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MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer
MLL3 在肝癌中调控 CDKN2A 抑癌基因位点

Changyu Zhu , Yadira M Soto-Feliciano , John P Morris , Chun-Hao Huang ,
Changyu Zhu , Yadira M Sotoo-Feliciano , John P Morris , Chun-Hao Huang
Richard P Koche , Yu-jui Ho', Ana Banito', Chun-Wei Chen', Aditya Shroff1,
Richard P Koche , Yu-jui Ho', Ana Banito', Chun-Wei Chen', Aditya Shroff1、
Sha Tian', Geulah Livshits , Chi-Chao Chen , Myles Fennell', Scott A Armstrong ,
沙田, Geulah Livshits , 陈志超 , Myles Fennell', Scott A Armstrong ,
C David Allis , Darjus F Tschaharganeh , Scott W Lowe
C David Allis {{0}, Darjus F Tschaharganeh 斯科特-W-洛 {{2}

Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer
斯隆-凯特琳癌症纪念医院癌症生物学和遗传学系
Center, New York, United States; Laboratory of Chromatin Biology and Epigenetics,
中心,美国纽约; 染色质生物学和表观遗传学实验室、
The Rockefeller University, New York, United States; Koch Institute for Integrative
美国纽约洛克菲勒大学; 科赫综合研究所
Cancer Research, Massachusetts Institute of Technology, Cambridge, United
美国剑桥麻省理工学院癌症研究中心
States; Department of Pharmacology, The University of North Carolina at Chapel
美国; 北卡罗来纳大学教堂分校药理学系
Hill, Chapel Hill, United States; Center for Epigenetics Research, Memorial Sloan
希尔,美国教堂山; 斯隆纪念医院表观遗传学研究中心
Kettering Cancer Center, New York, United States; Dana-Farber Cancer Institute,
美国纽约凯特琳癌症中心; 达纳-法伯癌症研究所、
Boston, United States; Helmholtz-University Group "Cell Plasticity and Epigenetic
美国波士顿; 亥姆霍兹大学 "细胞可塑性和表观遗传学 "小组
Remodeling", German Cancer Research Center, Heidelberg, Germany; Howard
重塑",德国海德堡德国癌症研究中心; 霍华德
Hughes Medical Institute, New York, United States
美国纽约休斯医学研究所

*For correspondence: ysoto@mit.edu (YMS-F); d.tschaharganeh@dkfz.de (DFT); lowes@mskcc.org (SWL)
*通信地址:ysoto@mit.edu (YMS-F); d.tschaharganeh@dkfz.de (DFT); lowes@mskcc.org (SWL)
TThese authors contributed equally to this work
这些作者对本研究做出了同等贡献
Competing interest: See page 18
竞争利益:参见第 18 页
Funding: See page 19
资金:见第 19 页
Received: 07 June 2022 Preprinted: 09 June 2022
收到:2022 年 6 月 7 日 预印:2022 年 6 月 9 日
Accepted: 31 May 2023
接受:接受: 2023 年 5 月 31 日
Published: 01 June 2023
出版日期:2023 年 6 月 1 日
Reviewing Editor: Hao Zhu, University of Texas Southwestern Medical Center, United States
审稿编辑:Hao Zhu,美国得克萨斯大学西南医学中心
(c) Copyright Zhu, Soto-Feliciano, Morris et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
(c) Zhu、Soto-Feliciano、Morris 等人版权所有。本文根据知识共享署名许可协议的条款发布,该协议允许在注明原作者和出处的前提下不受限制地使用和再发布。

Abstract 摘要

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arfdependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.
编码染色质修饰和重塑复合物成分的基因突变是人类癌症中最常见的体细胞事件之一。例如,针对混合系白血病家族成员 3(MLL3,由 KMT2C 编码)组蛋白甲基转移酶的错义突变和无义突变发生在一系列实体瘤中,而包含 KMT2C 的杂合缺失发生在侵袭性白血病的一个亚群中。虽然 MLL3 缺失会促进小鼠的肿瘤发生,但 MLL3 抑制肿瘤发生的分子靶点和生物学过程仍鲜为人知。在这里,我们结合遗传学、表观基因组学和动物模型方法证明,MLL3 将染色质重塑与肿瘤抑制联系起来的机制之一是通过共同激活 Cdkn2a 肿瘤抑制基因座。在小鼠肝细胞癌(HCC)的发病过程中, 的破坏与Myc的过度表达相互配合,MLL3与Cdkn2a基因座的结合被削弱,导致H3K4甲基化减少,基因座编码的肿瘤抑制因子p16/Ink4a和p19/Arf的表达水平降低。相反,KMT2C表达的升高会增加其与CDKN2A基因座的结合,并共同激活基因转录。内源性 的恢复会逆转这些染色质和转录效应,并引发依赖于Ink4a/Arf的细胞凋亡。我们发现,在人类 HCC 样本中,KMT2C 和 CDKN2A 基因组的改变与相似的转录谱相关,这强调了这种表观相关性的人类相关性。这些结果共同指出了在癌症发展过程中破坏 CDKN2A 活性的新机制,并由此将 MLL3 与已建立的肿瘤抑制因子网络联系起来。

Editor's evaluation 编辑评价

This paper convincingly shows that MLL3 regulates the CDKN2A tumor suppressor in MYC-driven liver cancers. The use of in vivo models and epigenomic analysis made the findings particularly robust. This work significantly advances our understanding of the function of MLL3 in cancer.
这篇论文令人信服地表明,在 MYC 驱动的肝癌中,MLL3 调节 CDKN2A 肿瘤抑制因子。活体模型和表观基因组分析的使用使研究结果尤为有力。这项工作极大地促进了我们对 MLL3 在癌症中功能的理解。

