementary figure S21. ChIP and WB analysis of the co-localization of


Chromatin of HeLa cells was digested with MNase, and was

immunoprecipitated with the indicated antibodies. The native ChIP samples

were subjected to WB with H2B S112 GlcNAc monoclonal antibody or H2B

K120 ub antibody. Asterisk indicates nonspecific band.

Supplementary figure S22. Genome-wide analysis of distributions of H2B

S112 GlcNAc peaks.

The results from Cis-regulatory Element Annotation System (CEAS). The

distributions of H2B S112 GlcNAc peaks in the promoter, downstream and

other gene loci are shown with a comparison to the whole genome



RESEARCH


DAPI Mergeα-H2B S112GlcNAc

10















S112 GlcNAc peaks.






WB:α-H2B S112GlcNAc

WB:α-H2B K120ub

H2B S112GlcNAc

H2B K120ub

H3 K9me2

H3 K27me3

H3 K4me2

IgG

*


10















S112 GlcNAc peaks.






RESEARCH

Promoter

<= 1000 bp <= 5000 bp <= 10000 bp

010

Per

cent

age

% 86

42

Genome

ChIP

0

Downstream

<= 1000 bp <= 5000 bp <= 10000 bp

10

Per

cent

age

% 86

42

Genome

ChIP

Gene

5’UTR 3’UTR Exon Intron All

Per

cent

age

%

8060

4020

0

Genome

ChIP


10















S112 GlcNAc peaks.




11

distribution. Analysis was performed on the web-based Cistrome analysis

pipeline module (http://cistrome.dfci.harvard.edu/).

Supplementary figure S23. H2B S112 GlcNAc was localized to the

transcribed genes.

a, The activity of the genes harboring H2B S112 GlcNAc in the promoter was

categorized into a high expression group (High) and a low expression group

(Low) based on the microarray datasets in Supplementary Table 2 [we

designated the ‘promoter’ as a region < 3 kbp upstream from the

transcription start site (TSS)]. b, The top five categories of gene ontology

analysis of biological processes of the genes harboring H2B S112 GlcNAc

peaks in the promoter are shown.

Supplementary figure S24. Genome-wide analysis of the H2B K120 ub

sites by ChIP-seq.


distributions of H2B K120 ub were averaged near TSS (left) and TTS (right)

(a). The average profiles of H2B S112 GlcNAc were overlaid. The distribution

of H2B K120 ub peaks in the promoter, downstream and other gene loci are

shown in comparison to the whole genome distribution (b). Analyses were

performed on the web-based Cistrome analysis pipeline module



a

50 10075250 (%)

H2B S112 GlcNAc

High Low Expression level

76.0 24.0

Percentage

b

(%)

Cellular process

Metabolic process

Biological regulation

Primary metabolic process

Cellular metabolic process

80 60 40 20 0Percentage

1,134 (0.00180)

833 (0.00882)

808 (0.00283)

767 (0.00215)

741 (0.00114)Analyzed1,891 genes


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transcribed genes.









sites by ChIP-seq.









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Average Profile near TTS

0 1000 2000 3000-1000-2000-3000

1.15

1.10

1.05

1.00

0.95

0.90

Aver

age

prof

ile

Relative Distance to TTS (bp)

H2B K120 ubH2B S112 GlcNAc

0 1000 2000 3000-1000-2000-3000

1.30

1.25

1.20

1.15

1.10

1.05

Aver

age

prof

ile

Average Profile near TSS

Relative Distance to TSS (bp)

H2B K120 ubH2B S112 GlcNAc

Promoter

<= 1000 bp <= 5000 bp <= 10000 bp

010

Per

cent

age

%8

64

2

GenomeChIP

0

Downstream

<= 1000 bp <= 5000 bp <= 10000 bp

10P

erce

ntag

e %

86

42

GenomeChIP

Gene

5’UTR 3’UTR Exon Intron All

Per

cent

age

% 8060

4020

0

GenomeChIP


a b

12

(http://cistrome.dfci.harvard.edu/).

Supplementary figure S25. ChIP-reChIP analysis of the co-localization of

H2B S112 GlcNAc and K120 ub.

a, The 3974 peaks co-localized with H2B S112 GlcNAc and K120 ub were

subdivided into five categories in terms of their overlapping length. b, The

top three peaks were picked from each category for a sequential ChIP

(α-H2B S112 GlcNAc antibody)-reChIP (α-H2B K120 ub antibody) analysis.

