Ales Vancura (ed.), Transcriptional Regulation: Methods and Protocols, Methods in Molecular Biology, vol. 809,
DOI 10.1007/978-1-61779-376-9_30, © Springer Science+Business Media, LLC 2012
Affi nity Purifi cation of MLL3/MLL4 Histone H3K4
Young-Wook Cho , SunHwa Hong , and Kai Ge
Methylation on histone H3 lysine 4 (H3K4) correlates with actively transcribed genes. In mammalian
cells, there exist multiple Set1-like histone H3K4 methyltransferase complexes, which have overlapping
but distinct subunit compositions. Developing methods to isolate each of these histone H3K4 methyl-
transferase complexes would help understand the molecular mechanisms by which histone H3K4 methyla-
tion regulates mammalian gene expression. In this chapter, we provide a one-step affi nity purifi cation
protocol on isolation of the MLL3/MLL4 histone H3K4 methyltransferase complex using FLAG-tagged
PA1, a unique subunit of the MLL3/MLL4 complex.
Key words: Histone H3K4 methyltransferase complex , MLL3 , MLL4 , PA1
Histone lysine methylation plays essential roles in chromatin
dynamics, transcription, and DNA repair. Histone lysine methyla-
tion is dynamically regulated by site-specifi c methyltransferases and
demethylases. In yeast, a single Set1 complex, also known as
COMPASS, is responsible for all methylations on histone H3K4
( 1– 3 ) . In mammalian cells, at least six Set1-like histone methyl-
transferase (HMT) complexes with robust H3K4 methyltransferase
activities have been isolated ( 4 ) . Each of these complexes contains
one SET domain-containing homolog of yeast Set1, such as Set1A
(also known as Setd1a, KMT2F) ( 5, 6 ) , Set1B (also known as
Setd1b, KMT2G) ( 7 ) , MLL1 (mixed-lineage leukemia 1, also
known as MLL, HRX, ALL1, KMT2A) ( 8– 10 ) , MLL2 (mixed-
lineage leukemia 2, also known as TRX2, MLL4, KMT2B) ( 10, 11 ) ,
466 Y.-W. Cho et al.
MLL3 (mixed-lineage leukemia 3, also known as KMT2C), and
MLL4 (mixed-lineage leukemia 4, also known as ALR, MLL2,
KMT2D) ( 4, 12– 14 ) , which carries the enzymatic activity for the
associated complex. Based on the homologies in both protein
sequences and domain structures, the six Set1-like HMTs fall into
three subgroups, Set1A and Set1B, MLL1 and MLL2, and MLL3
and MLL4. ASH2L, RbBP5, WDR5, and DPY30, which are
homologs of yeast Set1/COMPASS complex subunits Bre2, Swd1,
Swd3, and Sdc1, respectively, form a 4-subunit subcomplex that is
not only shared by all mammalian Set1-like HMT complexes, but
also critical for the H3K4 methyltransferase activities of these com-
plexes ( 4, 8 ) . In addition, each of these complexes contains distinct
but overlapping subunits (Fig. 1 ) ( 10, 11 ) . For example, WDR82
and CXXC1, which are homologs of the Swd2 and Spp1 subunits
of yeast Set1/COMPASS complex, selectively associate with
Set1A/B complexes ( 5 ) . Menin, a protein with no homology with
any of the yeast Set1/COMPASS complex components, selectively
associates with MLL1 and MLL2 complexes ( 10, 11 ) .
In cells, PTIP and a novel protein PA1 are both unique sub-
units of the MLL3/MLL4 histone H3K4 methyltransferase com-
plex that contains the enzymatic subunits MLL3 and MLL4, and
the histone H3K27 demethylase UTX ( 4, 15 ) . Methylation on
H3K4 is an activating epigenetic mark while methylation on
H3K27 is a repressive one. The fi nding that H3K4 methyltrans-
ferases MLL3/MLL4 physically associate with H3K27 demethy-
lase UTX suggests that by adding an activating epigenetic mark
and removing a repressive one, the MLL3/MLL4 complex may use
two distinct histone-modifying activities to synergistically activate
Fig. 1. Subunit compositions of the yeast Set1 and human Set1-like histone H3K4 meth-
467 30 Affi nity Purifi cation of MLL3/MLL4 Histone H3K4 Methyltransferase Complex
target gene expression. Recent evidence also suggests that MLL3
and MLL4 may exist in the HMT complex in a mutually exclusive
manner ( 16 ) . Here, we describe the one-step isolation of the
MLL3/MLL4 histone H3K4 methyltransferase complex from
nuclear extracts prepared from a HeLaS cell line stably expressing
FLAG-tagged PA1 ( 4 ) .