Introduction 导言

Hepatocellular carcinoma (HCC) is a deadly primary liver cancer with a 5 year survival rate of only 18% (Jemal et alo, 2017). HCC is currently the fourth most frequent cause of cancer-related mortality worldwide, and its incidence continues to grow (Llovet et al., 2021). Genomic alterations found in HCC are highly diverse and are characterized by promoter mutations in TERT (telomerase reverse transcriptase), amplifications, or chromosomal gains encompassing the MYC oncogene, activating hotspot mutations in CTNNB1 ( -catenin), and inactivating mutations and deletions in the TP53 and CDKN2A tumor suppressor genes (2017; Schulze et al., 2015).
肝细胞癌(HCC)是一种致命的原发性肝癌,5年生存率仅为18%(Jemal et alo, 2017)。目前,HCC 在全球癌症相关死亡率中排名第四,而且发病率还在持续增长(Llovet 等人,2021 年)。在 HCC 中发现的基因组改变非常多样化,其特点是 TERT(端粒酶逆转录酶)的启动子突变、扩增或染色体增益,包括 MYC 致癌基因、CTNNB1 ( -catenin) 的激活热点突变以及 TP53 和 CDKN2A 抑癌基因的失活突变和缺失(2017 年;Schulze 等人,2015 年)。
Among these alterations, genetic gain of MYC and inactivation of tumor suppressor p53 are known to cooperate to drive tumorigenesis in HCC (Molina-Sánchez et al., 2020). Mechanistically, oncogenic MYC activation triggers increased expression of the tumor suppressor ARF, one of two proteins encoded in CDKN2A in alternative reading frames. ARF binds to the E3 ubiquitin ligase MDM2 to prevent p53 degradation, leading to apoptosis to restrain MYC-driven tumorigenesis (Lowe and Sherr, 2003). However, it is unclear how the CDKN2A locus is regulated in response to MYC overexpression.
在这些改变中,已知 MYC 的遗传增殖和肿瘤抑制因子 p53 的失活共同推动了 HCC 的肿瘤发生(Molina-Sánchez 等人,2020 年)。从机理上讲,致癌的 MYC 激活会引发肿瘤抑制因子 ARF 的表达增加,ARF 是 CDKN2A 在替代阅读框中编码的两种蛋白之一。ARF 与 E3 泛素连接酶 MDM2 结合,阻止 p53 降解,导致细胞凋亡,从而抑制 MYC 驱动的肿瘤发生(Lowe 和 Sherr,2003 年)。然而,目前还不清楚 CDKN2A 基因座在 MYC 过表达时是如何调节的。
Beyond these well-studied drivers, HCC frequently harbors mutations in one or more chromatin modifying enzymes, including MLL3 (encoded by KMT2C; Fujimoto et al., 2012; Kan et al., 2013). MLL3 is a component of the COMPASS-like complex that has structural and functional similarities to the developmentally essential Drosophila Trithorax-related complex (Schuettengruber et al., 2017). This multiprotein complex controls gene expression through its histone H3 lysine 4 (H3K4) methyltransferase activity, which establishes chromatin modifications most often associated with transcriptional activation (Shilatifard, 2012). Most studies have shown that MLL3 and its paralog MLL4 (encoded by KMT2D) typically catalyze H3K4 monomethylation (H3K4me1) at enhancers (Herz et al., 2012; Hu et al., 2013), while the MLL1/2 complex is responsible for H3K4 trimethylation (H3K4me3) at promoters and enhancers in a locus-specific manner (Denissov et al., 2014; Rickels et al., 2016; Wang et al., 2009).
除了这些已被充分研究的驱动因素外,HCC 还经常携带一种或多种染色质修饰酶的突变,包括 MLL3(由 KMT2C 编码;Fujimoto 等人,2012 年;Kan 等人,2013 年)。MLL3 是 COMPASS-like 复合物的一个组成部分,该复合物与果蝇发育必需的 Trithorax 相关复合物具有结构和功能上的相似性(Schuettengruber 等人,2017 年)。这种多蛋白复合物通过其组蛋白 H3 赖氨酸 4(H3K4)甲基转移酶活性控制基因表达,这种活性可建立最常与转录激活相关的染色质修饰(Shilatifard,2012 年)。大多数研究表明,MLL3 及其同系物 MLL4(由 KMT2D 编码)通常在增强子处催化 H3K4 单甲基化(H3K4me1)(Herz 等人,2012 年;Hu 等人,2013 年),而 MLL1 及其同系物 MLL4(由 KMT2D 编码)则在增强子处催化 H3K4 单甲基化(H3K4me2)、2013),而 MLL1/2 复合物以基因座特异性的方式负责启动子和增强子的 H3K4 三甲基化(H3K4me3)(Denissov 等人,2014;Rickels 等人,2016;Wang 等人,2009)。
While less characterized, MLL3/4 regulation of promoter activity is emerging as an additional mechanism connecting the COMPASS-like complex to gene expression. Some publications report that H3K4me1 enrichment at promoters has been associated with gene repression (Cheng et al., 2014), and MLL3 inactivation decreases H3K4me3 levels at the promoters of metabolism-related genes in normal murine livers (Valekunja et al., 2013) and human liver cancer cells (Ananthanarayanan et al., 2011). Furthermore, a recent study in leukemia cells demonstrated that MLL3 and MLL4, in the absence of MLL 1/2 complex, are capable of binding to promoters to activate tumor suppressor genes (Soto-Feliciano et al., 2023). These divergent results suggest that the genomic binding pattern and functions of MLL3 are highly context dependent.
MLL3/4 对启动子活性的调控正在成为连接 COMPASS-like 复合物与基因表达的另一种机制,但这种机制的特征较少。一些文献报道,启动子上的 H3K4me1 富集与基因抑制有关(Cheng 等人,2014 年),MLL3 失活会降低正常鼠肝脏(Valekunja 等人,2013 年)和人类肝癌细胞(Ananthanarayanan 等人,2011 年)中代谢相关基因启动子上的 H3K4me3 水平。此外,最近在白血病细胞中进行的一项研究表明,在没有 MLL 1/2 复合物的情况下,MLL3 和 MLL4 能够与启动子结合,激活肿瘤抑制基因(Soto-Feliciano 等人,2023 年)。这些不同的结果表明,MLL3 的基因组结合模式和功能高度依赖于环境。
Notably, HCC also harbors mutations in KMT2D (Cleary et al., 2013), while KDM6A/UTX, an H3K27 demethylase within the COMPASS-like complex, has been functionally established as a potent tumor suppressor in pancreatic and liver cancers (Revia et al., 2022). These observations suggest that epigenetic-based mechanisms of gene regulation controlled by the MLL3 complex may constrain HCC development. However, because chromatin regulators such as ARID1A often exhibit contextspecific tumor suppressive and oncogenic roles in liver cancer development (Sun et al., 2017), it is unclear whether MLL3 is a bona fide tumor suppressor in HCC. We therefore employed mouse models of HCC to investigate the molecular targets of MLL3 and the biological processes it affects.
值得注意的是,HCC 也存在 KMT2D 突变(Cleary 等人,2013 年),而 COMPASS-like 复合物中的 H3K27 去甲基化酶 KDM6A/UTX 在功能上已被确定为胰腺癌和肝癌的有效肿瘤抑制因子(Revia 等人,2022 年)。这些观察结果表明,由 MLL3 复合物控制的基于表观遗传学的基因调控机制可能会制约 HCC 的发展。然而,由于ARID1A等染色质调控因子在肝癌发展过程中经常表现出特定的肿瘤抑制和致癌作用(Sun等人,2017年),因此目前还不清楚MLL3在HCC中是否是真正的肿瘤抑制因子。因此,我们利用HCC小鼠模型来研究MLL3的分子靶点及其影响的生物学过程。

Results
MLL3 is a tumor suppressor in Myc-driven liver cancer
结果 MLL3 是 Myc 驱动的肝癌中的肿瘤抑制因子

To better understand the functional significance of genes commonly inactivated in HCC, including a number of chromatin regulators, we selected 12 genes with recurrent inactivating mutations in human HCC (Cancer Genome Atlas Research Network, 2017; Ahn et al., 2014; Fujimoto et al., 2012; Figure 1-figure supplement 1A) and performed a CRISPR-based in vivo screen to determine whether they behave as tumor suppressors in HCC. Specifically, the screen tested whether loss of each of these 12 genes would drive hepatic tumorigenesis in cooperation with Myc-one of the most frequently gained and/or amplified oncogenes in HCC (Huang et al., 2014). We applied hydrodynamic tail vein injection (HTVI) in wild-type mice to directly introduce genetic manipulations into adult
为了更好地了解HCC中常见失活基因(包括一些染色质调控因子)的功能意义,我们选择了12个在人类HCC中反复发生失活突变的基因(癌症基因组图谱研究网络,2017;Ahn等人,2014;Fujimoto等人,2012;图1-图1A),并进行了基于CRISPR的体内筛选,以确定它们在HCC中是否表现为肿瘤抑制因子。具体来说,该筛选测试了这 12 个基因中每个基因的缺失是否会与 Myc--HCC 中最常增殖和/或扩增的癌基因之一(Huang 等人,2014 年)--共同驱动肝脏肿瘤发生。我们在野生型小鼠中应用流体动力尾静脉注射(HTVI)技术,将基因操作直接引入成年小鼠的肝脏中。

