Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation
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Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription.
Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1.
Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.
KeywordsChIP Assay Mixed Lineage Leukemia cAMP Response Element Binding Protein H3K18 Acetylation H3K27me3 Level
Di- or trimethylation (hypermethylation) of histone H3 at lysine 4 (H3K4) is found at actively transcribed genes [1, 2, 3, 4, 5], is functionally linked to activating acetylation events at promoters , and is mediated by the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS)-like complexes [7, 8]. MLL/COMPASS-like complexes contain several well-characterized, common subunits; Ash2L, WDR5 and RbBP5, which are conserved between yeast and humans [9, 10, 11]. These subunits are thought to constitute a common MLL/COMPASS subcomplex that forms a platform to mediate the Set1 enzyme and H3K4 substrate interaction [9, 11, 12, 13]. H3K4 hypermethylation also has been found to inhibit H3K9 methylation, and more recently has been associated with a release from repressive H3K27 methylation by MLL/COMPASS-associated histone demethylases [14, 15, 16, 17, 18, 19]. Observations that the deposition of activating methylation events on H3K4 correlates with the removal of silencing methylation events on H3K27 have important implications for the regulation of bivalent genes, including cytokine-inducible genes of the immune system.
Major histocompatibility class (MHC)-II molecules are cell surface glycoproteins that present antigenic peptides to CD4+ T lymphocytes of the adaptive immune system [20, 21]. MHC-II expression is regulated primarily at the level of transcription by several well-studied elements within the MHC-II proximal promoter, which bind a series of ubiquitously expressed transcription factors to form a basal enhanceosome complex (see Additional file 1) [22, 23, 24]. Once assembled, the enhanceosome recruits a master regulator, the class II transactivator (CIITA) . Transcription of CIITA is driven by one of four distinct promoters in a cell-type and cytokine specific manner. The well-characterized CIITA promoter (p)IV is responsible for interferon (IFN)-γ-inducible expression in nucleated cells [26, 27]. Upon cytokine stimulation, the Janus kinase/signal transducer and activator of transcription 1 (JAK/STAT) pathway is triggered, leading to pIV accumulation of the requisite transcription factors, and the initiation of CIITA transcription [28, 29, 30]. Once recruited to the MHC-II promoter, CIITA binding stabilizes the enhanceosome complex and recruits basal transcriptional components to initiate the switch to an elongation complex [24, 31, 32, 33, 34]. Although much is known about the requirement of transcription factors at these cytokine-inducible promoters, considerably less is understood about the coordination of the histone modifying events that allow these promoters to switch from a semi-poised state to a fully open, transcription-accessible structure.
The objective of this study was to investigate the importance of 19S ATPase-regulated activating histone H3 trimethylation at lysine 4 (H3K4me3) events in chromatin remodeling at MHC-II and CIITA promoters, and to determine if the 19S ATPase Sug1directly affects multiple promoter remodeling events. In this report, we show that common MLL/COMPASS subunits (Ash2L, RbBP5 and WDR5) bind to the MHC-II promoter and to CIITA pIV; that overexpressing a single common subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that WDR5 knockdown decreases H3K4me3 at promoters of both MHC-II and CIITA. Although we have previously shown H3K18 acetylation to be regulated by 19S ATPases, there were no observable changes in levels of H3K18 acetylation in the absence of WDR5, indicating that Sug1 independently regulates acetylation and methylation events. Further supporting our hypothesis that Sug1 regulates multiple independent events is our observation that knockdown of Sug1 promotes elevated H3K27me3 levels at cytokine-inducible promoters. Together these results implicate the 19S proteasome in the initial reorganization of chromatin to relax the repressive histone environment surrounding cytokine-inducible promoters.
Common MLL/COMPASS subunits associate with cytokine-inducible promoters
Overexpressing a single common subunit of MLL/COMPASS complexes enhances CIITA transactivity and MHC-II HLA-DRA expression
A WDR5-dependent complex trimethylates H3K4 at cytokine-inducible genes
WDR5 does not contribute to histone H3K18 acetylation
H3K27me3 is enhanced upon Sug1 knockdown
We previously demonstrated that the 19S proteasome regulates activating H3K4 trimethylation at cytokine-inducible promoters, potentially through interactions with common MLL/COMPASS subunits, as a loss of recruitment of WDR5 and Ash2L subunits is observed upon Sug1 knockdown . In the current study, we showed that these common MLL/COMPASS subunits including WDR5, Ash2L and RbBP5 bind to both the MHC-II promoter and to CIITA pIV. The enrichment of recruited subunits upon IFN-γ stimulation varies, and the subunits bind with intensities similar to constitutively expressed GAPDH. Whether the increases in recruitment of these common subunits reflects increases in binding or differences in antibody affinity remains to be determined. One plausible explanation is that a subcomplex is assembled on promoters before transcription initiation. Overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and gene expression, further implicating these subunits as co-activators for MHC-II. Consistent with this, we observed a loss of H3K4me3 at the MHC-II promoter and CIITA pIV when levels of endogenous WDR5 were diminished. However, as WDR5 has been found in complexes that lack SET domain histone methyltransferase enzymes, we cannot rule out the possibility that the loss of H3K4me3 observed upon WDR5 knockdown is due to other mechanisms .
