PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex - PubMed (original) (raw)
PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex
Young-Wook Cho et al. J Biol Chem. 2007.
Abstract
PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.
Figures
FIGURE 1. Ectopically expressed PTIP associates with two functionally distinct groups of proteins in cells
A, schematic representation of full-length mouse PTIP protein containing 1,056 amino acids. The six BRCT domains are marked in red. The glutamine-rich (Q-rich) region is marked in blue. B, silver staining of affinity-purified PTIP-associated proteins. Nuclear extracts prepared from HeLaS cells stably expressing FLAG-tagged PTIP (F-PTIP) or vector only (Vec) were incubated with anti-FLAG M2-agarose. After extensive washing, bound proteins were resolved on 4–15% SDS-PAGE and analyzed by mass spectrometry as described under “Experimental Procedures.” IP, immunoprecipitation. M, protein marker. C, Western blot confirmation of mass spectrometry results. PTIP-associated proteins purified as in B were analyzed by Western blot with antibodies indicated on the right.
FIGURE 2. PTIP associates with ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, MLL3, MLL4, and PA1 in one complex
A, PTIP exists in high molecular weight complex(es) associated with HMT activity. PTIP-associated proteins isolated as in Fig. 1_B_ were fractionated on Superose 6 gel filtration column (Amersham Biosciences) in buffer A containing 300 m
M
KCl. Fractions were analyzed by Western blot with antibodies indicated on the left (lower panel) and by HMT assay on unmodified histone H3 peptide (residues 1–20) (upper panel). Positions of protein standards are indicated at the bottom. Note that PTIP protein alone purified by gel filtration did not possess any HMT activity. B, the two functionally distinct groups of PTIP-associated proteins do not exist in the same complex. PTIP-associated proteins purified as in Fig. 1B were subjected to immuno-depletion with anti-53BP1 or anti-RBBP5 antibodies, followed by Western blot with antibodies indicated on the right. No Ab, no antibody. C, purification of PTIP complex. PTIP-associated proteins isolated as in Fig. 1_B_ were incubated with immobilized anti-RBBP5 antibody at 4 °C overnight. After washing with buffer A containing 300 m
M
KCl, bound proteins were eluted with 100 m
M
triethylamine (pH 11.5), neutralized with 1
M
Tris (pH 7.5), concentrated with microcon column, resolved on 4–15% SDS-PAGE, and stained with silver. Protein identities were confirmed by mass spectrometry and/or Western blot.
PTIP.com
, PTIP complex. IgG heavy chain (IgG H) and light chain (IgG L) are marked with asterisks.
FIGURE 3. One-step purification of PTIP complex through FLAG-tagged PA1
A, amino acid sequence of the novel human protein PA1. Peptide sequences identified by mass spectrometry analysis of PTIP-associated proteins as described in the legend Fig. 1B are underlined. B, PA1 binds directly to PTIP. GST or GST-PA1 was incubated with in vitro translated, [35S]methionine-labeled PTIP, ASH2L, RBBP5, WDR5, and NCOA6 in GST pull down assay. C, one-step purification of PTIP complex through FLAG-tagged PA1. Nuclear extracts prepared from HeLaS cells expressing FLAG-tagged PA1 (F-PA1)or vector only (Vec) were incubated with anti-FLAG M2-agarose. After extensive washing, bound proteins were resolved on 4–15% SDS-PAGE followed by silver staining (right panel) or by staining using Zinc Stain & Destain kit (Bio-Rad) (left panel). The identities of the proteins were determined by mass spectrometry analysis. The numbers of matching tryptic peptides are indicated in parentheses. The sequences and locations of tryptic peptides matching UTX are listed on the right side of the silver-stained gel. Note that the ∼540-kDa band containing both MLL3 and MLL4 could be well stained by zinc but not silver. D, PA1 requires PTIP for interaction with the rest of the PTIP complex. SV40T-immortalized PTIP conditional knock-out MEF cell line _PTIP_flox/flox was infected with a retroviral vector pMSCVhygro expressing F-PA1. After selection with hygromycin, cells were subsequently infected with retroviruses pMSCVpuro expressing vector (Vec) or Cre recombinase to delete PTIP gene. After selection with puromycin, cells were expanded, and nuclear extracts were prepared and subjected to immunoprecipitation with anti-FLAG M2-agarose. The immunoprecipitates were analyzed by Western blot with antibodies indicated on the left.