hepatocytes in vivo (Bell et al., 2007). We introduced both a transposon vector for stable genomic integration of oncogenic Myc cDNA and plasmids designed for transient expression of Cas9 and single guide RNAs (sgRNAs; a mix of two for each gene; Figure 1B; Largaespada, 2009; Moon et al., 2019; Tschaharganeh et al., 2014; Xue et al., 2014). 3 months after HTVI, only sgKmt2c resulted in liver tumor formation with high penetrance (Figure 1-figure supplement 1B), suggesting that MLL3 likely acts as a tumor suppressor to constrain Myc-driven liver cancer. Supporting this idea, KMT2C mutations co-occur with MYC genomic gains and amplifications in human HCC tumors (Figure 1A).
Bell等人,2007)。我们引入了用于致癌 Myc cDNA 稳定基因组整合的转座子载体,以及用于瞬时表达 Cas9 和单导 RNA(sgRNA;每个基因有两种;图 1B;Largaespada,2009 年;Moon 等人,2019 年;Tschaharganeh 等人,2014 年;Xue 等人,2014 年)的质粒。HTVI 3 个月后,只有 sgKmt2c 导致了肝肿瘤的形成,且具有高渗透性(图 1-图 1B),这表明 MLL3 很可能作为肿瘤抑制因子限制了 Myc 驱动的肝癌。支持这一观点的是,在人类 HCC 肿瘤中,KMT2C 突变与 MYC 基因组增益和扩增同时存在(图 1A)。
To validate and extend the results from the screen, we applied the same approach to test whether the screen phenotype could be recapitulated with oncogenic Myc and single Kmt2c-targeted sgRNAs (Figure 1B). Mice injected with an Myc cDNA transposon combined with either of two independent Cas9/Kmt2c sgRNAs (Myc; sgKmt2c. 1 or Myc; sgKmt2c.2) developed liver tumors, with a slightly later onset and slightly longer survival than mice receiving the Myc transposon combined with an sgRNA targeting Trp53 (Myc; sgTrp53; Figure 1C and D). In contrast, mice injected with Myc and a control sgRNA (sgChrom8) did not succumb to disease over the observation period (Figure 1C). These findings were confirmed in an independent cohort of mice (Figure 1-figure supplement 2b). Analyses of tumor-derived genomic DNA revealed insertions and deletions (indels) in either Kmt2c or Trp53 depending on the genotype of tumor-derived cells (Figure 1-figure supplement 2B). DNA sequencing of the CRISPR-targeted region from two independent Myc; sgKmt2c tumors revealed indels predicted to generate premature stop codons (Figure 1-figure supplement ). In one case, the indel was heterozygous, implying that even partial suppression of can promote tumorigenesis. In support of this, GFP-linked Kmt2c shRNAs efficiently cooperated with Myc overexpression to drive liver cancer, producing tumors with reduction in Kmt2c mRNA expression (Figure 1-figure supplement 2D-G). shKmt2c. 2 resulted in less potent knockdown than shKmt2c. 1 yet produced faster tumor formation, suggesting that, as in acute myeloid leukemia (Chen et al., 2014), MLL3 can likely act as a haploinsufficient tumor suppressor in liver cancer (Figure 1-figure supplement 2E, G).
为了验证和扩展筛选结果,我们用同样的方法测试了致癌 Myc 和单一 Kmt2c 靶向 sgRNA 是否能重现筛选表型(图 1B)。注射了 Myc cDNA 转座子和两个独立的 Cas9/Kmt2c sgRNAs(Myc;sgKmt2c. 1 或 Myc;sgKmt2c.2)的小鼠出现了肝肿瘤,与接受 Myc 转座子和靶向 Trp53 的 sgRNAs(Myc;sgTrp53;图 1C 和 D)的小鼠相比,发病时间稍晚,存活时间稍长。相比之下,注射了 Myc 和对照 sgRNA(sgChrom8)的小鼠在观察期内没有发病(图 1C)。这些发现在一个独立的小鼠群中得到了证实(图 1-图补充 2b)。对肿瘤衍生基因组 DNA 的分析表明,根据肿瘤衍生细胞的基因型,Kmt2c 或 Trp53 存在插入和缺失(indels)(图 1-图 2b)。对两个独立的Myc; sgKmt2c肿瘤的CRISPR靶向区域进行DNA测序,发现了预计会产生过早终止密码子的吲哚(图1-图补充 )。在其中一个病例中,嵌合体是杂合的,这意味着即使部分抑制 也会促进肿瘤发生。为证明这一点,与 GFP 相连的 Kmt2c shRNA 与 Myc 过表达有效合作,驱动肝癌的发生,产生 Kmt2c mRNA 表达 减少的肿瘤(图 1-图补充 2D-G)。2的敲除效果不如shKmt2c.1 的敲除效果较弱,但却能更快地形成肿瘤,这表明,与急性髓性白血病(Chen 等,2014 年)一样,MLL3 在肝癌中可能充当单倍体肿瘤抑制因子(图 1-图 2E、G)。
Apart from MYC, CTNNB1 ( -catenin) is a frequently mutated oncogene in human HCC (Rebouissou et al., 2016), although the co-occurrence between CTNNB1 and KMT2C mutations was not statistically significant (Figure 1A). To test whether MLL3 loss can cooperate with oncogenic CTNNB1 to promote liver tumorigenesis, we performed analogous HTVI of a transposon vector expressing constitutively active -catenin (Ctnnb1-N90; Tward et al., 2007) in combination with Kmt2c- or Trp53targeted sgRNAs (Figure 1-figure supplement 3A). However, no tumor formation was observed in mice that received the Kmt2c-targeted sgRNAs (Figure 1-figure supplement 3B), indicating that unlike p53, the tumor-suppressive role of MLL3 is specific to the oncogene and contexts.
除MYC外,CTNNB1( -catenin)也是人类HCC中经常发生突变的癌基因(Rebouissou等人,2016年),尽管CTNNB1和KMT2C突变之间的共同发生率没有统计学意义(图1A)。为了检验 MLL3 缺失是否能与致癌 CTNNB1 合作促进肝脏肿瘤发生,我们对表达组成型活性 -catenin(CtnNB1)的转座子载体进行了类似的 HTVI。-catenin(Ctnnb1-N90;Tward 等人,2007 年)的转座子载体,并结合 Kmt2c 或 Trp53 靶向 sgRNA(图 1-图 3A)。然而,在接受 Kmt2c 靶向 sgRNAs 的小鼠中没有观察到肿瘤形成(图 1-图 3B),这表明与 p53 不同,MLL3 的肿瘤抑制作用是特定于癌基因和环境的。

MLL3 loss alters the chromatin landscape of liver cancer cells
MLL3 缺失会改变肝癌细胞的染色质景观