We previously observed a loss in inducible WDR5 and Ash2L binding to CIITA pIV when endogenous Sug1 is diminished . These findings implied that the 19S proteasome plays a role in stabilizing a large multienzyme histone remodeling complex through an interaction dependent on WDR5 at cytokine-inducible genes. Therefore, we sought to determine if knockdown of WDR5 mirrors that of 19S ATPase knockdown, which results in a loss of H3K18ac in addition to H3K4me3 [36, 41, 45]. Knockdown of WDR5 did not mirror that of Sug1 knockdown as there was no effect on histone H3K18 acetylation. Thus, the initial chromatin reorganization event regulated by 19S ATPases remains to be determined.
Evidence now suggests that demethylation of silencing modifications is intimately linked to the deposition of histone-activating events to promote the relaxation of the repressive nature of chromatin [14, 15, 47, 48]. In addition to binding CBP and CARM-1, the common MLL/COMPASS subunit WDR5 is associated with histone H3K27me3 demethylases [14, 15]. Consistent with this, we observed a gain of one of the known H3K27me3 demethylases (UTX) and a loss of H3K27me3 at CIITA pIV, and at the MHC-II promoter, upon cytokine induction . As we previously observed that WDR5 and Ash2L binding to CIITA pIV is inhibited in the absence of Sug1, it was expected that histone demethylase recruitment would be negatively affected upon diminished Sug1 expression and would in turn affect regulation of H3K27me3. In fact, H3K27me3 levels are significantly elevated at each of these cytokine-inducible, bivalent promoters in Sug1 siRNA-transfected cells, but are unaffected at the constitutively active GAPDH promoter. As little is known regarding how inducible genes are relieved from and subsequently returned to a repressive state, the dynamic recruitment and activity of UTX and other histone H3K27me3 demethylases to cytokine-inducible promoters is not well understood. Although we show here that the 19S ATPase Sug1 functions to release chromatin from a repressive state, further studies are required to characterize the molecular interactions between 19S ATPases, UTX, JMJD3 and silenced chromatin.
To our knowledge, ours is the first report implicating the 19S proteasomal ATPases in regulating the initial chromatin remodeling events at cytokine-inducible promoters that releases surrounding chromatin from a more condensed, repressive state. Our observations that in the absence of Sug1, methylation of H3K27 is not only elevated, but is also persistent, strongly implicate the 19S ATPases in initial chromatin reorganization in response to stimuli. A full understanding of the contributions of Sug1 and the other 19S ATPases to the initial epigenetic regulation and chromatin reorganization at cytokine-inducible genes requires further studies into the molecular interactions occurring at these promoters.
HeLa (human epithelial) cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Mediatech Inc., Herndon, VA, USA) supplemented with 10% fetal calf serum, 5 mM L-glutamine and 5 mM penicillin-streptomycin at 37°C with 5% carbon dioxide.
Trimethylated H3K4, acetylated H3K18 and WDR5 antibodies were from Abcam (Cambridge, MA, USA). Ash2L and RbBP5 antibodies were from Bethyl Laboratories (Montgomery, TX, USA). Rabbit IgG isotype control and mouse IgG isotype control antibodies were from Upstate (Lake Placid, NY, USA). Sug1 antibody was from Novus Biologicals (Littleton, CO, USA). Horseradish peroxidase (HRP)-conjugated mouse antibody (W4021) was from Promega (Madison, WI, USA) and HRP-conjugated rabbit antibody (1858415) was from Pierce (Rockland, IL, USA).
