组蛋白去甲基酶UTX在发育和疾病中的作用

UTX(ubiquitously transcribed tetratricopeptide repeat,X chromosome)是抑制性组蛋白H3K27me3的特异性去甲基化酶,和甲基转移酶PRC2共同调控H3K27me3。此外,UTX也是组蛋白H3K4甲基转移酶MLL3/MLL4的组成部分。UTX参与胚胎发育、HOX基因的表达和重编程等生命过程。在歌舞伎综合征中,UTX突变是关键的致病因素。同时,UTX作为肿瘤抑制因子参与多种实体肿瘤和血液肿瘤的产生。该文总结了UTX在正常发育和疾病发生中的作用及近期研究的重大突破,并结合我们的研究探讨了UTX对体细胞重编程的影响。     

组蛋白去甲基化酶UTX与胚胎发育及疾病关系

组蛋白3赖氨酸27(histone 3 lysine 27, H3K27)去甲基化酶UTX(ubiquitously transcribed tetratricopeptide repeat on chromosome X, UTX)为X染色体上重复的转录三十四肽,是组蛋白3赖氨酸4(histone 3 lysine 4, H3K4)甲基转移酶复合物MLL2(mixed-lineage leukemia 2, MLL2)中的一员,可调节同源基因HOX(homeobox, HOX)和视网膜母细胞瘤基因RB(retinoblastoma, RB)转录谱系. UTX与BRG1-SWI/SNF重塑复合物(Brg1-containing ATPase-dependent Swi/Snf chromatin-remodeling complex, BRG1-SWI/SNF)相互作用促进染色质重塑. 因其在细胞的正确再编程、胚胎发育和组织特异性分化中扮演重要角色,UTX失活或缺失会导致癌症、胚胎发育缺陷等疾病的发生. 本文将对近年来UTX在胚胎发育及与疾病关系方面的研究进展做一综述.     

PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex

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.     

Histone methyltransferase MLL1 drives renal tubular cell apoptosis by p53-dependent repression of E-cadherin during cisplatin-induced acute kidney injury

Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102’s cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.Subject terms: Acute kidney injury, Methylation     

Molecular regulation of H3K4 trimethylation by Wdr82, a component of human Set1/COMPASS

In yeast, the macromolecular complex Set1/COMPASS is capable of methylating H3K4, a posttranslational modification associated with actively transcribed genes. There is only one Set1 in yeast; yet in mammalian cells there are multiple H3K4 methylases, including Set1A/B, forming human COMPASS complexes, and MLL1-4, forming human COMPASS-like complexes. We have shown that Wdr82, which associates with chromatin in a histone H2B ubiquitination-dependent manner, is a specific component of Set1 complexes but not that of MLL1-4 complexes. RNA interference-mediated knockdown of Wdr82 results in a reduction in the H3K4 trimethylation levels, although these cells still possess active MLL complexes. Comprehensive in vitro enzymatic studies with Set1 and MLL complexes demonstrated that the Set1 complex is a more robust H3K4 trimethylase in vitro than the MLL complexes. Given our in vivo and in vitro observations, it appears that the human Set1 complex plays a more widespread role in H3K4 trimethylation than do the MLL complexes in mammalian cells.     

The C. elegans H3K27 demethylase UTX-1 is essential for normal development, independent of its enzymatic activity

Epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. Here we report on the biological functions of UTX-1, the Caenorhabditis elegans homologue of mammalian UTX, a histone demethylase specific for H3K27me2/3. We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3 in C. elegans. Surprisingly, we found that the catalytic activity is not required for the developmental function of this protein. Biochemical analysis identified UTX-1 as a component of a complex that includes SET-16(MLL), and genetic analysis indicates that the defects associated with loss of UTX-1 are likely mediated by compromised SET-16/UTX-1 complex activity. Taken together, these results demonstrate that UTX-1 is required for many aspects of nematode development; but, unexpectedly, this function is independent of its enzymatic activity.     

Solution NMR Structure and Histone Binding of the PHD Domain of Human MLL5

Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide ‘reader’ domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.     

Automethylation Activities within the Mixed Lineage Leukemia-1 (MLL1) Core Complex Reveal Evidence Supporting a “Two-active Site” Model for Multiple Histone H3 Lysine 4 Methylation

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex.     

A Non-Active-Site SET Domain Surface Crucial for the Interaction of MLL1 and the RbBP5/Ash2L Heterodimer within MLL Family Core Complexes

The mixed lineage leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1–MLL4 and SETd1a,b. Dimethylation of H3K4 requires a sub-complex including WRAD (WDR5, RbBP5, Ash2L, and DPY-30), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active-site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5/Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or KIS, are required for formation of a second active site within SET1 family core complexes.