c, d, DNA library with ChIP (α-H2B S112 GlcNAc antibody, c) or reChIP

(α-H2B K120 ub antibody, d) was analyzed by qPCR. Error bars, means and

s.d. (n = 3). The sequences of the primers used for qPCR were as follows: 5’-

TTC AGG GGG AGA AAG CAA TG -3’ and 5’- TGC TGG GTA AAA GTG GAG

AGC -3’ (peak 4300); 5’- GAG AGA AGG CCT CCA AAA TCT C -3’ and 5’-

ACA TTG CCA CAT TGC CAC AG -3’ (peak 23556); 5’- TTT CTC AGC TTC

TGG CCA TC -3’ and 5’- AAT TTG AGT GGG GAC ACA GAG C -3’ (peak

817); 5’- AAG GAA AGG GAA AGG GAA GG -3’ and 5’- TTG CAT GCT GAT

GGT GAG TG -3’ (peak 25177); 5’- GCC CAT CTG ATT TGG GAA AC -3’ and

5’- ATA GGC ATG AAC CAC TGT GCT G -3’ (peak 42036); 5’- TCA TCC CAT

GAT GGC TTC AG -3’ and 5’- GAA GCA TTG GCA CTT TCT CG -3’ (peak

16699); 5’- ATT TCA ACC CAG CCA AGA GC -3’ and 5’- TGT GTC TTC CTC

CCA TGT TCT G -3’ (peak 28633); 5’- AGG CAG AAA TGG AAT CCT ACC C

11




transcribed genes.









sites by ChIP-seq.










> 10 10-100 100-500 500-1000 < 1000

ChIP: α-H2B S112GlcNAc

% in

put

0.0

0.2

0.4

0.6

0.8

1.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 N

% C

hIP

: α-H

2B S

112G

lcN

Ac

0

2

4

6

8

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 N

ChIP: α-H2B S112GlcNAcRe-ChIP: α-H2B K120Ub

bp peak number> 10 1210 -100 167100-500 2105500-1000 1544 < 1000 146total 3974

1 peak 43002 peak 235563 peak 8174 peak 251775 peak 420366 peak 166997 peak 28633 8 peak 427489 peak 743610 peak 151411 peak 575612 peak 2862513 peak 488814 peak 487815 peak 44128

> 10 10-100 100-500 500-1000 < 1000

n.d.n.d.n.d. n.d.

a c

db

12























RESEARCH

13

-3’ and 5’- AAT GGG GAT GCA GAG GAA AC -3’ (peak 42728); 5’- TGA AAA

GGA TGC TGC GAG TG -3’ and 5’- TCC CTC TTT GCC ACT ATG CTC -3’

(peak 7436); 5’- TTT GAT CAG GGA CCC AAA CC -3’ and 5’- TTC CCT TTG

GGT TGG TCA AG -3’ (peak 1514); 5’- TAT GGA CGG CAT GAT GGA TG -3’

and 5’- ATG TGA GCA ATG TGG GAA GG-3’ (peak 5756); 5’- ACT GCT TGC

AGC AAA TGT GG -3’ and 5’- ACT GCT TGC AGC AAA TGT GG -3’ (peak

28625); 5’- AGA CAA CGG CAA CCG AAA AG -3’ and 5’- CAT AGC CAT CCA

ATC GAA CG -3’ (peak 4888); 5’- TTT TCT CCT CCC AGG CCT TTA G -3’

and 5’- TGG GGA AAA TGT CTC CAA GG -3’ (peak 4878); 5’- AAC GTT GGA

AAT CCC AGA GC -3’ and 5’- CCA CCT CAA AAC GCA ACA AG -3’ (peak

44128); 5’- TCA CCA TTA GGG ACC TTC TTG C-3’ and 5’- TTC TGG GAT

TGC CTT TCC TG -3’ (GAPDH promoter as a negative control),

Supplementary table S1. List of identified GlcNAcylated proteins in

chromatin.

a, b, The eluted fractions were independently subjected to trypsin digestion,

and LC-MS/MS analysis to identify GlcNAcylated proteins in chromatin (the

mixture of elution #1 and #2 (a), and elution #3 (b), shown in Figure 1b).

Peptides were analyzed by Bioworks3.3 (SEQUEST algorithm) against a

non-redundant NCBI protein database and results were filtered according to

peptide probabilities, SEQUEST X-corr (XC) scores, peptide charge state,

12























13

-3’ and 5’- AAT GGG GAT GCA GAG GAA AC -3’ (peak 42728); 5’- TGA AAA

GGA TGC TGC GAG TG -3’ and 5’- TCC CTC TTT GCC ACT ATG CTC -3’