Flag-CIITA, HLA-DRA-Luc, pcDNA3 plasmids were as previously described . Renilla luciferase control vector (E2231) was from Promega (Madison, WI, USA). Myc-Sug1 was generously provided by A. A. Wani . Flag-WDR5, Flag-Ash2L and Flag-RbBP5 were graciously provided by A. Shilatifard .
siRNA constructs and transient transfections
Previously described siRNAs [41, 49] were used for transient knockdown of the Sug1 19S ATPase. Pooled siRNA duplexes for WDR5 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HeLa cells transfected with Allstar scrambled sequence control siRNA (Qiagen, Valencia, CA, USA) or specific siRNA were treated with IFN-γ as indicated. Cells were lysed in NP40 lysis buffer (1 M Tris pH 8.0, 1 M KCl, 10% NP40, 0.5 M EDTA, 5 M NaCl, 1 M dithiothreitol, distilled (d)H2O) supplemented with Complete EDTA-free Protease Inhibitors (Roche, Indianapolis, IN, USA), and knockdown efficiency was assessed by immunoblotting as described above.
Luciferase reporter assays
HeLa cells plated in six-well plates at a density of 5 × 104 cells/well were transfected with CIITA, HLA-DRA-Luc, Renilla, pcDNA3 and MLL/COMPASS subunit plasmids as indicated, using Fugene 6 (Roche) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were subjected to dual-luciferase assay (Promega) according to the manufacturer's protocol.
RNA transcript levels
HeLa cells were plated at a density of 8 × 105 cells/plate and transfected with 1 μg CIITA alone or in combination with MLL/COMPASS subunit plasmids (0.5 μg and 1 μg) as indicated, using Fugene 6 (Roche) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were harvested and total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Omniscript reverse transcription kit (Qiagen) was used to generate cDNA using gene-specific antisense primers for MHC-II HLA-DR and GAPDH . cDNA was quantified via real-time PCR using previously published primers and probes specific for MHC-II HLA-DR and GAPDH . Real-time PCR values were generated on the basis of standard curves generated for each gene and are presented as fold changes over non-transfected samples.
ChIP assays were performed as previously described . In brief, HeLa cells were stimulated with 500 U/ml IFN-γ (Peprotech, Rocky Hill, NJ, USA) as indicated. Crosslinked cells were lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0, dH20) with Complete EDTA-free Protease Inhibitors (Roche) for 20 minutes on ice, and sonicated to generate sheared DNA with an average length of 500-750 bp. Sonicated samples were precleared with salmon sperm-coated agarose beads (Upstate), and half of the lysate was immunoprecipitated with 5 μg of polyclonal antibody against H3K4me3, H3K18ac, WDR5 (all Abcam), Ash2L or RbBP5 (both Bethyl Laboratories) overnight at 4°C. The remaining lysate was immunoprecipitated with isotype control antibody (Upstate). After a 2 hour incubation with 60 μl of salmon sperm-coated agarose beads, samples were washed for 5 minutes at 4°C with the following buffers: low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl, dH20), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl, dH20), LiCl buffer (0.25 M LiCl, 1% NP40, 1% DOC, 1 mM EDTA, 10 mM Tris pH 8.0, dH20) and 1 × TE buffer, and were eluted with SDS elution buffer (1% SDS, 0.1 M NaHCO3, dH2O). After elution, crosslinks were reversed with 5 M NaCl at 65°C and immunoprecipitated, and control DNA was isolated using a phenol:chloroform:isoamyl alcohol mix (Invitrogen) according to the manufacturer's protocol. Isolated DNA was analyzed by real-time PCR using probes and primers spanning the W-X-Y box of the MHC-II HLA-DRA promoter and CIITA pIV [36, 41, 49]. Values graphed were calculated based on standard curves generated.
ChIP in siRNA knockdown cells
HeLa cells were transfected with specific siRNA or control siRNA using RNAiFect transfection reagent (all from Qiagen) according to the manufacturer's instructions. Cells were treated with 500 U/ml IFN-γ as indicated. 10% of the total cell volume was lysed in 1% Nonidet P-40 buffer (1 M Tris pH 8.0, 1 M KCl, 10% NP40, 0.5 M EDTA, 5 M NaCl, 1 M DTT, dH2O) with Complete EDTA-free Protease Inhibitors (Roche) and analyzed by immunoblotting for knockdown verification. The remaining fraction of cells was subjected to ChIP assay.
This work was supported by funding from the Molecular Basis of Disease Area of Focus at Georgia State University (in support of OIK and ADT), the HHMI Biotechnology Scholars Program (in support of RKD) and grants from the American Cancer Society; the Georgia Cancer Coalition; and the Georgia Research Alliance (to SFG).
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