MLL3 and MLL4 are histone methyltransferases that can deposit the H3K4 monomethylation mark at genomic enhancers and intergenic regions during organ development (Hu et al., 2013). However, more studies indicate that MLL3 and MLL4 are also capable of binding to promoter regions (Chen et al., 2014; Dhar et al., 2016; Wang et al., 2010), especially in the context of cancer (Soto-Feliciano et al., 2023). To determine the genomic binding patterns of MLL3 in HCC, we performed MLL3 chromatin immunoprecipitation (ChIP)-sequencing (ChIP-Seq) analysis in Myc; sgKmt2c (sgKmt2c. 1 which generates heterozygous or homozygous indels) and Myc; sgTrp53 liver cancer cell lines. Compared to the sgTrp53 cells, sgKmt2c cells had a marked reduction in MLL3 chromatin binding at a subset of genomic loci (Figure 2A). Approximately 40% of the peaks that were selectively lost in Kmt2cdeficient cells occurred at promoter regions, whereas unchanged MLL3 peaks between the two genotypes were more likely to be within intergenic regions (Figure 2B, Figure 2-figure supplement 1). Therefore, our data suggest that, beyond the canonical action of MLL3 at gene enhancers, MLL3 can also occupy promoter regions in Myc-induced liver cancer. Of note, the residual ChIP-seq signal observed in the sgKmt2c cells most likely reflects the binding of MLL4 and/or remnant MLL3 since the antibody used in these experiments can recognize both MLL3 and MLL4 proteins (Dorighi et al., 2017). Nonetheless, the downregulated peak signals in Myc; sgKmt2c cells were specifically due to MLL3 disruption.
MLL3和MLL4是组蛋白甲基转移酶,可在器官发育过程中将H3K4单甲基化标记沉积在基因组增强子和基因间区域(Hu等人,2013年)。然而,更多的研究表明,MLL3 和 MLL4 也能与启动子区域结合(Chen 等人,2014 年;Dhar 等人,2016 年;Wang 等人,2010 年),尤其是在癌症的背景下(Soto-Feliciano 等人,2023 年)。为了确定MLL3在HCC中的基因组结合模式,我们在Myc; sgKmt2c(sgKmt2c. 1,会产生杂合或同源嵌合)和Myc; sgTrp53肝癌细胞系中进行了MLL3染色质免疫共沉淀(ChIP)-测序(ChIP-Seq)分析。与 sgTrp53 细胞相比,sgKmt2c 细胞在部分基因组位点的 MLL3 染色质结合明显减少(图 2A)。在 Kmt2c 缺失的细胞中,选择性丢失的峰值约有 40% 出现在启动子区域,而两种基因型之间保持不变的 MLL3 峰值更有可能出现在基因间区域(图 2B,图 2-图补充 1)。因此,我们的数据表明,除了 MLL3 在基因增强子上的典型作用外,MLL3 还可以占据 Myc 诱导的肝癌的启动子区域。值得注意的是,在 sgKmt2c 细胞中观察到的残余 ChIP-seq 信号很可能反映了 MLL4 和/或残余 MLL3 的结合,因为这些实验中使用的抗体可以识别 MLL3 和 MLL4 蛋白(Dorighi 等人,2017 年)。然而,Myc; sgKmt2c 细胞中下调的峰值信号是由 MLL3 干扰引起的。
Similar to the Drosophila Trithorax-related complex (Schuettengruber et al., 2017), the mammalian MLL3 and MLL4 complexes facilitate gene transcription by establishing permissive modifications
与果蝇的 Trithorax 相关复合物相似(Schuettengruber 等人,2017 年),哺乳动物的 MLL3 和 MLL4 复合物通过建立允许的修饰来促进基因转录。
A
MYC  MYC {{0}
  • Inframe mutation (putative driver) Missense mutation (putative driver) Missense mutation (unknown significance) Splice mutation (putative driver)
    错义突变(可能的驱动因素) 错义突变(意义不明) 剪接突变(可能的驱动因素
  • Truncating mutation (putative driver) Structural variant (putative driver) Amplification Gain Deep deletion No alterations
    截断突变(推定驱动因子) 结构变异(推定驱动因子) {{1}扩增增益 {{2}深度缺失 无改变
Mutations Relationship -value
KMT2C and MYC Co-occurrence
TP53 and MYC Co-occurrence 0.025
KMT2C and CTNNB1 Not significant 0.178
TP53 and CTNNB1 Mutual exclusivity
B
Myc; sgTrp53
Figure 1. MLL3 constrains Myc-driven liver tumorigenesis. (A) Oncoprints displaying genomic mutations and deletions of KMT2C and TP53, gains and amplifications of MYC, and activating CTNNB1 mutations in merged publicly available datasets (TCGA, MSK, INSERM, RIKEN, AMC, and MERCi) of 1280 sequenced hepatocellular carcinomas, and the table showing their relationships. p-Values were calculated by Fisher exact tests. (B) Schematic for hydrodynamic tail vein injection (HTVI) of gene delivery into murine livers. Vectors permitting stable expression of Myc transposon (top) and transient expression of Cas9 and single guide RNAs (sgRNAs) targeting putative tumor suppressors (bottom) via sleeping beauty transposase were introduced into hepatocytes by HTVI. (C) Survival curves of mice injected with Myc transposon and pX330 expressing two independent sgRNAs targeting Kmt2c after HTVI (Myc; sgKmt2c.1, ; Myc; sgKmt2c.2, ). Myc; sgTrp53 ( ), and Myc; sgChrom8 ( ) serve as controls. Survival curves were compared using log-rank tests. (D) Representative images (left, liver macro-dissection, scale bar: ; right, H&E staining, scale bar: ) of mouse liver tumors generated by HTVI delivery of Myc transposon and in vivo gene editing. The dashed lines indicate the boundaries between liver tumors and non-tumor liver tissues.
图 1.MLL3 限制了 Myc 驱动的肝脏肿瘤发生。(A) 显示 1280 例已测序肝细胞癌的合并公开数据集(TCGA、MSK、INSERM、RIKEN、AMC 和 MERCi)中 KMT2C 和 TP53 的基因组突变和缺失、MYC 的增益和扩增以及 CTNNB1 的激活突变的肿瘤图谱,以及显示它们之间关系的表格。(B) 将基因递送到小鼠肝脏的水动力尾静脉注射(HTVI)示意图。通过 HTVI 将允许稳定表达 Myc 转座子(上图)以及通过睡美人转座酶瞬时表达 Cas9 和靶向假定肿瘤抑制因子(下图)的单导 RNA(sgRNA)的载体导入肝细胞。(C) 注射 Myc 转座子和表达两个独立的靶向 Kmt2c 的 sgRNAs 的 pX330 的小鼠在 HTVI 后的生存曲线(Myc; sgKmt2c.1, ; Myc; sgKmt2c.2, )。Myc; sgTrp53 ( ) 和 Myc; sgChrom8 ( ) 作为对照。生存曲线采用对数秩检验进行比较。(D) 代表性图像(左图,肝脏大切片,比例尺: ;右图,H&E 染色,比例尺: 小鼠肝脏肿瘤的代表性图像(左图:肝脏大切片,比例尺 ;右图:H&E 染色,比例尺 )。虚线表示肝肿瘤和非肿瘤肝组织之间的界限。
The online version of this article includes the following source data and figure supplement(s) for figure 1 :
本文的在线版本包括以下源数据和图 1 的补充图:
Figure supplement 1. In vivo screen identifies MLL3 as a tumor suppressor in Myc-driven liver cancer.
图 1.体内筛选确定 MLL3 是 Myc 驱动的肝癌的肿瘤抑制因子。
Figure supplement 2. Suppression of Kmt2c by CRISPR or RNAi promotes Myc-driven liver cancer.
图 2.通过 CRISPR 或 RNAi 抑制 Kmt2c 会促进 Myc 驱动的肝癌。
Figure supplement 2-source data 1. Original gel for surveyor assays in Figure 1-figure supplement 2B.
图 2-源数据 1。图 1-图 2B 中测量器检测的原始凝胶。
Figure supplement 3. MLL3 loss does not cooperate with CTNNB1 oncogene to drive liver cancer.
图 3.MLL3 缺失不会与 CTNNB1 致癌基因合作驱动肝癌。
A
Peak distance (kb) Peak distance (kb)
峰值距离(千字节) 峰值距离(千字节)
B
Figure 2. MLL3 disruption alters the chromatin and transcriptional landscape of liver cancer cells. (A) Tornado plots showing MLL3 chromatin immunoprecipitation-sequencing (ChIP-Seq) signal (peaks) that were down or remained unchanged in Myc; sgKmt2c cells relative to Myc; sgTrp53 cells. Center: transcriptional start site (TSS). (B) Alluvial plot showing the percentages of MLL3 ChIP-Seq peaks in different genomic elements in Myc; sgKmt2c vs Myc; sgTrp53 cells, including 16,999 peaks down, 48,815 peaks unchanged, and 265 peaks up in sgKmt2c cells. Promoter regions were defined as Figure 2 continued on next page
图 2.MLL3 干扰改变了肝癌细胞的染色质和转录格局。(A)龙卷风图显示相对于 Myc; sgTrp53 细胞,MLL3 染色质免疫沉淀-测序(ChIP-Seq)信号(峰)下降或保持不变。中心:转录起始位点(TSS)。 (B) 冲积图显示了在 Myc; sgKmt2c 与 Myc; sgTrp53 细胞中不同基因组元件中 MLL3 ChIP-Seq 峰的百分比,包括在 sgKmt2c 细胞中下降的 16,999 个峰、不变的 48,815 个峰和上升的 265 个峰。启动子区域被定义为 图 2 下一页继续
Figure 2 continued 图 2 续
TSS kb. (C) Heatmaps of histone modification ChIP-Seq signals (H3K4me3, H3K4me1, H3K27ac, left panel) and MLL3 ChIP-Seq signal (right panel) at promoter or intergenic regions in three independent Myc; sgKmt2c and Myc; sgTrp53 liver tumor-derived cell lines. Cluster 1: loss of promoter and enhancer activity (loss of H3K4me3, H3K4me1, and H3K27ac); cluster 2: gain of enhancer activity (gain of H3K4me1 and H3K27ac); and cluster 3: gain of promoter activity (increase of H3K4me3). Representative top five loci for each cluster were listed on the right. (D) Volcano plot of differentially expressed genes revealed by RNA-sequencing of three independent Myc; sgKmt2c and Myc; sgTrp53 hepatocellular carcinoma (HCC) cell lines. Genes in sgKmt2c cells with more than twofold expression change and exceeding adjusted p-value are color-labeled (orange: upregulated; green: downregulated).
TSS kb。(C) 三个独立的 Myc; sgKmt2c 和 Myc; sgTrp53 肝肿瘤衍生细胞系启动子或基因间区域组蛋白修饰 ChIP-Seq 信号(H3K4me3、H3K4me1、H3K27ac,左图)和 MLL3 ChIP-Seq 信号(右图)的热图。第 1 组:启动子和增强子活性缺失(H3K4me3、H3K4me1 和 H3K27ac 缺失);第 2 组:增强子活性增强(H3K4me1 和 H3K27ac 增强);第 3 组:启动子活性增强(H3K4me3 增加)。右侧列出了每个群组中具有代表性的前五个位点。(D) 三个独立的 Myc; sgKmt2c 和 Myc; sgTrp53 肝细胞癌(HCC)细胞系通过 RNA 测序发现的差异表达基因的火山图。sgKmt2c细胞中表达量变化超过两倍且超过调整后p值 的基因用颜色标记(橙色:上调;绿色:下调)。
Some differentially expressed genes are labeled with gene symbols, and p53 targets are bolded.
一些差异表达基因用基因符号标出,p53 靶标用粗体标出。
The online version of this article includes the following figure supplement(s) for figure 2 :
本文的在线版本包括以下图 2 的补充图:
Figure supplement 1. MLL3 deficiency disrupts its binding at promoters in liver cancer cells.
图 1.缺乏 MLL3 会破坏其与肝癌细胞启动子的结合。
Figure supplement 2. MLL3 disruption impacts transcriptional and histone modification profiles in liver tumors.
图 2.MLL3 干扰会影响肝脏肿瘤的转录和组蛋白修饰谱。
on histone H3K4 via the MLL3 and MLL4 methyltransferase (Shilatifard, 2012). To determine whether MLL3 disruption impacts the local or global chromatin landscape of HCC cells, we performed ChIP-Seq analyses for H3K4 methylation and H3K27 acetylation in six independently derived tumor cell lines: three each for Myc; sgKmt2c and Myc; sgTrp53 (Figure 2C). Cluster analysis on genomic areas revealed three clusters of genomic loci that showed enrichment or depletion between Myc; sgKmt2c and Myc; sgTrp53 tumor cells for each tested histone modification (Figure 2C, Figure 2figure supplement 2A-C). Loci in cluster 1 (reduced H3K4me3, H3K4me1, and H3K27ac in sgKmt2c cells) showed the most pronounced differences in chromatin modifications between the two liver tumor genotypes. In contrast, the loci in cluster 2 showed increased H3K4me1 and H3K27ac marks, most of which mapped to intergenic regions. The loci in cluster 3 showed increased and included some p53 target genes such as Cdkn1a and Eda2r.
通过 MLL3 和 MLL4 甲基转移酶作用于组蛋白 H3K4(Shilatifard,2012 年)。为了确定 MLL3 干扰是否会影响 HCC 细胞的局部或全局染色质景观,我们在六个独立衍生的肿瘤细胞系(Myc; sgKmt2c 和 Myc; sgTrp53 各三个)中对 H3K4 甲基化和 H3K27 乙酰化进行了 ChIP-Seq 分析(图 2C)。对基因组区域的聚类分析显示,在Myc; sgKmt2c和Myc; sgTrp53肿瘤细胞中,有三个基因组位点群对每种测试的组蛋白修饰显示出富集或缺失(图2C,图2图补充2A-C)。第 1 组基因座(sgKmt2c 细胞中的 H3K4me3、H3K4me1 和 H3K27ac 减少)显示出两种肝脏肿瘤基因型之间染色质修饰的最明显差异。相比之下,群集2中的基因位点显示出更多的H3K4me1和H3K27ac标记,其中大部分映射到基因间区域。第3群组中的位点显示 增加,其中包括一些p53靶基因,如Cdkn1a和Eda2r。
To determine whether these drastic changes in the chromatin landscape were associated with changes in MLL3 binding, we integrated the chromatin modifications results with our MLL3 ChIP-Seq results (Figure 2C). Interestingly, the loci in cluster 1, which displayed the most substantial changes in histone modifications, involved genes that showed enriched MLL3 binding in Myc; sgTrp53 cells compared to the Myc; sgKmt2c genotype. These data support a model whereby MLL3 binding to these loci facilitates the acquisition of a chromatin environment conducive for active gene transcription.
为了确定染色质景观的这些剧烈变化是否与 MLL3 结合的变化有关,我们将染色质修饰结果与 MLL3 ChIP-Seq 结果进行了整合(图 2C)。有趣的是,与 Myc; sgKmt2c 基因型相比,群组 1 中组蛋白修饰变化最大的基因位点涉及的基因在 Myc; sgTrp53 细胞中显示出丰富的 MLL3 结合。这些数据支持一种模型,即 MLL3 与这些基因位点的结合有助于获得有利于活跃基因转录的染色质环境。