All cells are routinely cultured in Dulbecco’s modifi ed Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
1. Phoenix-Ampho retrovirus packaging cell line (ATCC product
# SD 3443) and HeLaS cell line.
2. GenJet™ in vitro DNA transfection reagent (SignaGen
3. Bovine serum (for large-scale cell culture only).
4. Retroviral infection medium (RIM) (DMEM + 10% heat-
5. Sterile 0.45- μ m syringe fi lter (Millipore).
6. Polybrene (Sigma H9268).
7. G418 (Invitrogen).
8. 1× PBS.
9. Anti-FLAG M2 antibody (Sigma F3165).
1. Retrovirus plasmid pWZLneo-F-PA1 expressing FLAG-tagged
PA1 (available from the authors upon request).
2. HeLaS cells expressing FLAG-tagged PA1 (available from the
authors upon request).
3. 15- and 50-ml conical tubes.
4. 200- μ l and 1-ml wide-orifi ce tips (see Note 1).
5. Mouse IgG-agarose (Sigma A0919).
6. M2 agarose (anti-FLAG antibody conjugated to agarose,
7. 5 mg/ml FLAG Peptide (Sigma F3290).
8. Buffer A: 20 mM HEPES, pH 7.9, 180 mM KCl, 0.2 mM
EGTA, 1.5 mM MgCl 2 , 20% (v/v) glycerol, 0.1% (v/v)
9. Elution buffer: 20 mM HEPES, pH 7.9, 180 mM KCl,
0.2 mM EGTA, 1.5 mM MgCl 2 .
of Stable Cell Lines
2.2. Isolation of MLL3/
MLL4 Complex by
468 Y.-W. Cho et al.
10. Ultrafree-MC centrifugal fi lter units with microporous mem-
brane 0.45 μ m (Millipore).
11. Vivaspin 500 centrifugal fi lter units, MWCF 10 kDa (Fisher
12. 4–15% SDS-PAGE gel (Bio-Rad).
13. Protease inhibitors: Aprotinin, leupeptin, and pepstatin
14. Dithiothreitol (DTT, Bio-Rad, stock solution 1 M).
15. PMSF (Sigma, stock solution 200 mM).
PTIP and PA1 both associate with the MLL3/MLL4 histone
H3K4 methyltransferase complex ( 4 ) . However, ectopically
expressed FLAG-tagged PTIP associates not only with the MLL3/
MLL4 complex, but also with proteins involved in DNA damage
response and repair. In contrast, FLAG-tagged PA1 selectively
associates with the MLL3/MLL4 complex ( 4 ) . Using retrovirus-
mediated gene transfer, a HeLaS cell line stably expressing FLAG-
tagged, full-length, human PA1 (F-PA1) is established. The
MLL3/MLL4 complex can be purifi ed in one step using anti-
FLAG antibody immunoprecipitation from nuclear extracts pre-
pared from this cell line.
1. In the afternoon, plate 1 × 10 6 Phoenix-Ampho amphotropic
retrovirus packaging cells in 4 ml of culture medium in a 6-cm
dish. Incubate cells in a 37°C incubator for 24 h.
2. In the afternoon, dilute 2.5 μ g pWZLneo-F-PA1 plasmid in
100 μ l of plain DMEM without serum and antibiotics. Dilute
7.5 μ l of GenJet™ transfection reagent in 100 μ l of plain
DMEM. Mix the two solutions and incubate at room tempera-
ture for 15 min. Add the 200 μ l of mixture to the dish of cells
from step 1. Incubate cells in a 37°C incubator for 2 days.
3. In the afternoon, replace the medium with 4 ml of RIM. Plate
3.5 × 10 5 HeLaS cells in another 6-cm dish.