(peak 7436); 5’- TTT GAT CAG GGA CCC AAA CC -3’ and 5’- TTC CCT TTG

GGT TGG TCA AG -3’ (peak 1514); 5’- TAT GGA CGG CAT GAT GGA TG -3’

and 5’- ATG TGA GCA ATG TGG GAA GG-3’ (peak 5756); 5’- ACT GCT TGC

AGC AAA TGT GG -3’ and 5’- ACT GCT TGC AGC AAA TGT GG -3’ (peak

28625); 5’- AGA CAA CGG CAA CCG AAA AG -3’ and 5’- CAT AGC CAT CCA

ATC GAA CG -3’ (peak 4888); 5’- TTT TCT CCT CCC AGG CCT TTA G -3’

and 5’- TGG GGA AAA TGT CTC CAA GG -3’ (peak 4878); 5’- AAC GTT GGA

AAT CCC AGA GC -3’ and 5’- CCA CCT CAA AAC GCA ACA AG -3’ (peak

44128); 5’- TCA CCA TTA GGG ACC TTC TTG C-3’ and 5’- TTC TGG GAT

TGC CTT TCC TG -3’ (GAPDH promoter as a negative control),

Supplementary table S1. List of identified GlcNAcylated proteins in

chromatin.

a, b, The eluted fractions were independently subjected to trypsin digestion,

and LC-MS/MS analysis to identify GlcNAcylated proteins in chromatin (the

mixture of elution #1 and #2 (a), and elution #3 (b), shown in Figure 1b).

Peptides were analyzed by Bioworks3.3 (SEQUEST algorithm) against a

non-redundant NCBI protein database and results were filtered according to

peptide probabilities, SEQUEST X-corr (XC) scores, peptide charge state,

14

and two or more unique peptide hits per protein to eliminate false or

low-quality identifications. Protein name, p-value, SEQUEST Full (Sf) score,

SEQUEST X-corr (XC) score, molecular weight, accession no., and number of

peptides are indicated for each. c, 257 proteins were successfully converted

from 284 identified genes, listed in (a) and (b), and with the DAVID

bioinformatic database.

Supplementary table S2. Microarray analysis of HeLa cells cultured under

specific experimental conditions.

Total RNA was prepared from HeLa cells, which were cultured in DMEM

supplemented with 10% FBS (FBS), or glucose-free DMEM for 24 hrs

[Glucose (-)], or 4.5 g/L glucose re-treatment for 24 hrs after a 24 hr glucose

depletion [Glucose (+)] (n = 3, in each culture condition). The labeled cRNA

samples, which were obtained according to the standard protocols, were then

hybridized to a human U133 plus 2.0 microarray (Affymetrix).

Supplementary table S3. List of genes harboring H2B S112 GlcNAc in the

promoter or 50 kbp within the gene body, and the expression levels.

a, d, Gene symbols, which have H2B S112 GlcNAc peaks, H2B K120 ub

peaks and both in the promoter regions (a) or the regions between 50 kbp

upstream from the TSS and 50 kbp downstream from the TTS of each gene



RESEARCH

14

and two or more unique peptide hits per protein to eliminate false or

low-quality identifications. Protein name, p-value, SEQUEST Full (Sf) score,

SEQUEST X-corr (XC) score, molecular weight, accession no., and number of

peptides are indicated for each. c, 257 proteins were successfully converted

from 284 identified genes, listed in (a) and (b), and with the DAVID

bioinformatic database.

Supplementary table S2. Microarray analysis of HeLa cells cultured under

specific experimental conditions.

Total RNA was prepared from HeLa cells, which were cultured in DMEM

supplemented with 10% FBS (FBS), or glucose-free DMEM for 24 hrs

[Glucose (-)], or 4.5 g/L glucose re-treatment for 24 hrs after a 24 hr glucose

depletion [Glucose (+)] (n = 3, in each culture condition). The labeled cRNA

samples, which were obtained according to the standard protocols, were then

hybridized to a human U133 plus 2.0 microarray (Affymetrix).

Supplementary table S3. List of genes harboring H2B S112 GlcNAc in the

promoter or 50 kbp within the gene body, and the expression levels.

a, d, Gene symbols, which have H2B S112 GlcNAc peaks, H2B K120 ub

peaks and both in the promoter regions (a) or the regions between 50 kbp

upstream from the TSS and 50 kbp downstream from the TTS of each gene

15

(d), are listed. b, The expression signals of 1,702 genes out of 2,048 genes

listed in (a), which have H2B S112 GlcNAc peaks in the promoter, are shown.

In the analysis, 346 genes were omitted due to gene conversion processes, in

which official gene symbols were converted from the Affymetrix probe ID for

expression levels (Supplementary table S2) and from the RefSeq number of

the genes with H2B S112 GlcNAc in the promoter by using DAVID

Bioinformatics Resources (http://david.abcc.ncifcrf.gov/). c, The 1,891 genes

were successfully converted from 2,048 genes harboring H2B S112 GlcNAc in

the promoter listed in (a), and were categorized by Gene Ontology analysis of

biological processes.

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