MLL3 regulates specific tumor suppression programs in liver cancer cells
MLL3 在肝癌细胞中调节特定的肿瘤抑制程序

Transcriptional profiling helped hone in on potentially critical targets of MLL3. Specifically, we determined the output of these chromatin landscape changes by transcriptional profiling of the same set of Myc; sgTrp53 and Myc; sgKmt2c liver cancer cell lines described above. Despite the broad binding of MLL3 across the genome, we found only 248 differentially expressed genes (DEGs): 132 significantly upregulated fold-change ) and 116 significantly downregulated foldchange ) in Myc; sgKmt2c liver tumor cells compared to Myc; sgTrp53 controls.
转录谱分析有助于确定 MLL3 的潜在关键靶标。具体来说,我们通过对上述同一组 Myc; sgTrp53 和 Myc; sgKmt2c 肝癌细胞系进行转录谱分析,确定了这些染色质景观变化的结果。尽管 MLL3 在整个基因组中的结合范围很广,但我们只发现了 248 个差异表达基因(DEGs):132 个显著上调 折叠变化 )和 116 个显著下调 折叠变化 折叠变化 )和 116 个明显下调 折叠变化 )。与 Myc; sgTrp53 对照组相比,Myc; sgKmt2c 肝肿瘤细胞中有 132 个表达量明显增加 ,116 个表达量明显减少
As predicted, transcripts encoding p53 and p53 target genes such as Ccng1, Cdkn1a, and Zmat3 (Bieging-Rolett et al., 2020) were upregulated in Myc; sgKmt2c cells, consistent with nonsensemediated decay of truncated p53 transcripts and a concomitant reduction in p53 effector genes. Strikingly, some of the downregulated genes in Myc; sgKmt2c lines mapped to loci enriched in cluster 1, including Cdkn2a, Bmp6, and Lrp2 (Figure 2C-D, Figure 2-figure supplement 2D, E). Of note, sgKmt2c did not lead to compensatory changes in the transcript levels of other major components of the COMPASS-like complexes, including MII4 (Kmt2d), Utx (Kdm6a), Mll1 (Kmt2a), and MII2 (Kmt2b; Figure 3-figure supplement 1A), suggesting that the alterations in MLL3 binding, histone modification, and transcription were specifically attributed to MLL3 disruption.
正如预测的那样,编码 p53 和 p53 靶基因如 Ccng1、Cdkn1a 和 Zmat3(Bieging-Rolett 等人,2020 年)的转录本在 Myc; sgKmt2c 细胞中上调,这与截短的 p53 转录本的非介导衰变以及 p53 效应基因的相应减少一致。引人注目的是,Myc; sgKmt2c 株系中一些下调的基因映射到了第 1 组中富集的位点,包括 Cdkn2a、Bmp6 和 Lrp2(图 2C-D,图 2-图补充 2D,E)。值得注意的是,sgKmt2c 并未导致 COMPASS-like 复合物其他主要成分(包括 MII4 (Kmt2d)、Utx (Kdm6a)、Mll1 (Kmt2a) 和 MII2 (Kmt2b;图 3-图 1A))的转录水平发生补偿性变化,这表明 MLL3 结合、组蛋白修饰和转录的改变是由 MLL3 破坏引起的。
We reason that the mediators of MLL3 actions in tumor suppression should be within cluster 1 with reduced transcription and MLL3 binding in Kmt2c-deficient cells. To further characterize the gene repertoire directly regulated by MLL3 genomic binding, we integrated the results of MLL3 ChIP-seq and RNA-seq. Specifically, we selected downregulated DEGs that show concordant decreased binding of MLL3 in Myc; sgKmt2c lines and subjected them to gene ontology analysis. Apart from the cluster 1 genes noted above, the integrative analysis revealed multiple MLL3-regulated tumor suppressive programs (Figure 3A, Figure 3-figure supplement 1B), including both cell-autonomous
我们推断,在 Kmt2c 缺失的细胞中,转录和 MLL3 结合减少,因此 MLL3 在抑制肿瘤方面的作用介质应属于第 1 组。为了进一步确定受 MLL3 基因组结合直接调控的基因库的特征,我们整合了 MLL3 ChIP-seq 和 RNA-seq 的结果。具体来说,我们选择了在 Myc; sgKmt2c 株系中与 MLL3 结合减少的下调 DEGs,并对它们进行了基因本体分析。除了上述第 1 组基因外,综合分析还发现了多个由 MLL3 调控的肿瘤抑制程序(图 3A,图 3-图补充 1B),包括细胞自主的
A
Figure 3. MLL3 regulates specific transcription programs including tumor suppressor CDKN2A. (A) Network plot showing the major biological processes and related genes directly regulated by MLL3 binding. p-Values and cluster sizes were calculated by the integrative analyses of RNA-seq and MLL3 chromatin immunoprecipitation-sequencing (ChIP-Seq), as detailed in the Materials and methods. (B) Gene set enrichment analysis (GSEA) plots of transcriptional signatures derived from mouse hepatocellular carcinomas (HCCs; Myc; sgKmt2c vs Myc; sgTrp53) against transcriptomics of HCCs Figure 3 continued on next page
图 3.MLL3 调控包括肿瘤抑制因子 CDKN2A 在内的特定转录程序。(p 值和聚类大小是通过 RNA-seq 和 MLL3 染色质免疫沉淀测序(ChIP-Seq)的综合分析计算得出的,详见 "材料与方法"。(B)小鼠肝细胞癌(HCCs;Myc;sgKmt2c vs Myc;sgTrp53)转录特征与 HCCs 转录组学的基因组富集分析(GSEA)图。
Figure 3 continued 图 3 续
with CDKN2A vs TP53 mutations. (C) GSEA plots of transcriptional signatures derived from KMT2C mutated/deleted human HCCs against the ones with CDKN2A mutations or homozygous deletions. HCCs with TP53 mutations were used as the controls for both comparisons. Normalized enrichment scores (NES) and false discovery rate (FDR) q-values were calculated by GSEA.
CDKN2A突变与TP53突变的对比图。(C) KMT2C突变/缺失的人类HCC与CDKN2A突变或同源缺失的HCC转录特征的GSEA图。两组比较均以 TP53 突变的 HCC 为对照。通过 GSEA 计算归一化富集分(NES)和误诊率(FDR)q 值。
The online version of this article includes the following figure supplement(s) for figure 3 :
本文的在线版本包括以下图 3 的补充图:
Figure supplement 1. MLL3 loss impacts specific transcription programs.
图 1.MLL3 缺失会影响特定的转录程序。
Figure supplement 2. CDKN2A and KMT2C mutations cause similar transcriptional changes in human hepatocellular carcinoma (HCC).
图 2.CDKN2A 和 KMT2C 突变会导致人类肝细胞癌(HCC)发生类似的转录变化。
mechanisms (cellular metabolism) and non-autonomous mechanisms (interaction with extracellular matrix and immune system).
机制(细胞代谢)和非自主机制(与细胞外基质和免疫系统的相互作用)。