FIGURE 4. In cells without DNA damage agent treatment, endogenous PTIP complex components do not associate with 53BP1 and other DNA damage response proteins
A, rabbit IgG (as control), anti-PTIP, ASH2L, RBBP5, NCOA6, 53BP1, MRE11, and RAD50 antibodies were incubated with 1 mg of HeLaS nuclear extracts. The immunoprecipitates were subjected to Western blot with antibodies indicated on the left. B—D, endogenous interactions among PTIP complex components. Experiments were done as described for A.
FIGURE 5. PTIP complex methylates histone H3 on K4
A, PTIP complex methylates histone H3. Proteins immunoprecipitated by anti-FLAG M2-agarose from nuclear extracts of HeLaS cells expressing F-PTIP (left panel) or F-PA1 (right panel) were subjected to HMT assay using recombinant histone H3 or core histones as substrates. Assay products were separated on SDS-PAGE, stained with Coomassie Blue, followed by fluorography. B, PTIP complex methylates histone H3 on K4. Four _μ_g of histone H3 methylated as described for A was subjected to 40 cycles of Edman degradation and scintillation counting. Positions of histone H3 residues are indicated at the bottom. C, endogenous PTIP complex subunits are associated with robust HMT activity. HeLaS nuclear extracts were immunoprecipitated with indicated antibodies. Immunoprecipitates were subjected to HMT assay on unmodified histone H3 peptide (residues 1–20). D, nuclear extracts prepared from HeLaS cells expression F-PA1 or vector were immunoprecipitated with anti-FLAG M2-agarose followed by HMT assay. E, PTIP is not required for the enzymatic activity of the associated HMT complex. SV40T-immortalized _PTIP_flox/flox MEF cell line was infected with retroviruses expressing vector (Vec) or Cre to delete PTIP gene. Nuclear extracts were prepared and subjected to immunoprecipitation with indicated antibodies. The immunoprecipitates were subjected to HMT assay on unmodified histone H3 peptide (residues 1–20) (left panel) and Western blot analysis (right panel).
FIGURE 6. PA1 and the JmjC domain-containing putative histone demethylase UTX specifically associate with PTIP complex
A, Western blot characterization of subunit composition of PTIP complex isolated through F-PA1. Nuclear extracts prepared from HeLaS cells stably expressing F-PA1 were incubated with anti-FLAG M2-agarose. Immunoprecipitates were analyzed by Western blot using antibodies indicated on the left. B, nuclear extracts prepared from HeLaS cells expressing FLAG-tagged UTX (F-UTX) were incubated with anti-FLAG M2-agarose. Immunoprecipitates were analyzed by Western blot against distinct subunits (hSet1, Menin, and PTIP) or the shared RBBP5 subunit of various human Set1-like complexes.
FIGURE 7. hDPY-30 binds ASH2L directly and is shared by all human Set1-like complexes
A, hDPY-30 binds ASH2L directly. To characterize the direct interactions among PTIP complex components, GST or GST-ASH2L, -RBBP5, -WDR5, and -hDPY-30 were incubated with in vitro translated, [35S]methionine-labeled ASH2L, RBBP5, WDR5, hDPY-30, and the COOH-terminal SET domain-containing fragments of MLL3 and MLL4 in GST pull-down assay. B, WDR5 interacts with the FYRN- and FYRC-domain-containing region of MLL3. Top panel, schematic diagram of deletion mutations of the COOH-terminal 3,993– 4,911 amino acids of MLL3. Lower panel, GST or GST-WDR5 was incubated with in vitro translated, [35S]methionine-labeled deletion mutations of the COOH-terminal fragment of MLL3 in GST pull-down assay. C, hDPY-30 is shared by all human Set1-like complexes. Nuclear extracts prepared from HeLaS cells stably expressing FLAG-tagged hDPY-30 (F-hDPY-30) were incubated with anti-FLAG M2-agarose. Immunoprecipitates were analyzed by Western blot using antibodies against distinct subunits (hSet1, MLL, Menin, NCOA6, PTIP, and PA1) or shared subunits (ASH2L, RBBP5, and WDR5) of various human Set1-like complexes.
References
- Santos-Rosa H, Schneider R, Bannister AJ, Sherriff J, Bernstein BE, Emre NC, Schreiber SL, Mellor J, Kouzarides T. Nature. 2002;419:407–411. - PubMed
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