4. In the morning, collect the 4 ml of retrovirus-containing
supernatant, fi lter through 0.45- μ m syringe fi lter, dilute with
an equal volume of fresh RIM, and add 8 μ g/ml polybrene.
of Stable Cell Lines
469 30 Affi nity Purifi cation of MLL3/MLL4 Histone H3K4 Methyltransferase Complex
5. Replace the supernatant of the HeLaS cells with 3–4 ml of the
diluted virus-containing supernatant. Incubate cells at 37°C
for 2 days.
6. Trypsinze the infected HeLaS cells in the 6-cm dish, transfer
1/5, 1/25, 1/125, and 1/625, respectively, into four 15-cm
dishes (i.e., 1:35, 1:175, 1:875, and 1:4,375 dilutions). Add
1 mg/ml G418. Every 3 days, change to fresh medium supple-
mented with 1 mg/ml G418.
7. 2 weeks since the splitting of the infected HeLaS cells, pick
24 well-isolated single colonies using 200- μ l pipette tips (see
Note 2). Transfer each colony into one well of a 24-well plate
fi lled with 1 ml of culture medium containing 0.5 mg/ml G418.
Pipette up and down with 1-ml tips to disperse the cells.
8. Choose 12–18 fast-growing colonies in the 24-well plate.
When cells reach confl uency, trypsinize and split cells in one
well of the 24-well plate into two wells of 6-well plates (one for
freezing down and the other for western blotting). Add
0.5 mg/ml G418.
9. After cells reach confl uency in 6-well plates, trypsinize cells
from one well and freeze down in −80°C freezer (see Note 3).
Collect cells from the second well and prepare whole cell
extracts for western blot analysis using anti-FLAG M2 anti-
body. The extracts from the parental HeLaS cells serve as the
negative control in western blot. PA1 runs at ~42 kDa on SDS-
After identifying HeLaS cell lines expressing FLAG-tagged PA1
(HeLaS/F-PA1) by western blot, the HeLaS/F-PA1 cells are cul-
tured till confl uence in 100 × 15-cm dishes in DMEM containing
8% bovine serum, 2% FBS, and 0.1 mg/ml G418. Cells are col-
lected, and nuclear extracts (N.E.) are prepared exactly as described
( 17 ) .
Next, we describe one-step purifi cation of the MLL3/MLL4
complex by immunoprecipitation with anti-FLAG antibody conju-
gated to agarose (M2 agarose) from N.E. prepared from HeLaS/
F-PA1 cells. All steps described below are performed in cold room
or on ice. All buffers are freshly supplemented with 1 mM DTT
and protease inhibitors 0.5 mM PMSF, 1 μ g/ml aprotinin, 2 μ g/ml
leupeptin, and 0.7 μ g/ml pepstatin.
1. Dilute 100 mg of N.E. protein in buffer A to get a fi nal protein
concentration of 2–3 mg/ml. Centrifuge at 25,000 × g for
30 min at 4°C to remove denatured proteins and cell debris.
Transfer the supernatant to a 50-ml conical tube.
Complex by Anti-FLAG
470 Y.-W. Cho et al.
2. To remove proteins that nonspecifi cally bind to M2 agarose
from the diluted N.E., the supernatant from step 1 is pre-
cleared by adding 0.2 ml of mouse IgG–agarose that has been
washed twice with 1 ml of buffer A. Rotate the tube for 2 h.
3. Centrifuge at 1,500 × g for 5 min in a swinging bucket rotor,
and transfer the supernatant into a new 50-ml conical tube (see
4. Repeat steps 2 and 3 once.
5. While doing steps 2–4, equilibrate 0.2 ml of anti-FLAG M2
agarose by washing three times with 1 ml of buffer A for
6. Incubate precleared N.E. from step 4 with 0.2 ml of anti-
FLAG M2 agarose for overnight immunoprecipitation. Rotate
the tube in the cold room.
7. The next morning, centrifuge the tube at 1,500 × g for 10 min
in a swinging bucket rotor. Transfer the supernatant to a new
tube. Keep the agarose beads.
8. Wash the agarose beads by adding 40 ml of buffer A, rotate for
30 min, spin the tube at 1,500 × g for 5 min, and remove the
9. Add 10 ml of buffer A to the agarose beads, resuspend, and
transfer to a 15-ml conical tube. Rotate for 5 min. Centrifuge
the tube at 1,500 × g for 2 min and remove the supernatant.