KMT2C and CDKN2A mutations result in similar transcriptomes in human HCC
KMT2C 和 CDKN2A 突变导致人类 HCC 中出现相似的转录组

One genomic locus that stood out in our integrative analysis was Cdkn2a, which encompasses both the p16/Ink4a and p19/Arf (p14 in human) tumor suppressors (Gil and Peters, 2006). CDKN2A is located on the human chromosome and is deleted or epigenetically silenced in many cancer types (Sherr, 2012), including HCC (2017). While MLL3 likely regulates a plethora of genes that contribute to its tumor-suppressive potential, the well-defined and potent antitumor functions of Cdkn2a-encoded proteins make them attractive candidates as functionally relevant MLL3 effectors.
在我们的综合分析中,一个突出的基因组位点是Cdkn2a,它包含p16/Ink4a和p19/Arf(人类为p14)肿瘤抑制因子(Gil和Peters,2006年)。CDKN2A 位于人类染色体 上,在许多癌症类型中被删除或表观遗传沉默(Sherr,2012),包括 HCC(2017)。虽然 MLL3 可能调控了大量有助于发挥其肿瘤抑制潜能的基因,但 Cdkn2a 编码的蛋白具有明确且有效的抗肿瘤功能,这使它们成为具有吸引力的候选功能相关 MLL3 效应子。
Furthermore, in our analysis publicly available genomic data on samples, we found that CDKN2A alterations, like KMT2C alterations, showed significant co-occurrence with MYC gains and amplifications. However, we were unable to conduct a meaningful test of mutual exclusivity between CDKN2A and KMT2C alterations (Figure 3-figure supplement 2A), given the constraints of sample size and the modest frequencies of alteration in each gene. Further dissection of transcriptional profiling datasets from human and mouse HCCs harboring known gene alterations using gene set enrichment analysis (GSEA) revealed that human tumors with CDKN2A deletions transcriptionally resembled both mouse and human HCC harboring KMT2C alterations (Figure and ) but not those harboring RB1 loss (Figure 3-figure supplement 2B), even though the tumor suppressor RB1 is regulated by CDKN2A/p16 and their genomic alterations exhibit mutual exclusivity in multiple cancer types (Knudsen et alo, 2020). While we cannot rule out the possibility that other factors drive these associations, our results support a biologically meaningful relationship between MLL3 and CDKN2A.
此外,在分析 样本的公开基因组数据时,我们发现CDKN2A的改变与KMT2C的改变一样,与MYC的增益和扩增有显著的共存性。然而,由于样本量的限制以及每个基因的改变频率都不高,我们无法对CDKN2A和KMT2C改变之间的互斥性进行有意义的检验(图3-图补充2A)。利用基因组富集分析(GSEA)对携带已知基因改变的人类和小鼠HCC的转录剖析数据集进行进一步分析后发现,CDKN2A缺失的人类肿瘤在转录上与携带KMT2C改变的小鼠和人类HCC相似(图 ),但与携带RB1缺失的HCC不相似(图3-图补充2B)、尽管肿瘤抑制因子RB1受CDKN2A/p16 调控,而且它们的基因组改变在多种癌症类型中表现出互斥性(Knudsen et alo,2020)。虽然我们不能排除其他因素驱动这些关联的可能性,但我们的结果支持 MLL3 和 CDKN2A 之间存在生物学意义上的关系。

Cdkn2a locus is a genomic and transcriptional target of MLL3 in liver cancer
Cdkn2a 基因座是肝癌中 MLL3 的基因组和转录靶标

