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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 methyltransferase complexes would help understand the molecular mechanisms by which histone H3K4 methylation regulates mammalian gene expression. In this chapter, we provide a one-step affinity purification protocol on isolation of the MLL3/MLL4 histone H3K4 methyltransferase complex using FLAG-tagged PA1, a unique subunit of the MLL3/MLL4 complex.
Histone lysine methylation plays essential roles in chromatin dynamics, transcription, and DNA repair. Histone lysine methylation is dynamically regulated by site-specific 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 methyltransferase (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), 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 complexes (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 subunits of the MLL3/MLL4 histone H3K4 methyltransferase complex 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 finding that H3K4 methyltransferases MLL3/MLL4 physically associate with H3K27 demethylase 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 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 modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
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 purified in one step using anti-FLAG antibody immunoprecipitation from nuclear extracts prepared from this cell line.
After identifying HeLaS cell lines expressing FLAG-tagged PA1 (HeLaS/F-PA1) by western blot, the HeLaS/F-PA1 cells are cultured till confluence in 100 × 15-cm dishes in DMEM containing 8% bovine serum, 2% FBS, and 0.1 mg/ml G418. Cells are collected, and nuclear extracts (N.E.) are prepared exactly as described (17).
Next, we describe one-step purification of the MLL3/MLL4 complex by immunoprecipitation with anti-FLAG antibody conjugated 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 μg/ml aprotinin, 2 μg/ml leupeptin, and 0.7 μg/ml pepstatin.
1Wide-orifice tips have a larger opening to allow the pipetting of viscous solutions. Wide-orifice tips can be homemade by simply cutting off the end of regulator tips with a razor blade. Use wide-orifice tips to pipette antibody-conjugated agarose.
2To pick single HeLaS cell colonies, first 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 filled 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-μl pipette tips in a way similar to picking bacteria colonies from agar plates.
3One day before freezing down cells, remove G418-containing medium and change to fresh culture medium without G418.
4After centrifugation, the pellet is loose in 50-ml conical tubes. Do not disturb the pellet when transferring the supernatant.