Repeat the wash three more times.
10. Add 1 ml of buffer A to the agarose beads, resuspend, and
transfer to a 1.5-ml tube. Rotate for 2 min. Spin at 1,500 × g in
a microcentrifuge for 2 min and remove the supernatant.
Repeat the wash three more times. After the fi nal wash, spin
down the agarose beads and completely remove the superna-
tant with a 27-G needle.
11. To elute proteins bound to the M2 agarose, dilute 5 mg/ml
FLAG peptide solution 20-fold in 1 ml of elution buffer to get
a fi nal concentration of 0.25 mg/ml.
12. Elution: Add 0.5 ml of diluted FLAG peptide to the agarose
beads after the fi nal wash at step 10. Rotate for 30 min, spin at
1,500 × g in a microcentrifuge for 2 min, and transfer the super-
natant to a new 1.5-ml tube. Add 0.5 ml of diluted FLAG
peptide to repeat the elution once.
13. Combine the fi rst and second eluates and fi lter through an
Ultrafree-MC centrifugal fi lter unit by a quick spin to com-
pletely remove agarose beads.
14. Concentrate with a Vivaspin 500 (10-kDa cutoff) by centrifug-
ing at 15,000 × g in a microcentrifuge for 4× 5 min until the
volume is less than 100 μ l.
471 30 Affi nity Purifi cation of MLL3/MLL4 Histone H3K4 Methyltransferase Complex
15. Run the concentrated sample on a 4–15% SDS-PAGE gel and
analyze by western blot or mass spectrometry. Alternatively,
the purifi ed MLL3/MLL4 complex is subjected to HMT
1. Wide-orifi ce tips have a larger opening to allow the pipetting
of viscous solutions. Wide-orifi ce tips can be homemade by
simply cutting off the end of regulator tips with a razor blade.
Use wide-orifi ce tips to pipette antibody-conjugated agarose.
2. To pick single HeLaS cell colonies, fi rst identify well-isolated
single colonies under the microscope and draw circles around
the single colonies by labeling at the bottom of the 15-cm
dishes. Prepare a 24-well cell culture plate fi lled with 1 ml of
culture medium per well. Remove the majority of the culture
medium from the 15-cm dishes. In the tissue culture hood,
quickly pick single HeLaS cell colonies using 200- μ l pipette
tips in a way similar to picking bacteria colonies from agar
3. One day before freezing down cells, remove G418-containing
medium and change to fresh culture medium without G418.
4. After centrifugation, the pellet is loose in 50-ml conical tubes.
Do not disturb the pellet when transferring the supernatant.
1. Roguev, A., Schaft, D., Shevchenko, A.,
Pijnappel, W. W., Wilm, M., Aasland, R. &
Stewart, A. F. (2001). The Saccharomyces cere-
visiae Set1 complex includes an Ash2 homo-
logue and methylates histone 3 lysine 4. EMBO
J 20 , 7137–48.
2. Briggs, S. D., Bryk, M., Strahl, B. D., Cheung,
W. L., Davie, J. K., Dent, S. Y., Winston, F. &
Allis, C. D. (2001). Histone H3 lysine 4
methylation is mediated by Set1 and required
for cell growth and rDNA silencing in
Saccharomyces cerevisiae . Genes Dev 15 ,
3. Miller, T., Krogan, N. J., Dover, J., Erdjument-
Bromage, H., Tempst, P., Johnston, M.,
Greenblatt, J. F. & Shilatifard, A. (2001).
COMPASS: a complex of proteins associated
with a trithorax-related SET domain protein.
Proc Natl Acad Sci USA 98 , 12902–7.
4. Cho, Y.-W., Hong, T., Hong, S., Guo, H., Yu,
H., Kim, D., Guszczynski, T., Dressler, G. R.,
Copeland, T. D., Kalkum, M. & Ge, K. (2007).
PTIP Associates with MLL3- and MLL4-
Methyltransferase Complex. J. Biol. Chem.
282 , 20395–20406.
5. Lee, J. H. & Skalnik, D. G. (2005). CpG-
binding protein (CXXC fi nger protein 1) is a
component of the mammalian Set1 histone
H3-Lys4 methyltransferase complex, the ana-
logue of the yeast Set1/COMPASS complex. J
Biol Chem 280 , 41725–31.