To explore the relationship between MLL3 and Cdkn2a locus in more detail, we tested whether genes encoded by Cdkn2a were direct targets of MLL3-regulated transcription. Indeed, Cdkn2a is a cluster 1 locus that, in Myc; sgKmt2c cancer cells, displays significant reduction in (1) expression, (2) H3K4me1/3 and H3K27ac levels, and (3) MLL3 binding at the Cdkn2a promoter compared with Myc; sgTrp53 cells (Figure 2C-D, Figure 4A). Of note, MLL3 binding peaks were also observed within the gene body of Cdkn2a. The differential expression of Ink4a and Arf was confirmed by qPCR, immunoblotting, and ChIP-qPCR analyses on multiple Myc; sgKmt2c and Myc; sgTrp53 liver cancer lines (Figure 4B, Figure 4-figure supplement 1A, B). These results imply that Cdkn2a locus is a genomic and transcriptional target of MLL3 in liver cancer cells.
为了更详细地探讨 MLL3 与 Cdkn2a 基因座之间的关系,我们测试了 Cdkn2a 编码的基因是否是 MLL3 调控转录的直接靶标。事实上,与 Myc; sgTrp53 细胞相比,Cdkn2a 是第 1 组基因位点,在 Myc; sgKmt2c 癌细胞中,(1) 表达、(2) H3K4me1/3 和 H3K27ac 水平以及 (3) MLL3 与 Cdkn2a 启动子的结合均显著降低(图 2C-D,图 4A)。值得注意的是,在 Cdkn2a 基因体内也观察到了 MLL3 结合峰。在多个 Myc; sgKmt2c 和 Myc; sgTrp53 肝癌株上进行的 qPCR、免疫印迹和 ChIP-qPCR 分析证实了 Ink4a 和 Arf 的差异表达(图 4B,图 4-图 1A,B)。这些结果表明,Cdkn2a 基因座是肝癌细胞中 MLL3 的基因组和转录靶标。
Since the Myc; sgTrp53 and Myc; sgKmt2c cells we studied above are not isogenic, we performed a series of additional experiments to demonstrate a direct transcriptional effect of MLL3 on the Cdkn2a locus. Because p53 inactivation can lead to compensatory increases in Ink4a and Arf expression (Stott et al., 1998), representing an alternative possibility accounting for the observed difference of Cdkn2a expression in sgTrp53 vs sgKmt2c cells. However, p53 suppression in sgKmt2c cells produced only a subtle and inconsistent effect on the expression of Ink4a and Arf, whereas Kmt2c suppression in sgTrp53 cells consistently attenuated p16 and p19 Arf protein levels (Figure 4-figure supplement 1C, D). As another means of ruling out the p53 pathway as an explanation for altered Cdkn2a expression in Myc; sgKmt2c cells, we tested the ability of MLL3 to regulate Cdkn2a transcripts in an orthogonal liver cancer model driven by Myc and inactivation of Axin1, which is a well-defined
由于我们上述研究的 Myc; sgTrp53 和 Myc; sgKmt2c 细胞不是同源的,因此我们进行了一系列额外的实验,以证明 MLL3 对 Cdkn2a 基因座的直接转录效应。由于 p53 失活可导致 Ink4a 和 Arf 表达的代偿性增加(Stott 等人,1998 年),这代表了另一种可能,即在 sgTrp53 与 sgKmt2c 细胞中观察到的 Cdkn2a 表达差异。然而,在 sgKmt2c 细胞中抑制 p53 只对 Ink4a 和 Arf 的表达产生了微妙且不一致的影响,而在 sgTrp53 细胞中抑制 Kmt2c 则持续降低 p16 和 p19 Arf 蛋白水平(图 4-图 1C、D)。作为排除 p53 通路解释 Myc; sgKmt2c 细胞中 Cdkn2a 表达改变的另一种方法,我们在一个由 Myc 和 Axin1 失活驱动的正交肝癌模型中测试了 MLL3 调节 Cdkn2a 转录物的能力。
Figure 4. CDKN2A locus is a genomic and transcriptional target of MLL3 in liver cancer. (A) Genome browser tracks for MLL3 and H3K4me1 chromatin immunoprecipitation-sequencing (ChIP-Seq) in Myc; sgTrp53 (red) and Myc; sgKmt2c (blue) hepatocellular carcinoma (HCC) cell lines at the Cdkn2a locus. (B) qPCR analysis for mRNA expression of Ink4a and Arf from three independent Myc; sgKmt2c and Myc; sgTrp53 HCC lines ( cell lines each genotype). Values are shown as mean SD. (unpaired two-tailed t-test). (C) Schematic for CRISPR activation (CRISPRa) system of nuclease-
图 4.CDKN2A 基因座是肝癌中 MLL3 的基因组和转录靶标。(A) 基因组浏览器追踪 Myc; sgTrp53(红色)和 Myc; sgKmt2c(蓝色)肝细胞癌(HCC)细胞系中 Cdkn2a 基因座上的 MLL3 和 H3K4me1 染色质免疫沉淀测序(ChIP-Seq)。(B) qPCR 分析三个独立的 Myc; sgKmt2c 和 Myc; sgTrp53 HCC 细胞系(每个基因型 个细胞系)中 Ink4a 和 Arf 的 mRNA 表达。数值以平均值 表示。SD. (非配对双尾 t 检验)。(C) CRISPR激活(CRISPRa)系统的核酸酶激活示意图。
Figure 4 continued on next page
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Figure 4 continued 图 4 续
dead Cas9 (dCas9) and VP64-p65-Rta (VPR) guided by sgKMT2C to activate KMT2C expression in human HLE HCC cell line. (D) qPCR analysis for mRNA expression of KMT2C in HLE cells with sgGFP (control) or two different CRISPRa single guide RNAs (sgRNAs) targeting KMT2C ( cell lines each genotype). Each data point represents the average of technical duplicates. Data are shown as mean SEM. (one-way ANOVA followed by post-hoc t-tests). (E) Genome browser tracks for MLL3 ChIP-Seq at the CDKN2A locus in HLE cells with sgGFP (control, black) or sgKmt2c. 1 (blue). (F and G) qPCR analysis for mRNA expression of (F) INK4A and (G) ARF in HLE cells with sgGFP (control) or sgKMT2C ( ). Each data point represents the average of technical duplicates. Data are shown as mean SEM. (one-way ANOVA followed by post-hoc t-tests).
死 Cas9 (dCas9) 和 VP64-p65-Rta (VPR) 在 sgKMT2C 引导下激活人 HLE HCC 细胞系中 KMT2C 的表达。(D) qPCR分析sgGFP(对照)或两种不同的靶向KMT2C的CRISPRa单导RNA(sgRNA)(每种基因型 细胞系)在HLE细胞中KMT2C的mRNA表达。每个数据点代表技术重复的平均值。数据以平均值 表示。SEM. (单因素方差分析,然后进行事后 t 检验)。(E) 基因组浏览器对带有 sgGFP(对照,黑色)或 sgKmt2c.1(蓝色)。(F和G) sgGFP(对照)或sgKMT2C( )的HLE细胞中(F) INK4A和(G) ARF的mRNA表达的qPCR分析。每个数据点代表技术重复的平均值。数据显示为平均值 SEM. (单因素方差分析,然后进行事后 t 检验)。
The online version of this article includes the following source data and figure supplement(s) for figure 4:
本文的在线版本包括以下源数据和图 4 的补充图:
Figure supplement 1. MLL3 directly regulates Cdkn2a expression in liver cancer cells.
图 1.MLL3 直接调控肝癌细胞中 Cdkn2a 的表达。
Figure supplement 1-source data 1. Original western blots for Figure 4-figure supplement 1A,C,D,E.
图补 1-原始数据 1。图 4 图 1A,C,D,E 的原始 Western 印迹。
tumor suppressor that negatively regulates -catenin activity in HCC (Satoh et al., 2000). Liver cancer cells produced by hydrodynamic delivery of the Myc transposon vector and Axin1 sgRNAs displayed reduced Ink4a and Arf expression upon Kmt2c knockdown without targeting p53 (Figure 4-figure supplement 1E,F). Importantly, MLL3 binding peaks at the Cdkn2a locus were also detected in Myc; sgAxin1 liver cancer cells (Figure 4-figure supplement 1G), suggesting that Cdkn2a transcription is directly regulated by MLL3 rather than an indirect outcome of p53 loss. These data imply that MLL3 supports a chromatin environment at the Cdkn2a locus that facilitates the transcription of both Ink4a and Arf and raises the possibility that these factors contribute to the tumor suppressor activity of MLL3 in liver cancer.
肿瘤抑制因子,负向调节 HCC 中 -catenin 的活性(Satoh 等人,2000 年)。-catenin 的活性(Satoh 等人,2000 年)。通过水动力递送 Myc 转座子载体和 Axin1 sgRNAs 产生的肝癌细胞在 Kmt2c 敲除后显示出 Ink4a 和 Arf 表达减少,而没有靶向 p53(图 4-图 1E、F)。重要的是,在 Myc; sgAxin1 肝癌细胞中也检测到了 Cdkn2a 基因座上的 MLL3 结合峰(图 4-图 1G),这表明 Cdkn2a 的转录是由 MLL3 直接调控的,而不是 p53 缺失的间接结果。这些数据表明,MLL3 在 Cdkn2a 基因座上支持一种染色质环境,这种环境有利于 Ink4a 和 Arf 的转录,这就提出了一种可能性,即这些因子有助于 MLL3 在肝癌中的肿瘤抑制活性。
We next set out to determine whether MLL3 binding is sufficient to induce transcriptional activation of the CDKN2A locus and, in doing so, extend our analysis to human liver cancer cells. As the KMT2C transcript is too large (14,733 bp) for cDNA transduction, we turned to the CRISPR activation (CRISPRa) system (Chavez et al., 2015) in a human hepatocellular carcinoma cell line (HLE). Following stable integration of the nuclease dead Cas9 fused to the VP64-p65-Rta (VPR) transcriptional activator, cells were transduced with two orthogonal sgRNAs targeting the human KMT2C promoter (or, as control, transduced with sgRNA against GFP; Figure 4C). Cells expressing the KMT2C sgRNAs showed a marked and specific increase in the expression of endogenous KMT2C, but not of KMT2D or TP53 (Figure 4D, Figure 5-figure supplement 1A, B), which was accompanied by an increase in MLL3 binding to the CDKN2A locus (Figure 4E) and transcriptional upregulation of both CDKN2A transcripts (Figure 4F and G). Therefore, MLL3 directly binds and co-activates transcription of the CDKN2A locus in human liver cancer cells.
接下来,我们开始确定 MLL3 结合是否足以诱导 CDKN2A 基因座的转录激活,并将我们的分析扩展到人类肝癌细胞。由于 KMT2C 转录本太大(14,733 bp),无法进行 cDNA 转导,我们转而在人肝癌细胞系(HLE)中使用 CRISPR 激活(CRISPRa)系统(Chavez 等人,2015 年)。将核酸酶死体 Cas9 与 VP64-p65-Rta (VPR)转录激活因子稳定整合后,用两个靶向人 KMT2C 启动子的正交 sgRNA 转导细胞(或作为对照,用针对 GFP 的 sgRNA 转导细胞;图 4C)。表达 KMT2C sgRNAs 的细胞显示内源性 KMT2C 的表达明显和特异性增加,但 KMT2D 或 TP53 的表达没有增加(图 4D,图 5-图补充 1A、B),与此同时,MLL3 与 CDKN2A 基因座的结合增加(图 4E),两个 CDKN2A 转录本的转录上调(图 4F 和 G)。因此,在人类肝癌细胞中,MLL3 直接结合并共同激活 CDKN2A 基因座的转录。