6. Wysocka, J., Myers, M. P., Laherty, C. D.,
Eisenman, R. N. & Herr, W. (2003). Human
Sin3 deacetylase and trithorax-related Set1/
Ash2 histone H3-K4 methyltransferase are
tethered together selectively by the cell-prolif-
eration factor HCF-1. Genes Dev 17 ,
7. Lee, J. H., Tate, C. M., You, J. S. & Skalnik, D.
G. (2007). Identification and characteriza-
tion of the human Set1B histone H3-Lys4
H3 Lysine 4
472 Y.-W. Cho et al.
methyltransferase complex. J Biol Chem 282 ,
8. Dou, Y., Milne, T. A., Ruthenburg, A. J., Lee,
S., Lee, J. W., Verdine, G. L., Allis, C. D. &
Roeder, R. G. (2006). Regulation of MLL1
H3K4 methyltransferase activity by its core
components. Nat Struct Mol Biol 13 , 713–9.
9. Milne, T. A., Briggs, S. D., Brock, H. W.,
Martin, M. E., Gibbs, D., Allis, C. D. & Hess,
J. L. (2002). MLL targets SET domain methyl-
transferase activity to Hox gene promoters. Mol
Cell 10 , 1107–17.
10. Yokoyama, A., Wang, Z., Wysocka, J., Sanyal,
M., Aufi ero, D. J., Kitabayashi, I., Herr, W. &
Cleary, M. L. (2004). Leukemia proto-onco-
protein MLL forms a SET1-like histone meth-
yltransferase complex with menin to regulate
Hox gene expression. Mol Cell Biol 24 ,
11. Hughes, C. M., Rozenblatt-Rosen, O., Milne,
T. A., Copeland, T. D., Levine, S. S., Lee, J. C.,
Hayes, D. N., Shanmugam, K. S., Bhattacharjee,
A., Biondi, C. A., Kay, G. F., Hayward, N. K.,
Hess, J. L. & Meyerson, M. (2004). Menin
associates with a trithorax family histone meth-
yltransferase complex and with the hoxc8 locus.
Mol Cell 13 , 587–97.
12. Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky,
K., Croce, C. M., Nakamura, T., Mazo, A.,
Eisenbach, L. & Canaani, E. (2007). Knockdown
of ALR (MLL2) reveals ALR target genes
and leads to alterations in cell adhesion and
growth. Mol Cell Biol 27 , 1889–903.
13. Patel, S. R., Kim, D., Levitan, I. & Dressler, G.
R. (2007). The BRCT-Domain Containing
Protein PTIP Links PAX2 to a Histone H3,
Lysine 4 Methyltransferase
Developmental Cell 13 , 580.
14. Goo, Y. H., Sohn, Y. C., Kim, D. H., Kim, S.
W., Kang, M. J., Jung, D. J., Kwak, E., Barlev,
N. A., Berger, S. L., Chow, V. T., Roeder, R.
G., Azorsa, D. O., Meltzer, P. S., Suh, P. G.,
Song, E. J., Lee, K. J., Lee, Y. C. & Lee, J. W.
(2003). Activating signal cointegrator 2 belongs
to a novel steady-state complex that contains a
subset of trithorax group proteins. Mol Cell
Biol 23 , 140–9.
15. Hong, S., Cho, Y.-W., Yu, L.-R., Yu, H.,
Veenstra, T. D. & Ge, K. (2007). Identifi cation
of JmjC domain-containing UTX and JMJD3
as histone H3 lysine 27 demethylases. Proc
Natl Acad Sci USA 104 , 18439–18444.
16. Lee, S., Lee, D.-K., Dou, Y., Lee, J., Lee, B.,
Kwak, E., Kong, Y.-Y., Lee, S.-K., Roeder, R.
G. & Lee, J. W. (2006). Coactivator as a target
gene specifi city determinant for histone H3
lysine 4 methyltransferases. Proc Natl Acad Sci
U S A 103 , 15392–15397.
17. Abmayr, S. M., Yao, T., Parmely, T. & Workman,
J. L. (2006). Preparation of nuclear and cyto-
plasmic extracts from mammalian cells. Curr
Protoc Mol Biol Chapter 12 , Unit 12 1.