MLL3 mediates oncogene-induced apoptosis in a Cdkn2a-dependent manner
MLL3 以依赖 Cdkn2a 的方式介导癌基因诱导的细胞凋亡

The above results raise the possibility that the Cdkn2a products, INK4A and ARF, may contribute to the tumor suppressive activity of MLL3. In this regard, Myc overexpression in primary cells (mouse embryonic fibroblasts; MEFs) often triggers apoptosis (Evan et al., 1992), and this in turn limits tumorigenesis in a manner that is dependent on Cdkn2a (Zindy et al., 1998). This pathway also suppresses liver tumorigenesis since concomitant disruption of Ink4a and Arf using CRISPR, or germline deletion of Arf alone, cooperated with Myc overexpression to rapidly promote tumor development (Figure 5-figure supplement ). Similarly, Kmt2c suppression also attenuated MYC-induced apoptosis, as shown by tumor histology and apoptosis by TUNEL assay (Negoescu et al., 1997), 5 days after hydrodynamic delivery of transposon vectors encoding Myc together with GFP-linked shRNAs targeting Kmt2c (or Renilla luciferase as a control; Figure 5A and B). This difference in apoptosis correlated with an increase in retention of GFP-shKmt2c expressing cells 10 days after injection (Figure 5-figure supplement 1D, E). Altogether, these results show that suppression impairs Myc-induced apoptosis in vivo in a manner that is reminiscent of the anti-apoptotic effects of Cdkn2a loss in the context of aberrant Myc activation (Eischen et al., 1999; Jacobs et al., 1999; Schmitt et al., 1999).
上述结果提出了一种可能性,即 Cdkn2a 产物 INK4A 和 ARF 可能有助于 MLL3 的肿瘤抑制活性。在这方面,Myc 在原代细胞(小鼠胚胎成纤维细胞;MEFs)中的过度表达通常会引发细胞凋亡(Evan 等人,1992 年),这反过来又以依赖 Cdkn2a 的方式限制了肿瘤的发生(Zindy 等人,1998 年)。这一途径也能抑制肝脏肿瘤的发生,因为使用 CRISPR 同时破坏 Ink4a 和 Arf,或单独种系缺失 Arf,都会与 Myc 的过表达合作,迅速促进肿瘤的发展(图 5-图补充 )。同样,在通过流体动力递送编码 Myc 的转座子载体和靶向 Kmt2c 的 GFP 连接 shRNA(或雷尼拉荧光素酶作为对照;图 5A 和 B)5 天后,肿瘤组织学和 TUNEL 检测(Negoescu 等人,1997 年)显示,Kmt2c 抑制也会减轻 MYC 诱导的细胞凋亡。这种细胞凋亡的差异与注射 10 天后 GFP-shKmt2c 表达细胞的保留率增加有关(图 5-图 1D、E)。总之,这些结果表明, 抑制会损害体内Myc诱导的细胞凋亡,其方式与异常Myc活化背景下Cdkn2a缺失的抗凋亡效应相似(Eischen等人,1999;Jacobs等人,1999;Schmitt等人,1999)。
To model the interaction between Myc overexpression, MLL3 function, and Cdkn2a regulation, we transduced liver progenitor cells (LPCs) with retroviral vectors encoding Myc linked to a reverse tetracycline transactivator (rtTA3), together with doxycycline (dox)-inducible Kmt2c shRNAs to enable reversible Kmt2c silencing (Figure 5-figure supplement 2A). Infection of LPCs with Myc in the
为了模拟Myc过表达、MLL3功能和Cdkn2a调控之间的相互作用,我们用编码与反向四环素转座子(rtTA3)相连的Myc的逆转录病毒载体以及强力霉素(dox)诱导的Kmt2c shRNA转导肝祖细胞(LPCs),以实现可逆的Kmt2c沉默(图5-图补充2A)。用Myc感染LPCs在
Figure 5. MLL3 mediates oncogene-induced apoptosis in a Cdkn2a-dependent manner. (A) Representative images of TUNEL-positive nuclei (red staining) in murine livers 5 days after hydrodynamic injection of the indicated vector combinations. DAPI(4',6-diamidino-2-phenylindole) was used to visualize nuclei. (B) Quantification of TUNEL-positive nuclei in mouse livers 5 days after hydrodynamic tail vein injection (HTVI) of the indicated vector combinations. Data points represent the number of TUNEL-positive cells in five different high-power fields in three independent murine livers per group. (one-way ANOVA followed by post hoc t-tests). (C) Chromatin immunoprecipitation (ChIP)-qPCR analysis for H3K4me3 signals at Arf Figure 5 continued on next page
图 5.MLL3 以 Cdkn2a 依赖性方式介导癌基因诱导的细胞凋亡。(A) 水动力注射所述载体组合 5 天后,小鼠肝脏中 TUNEL 阳性细胞核(红色染色)的代表性图像。DAPI(4',6-二脒基-2-苯基吲哚)用于观察细胞核。(B) 水动力尾静脉注射(HTVI)指定载体组合 5 天后小鼠肝脏中 TUNEL 阳性细胞核的定量。数据点代表每组三只独立小鼠肝脏中五个不同高倍视野中 TUNEL 阳性细胞的数量。 (单因素方差分析,然后进行事后 t 检验)。(C) 染色质免疫沉淀(ChIP)-qPCR 分析 Arf 处的 H3K4me3 信号。
Figure 5 continued 图 5 续
and Ink4a promoters 4 days after doxycycline (dox) withdrawal in Myc-rtTA3; TRE-shKmt2c cells. Values are mean SD from technical replicates ( ), and the experiments were conducted in two independent liver progenitor cell (LPC) lines with different shKmt2c. (D) qPCR analysis for mRNA expression of Arf and Ink4a 4 days after dox withdrawal in two independent LPC lines with different shKmt2c. Values are mean SD from technical replicates ( ).
和 Ink4a 启动子。数值为技术重复的平均值 。SD,来自技术重复( ),实验在两个独立的肝祖细胞(LPC)系中进行,shKmt2c 不同。(D) qPCR 分析不同 shKmt2c 的两个独立 LPC 株系在停药 4 天后 Arf 和 Ink4a 的 mRNA 表达。数值为技术重复( )的平均值 。SD from technical replicates ( )。
and (unpaired two-tailed t-test). (E) Representative images of colony formation assay of the indicated cell lines 5 days after dox withdrawal. (F) Quantification of colony formation assay. Values are mean SD of three independent experiments with two independent LPC lines. (unpaired two-tailed t-test). (G) Time course analysis of Draq7-positive (dead or permeabilized) cells as a fraction of Venus-positive, Myc-rtTA3; TRE-shKmt2c cells expressing constitutive shRNAs targeting both Ink4a and Arf (shCdkn2a) or Renilla luciferase (shRen) off and on dox. Values represent mean SEM of triplicate wells of each genotype at each timepoint of two independently derived LPC lines, infected with either shRen or shCdkn2a.
。(非配对双尾 t 检验)。(E)停药 5 天后,上述细胞系的菌落形成检测的代表性图像。(F)集落形成测定的定量。数值为平均值 。SD 的平均值。 (非配对双尾 t 检验)。(G)Draq7 阳性(死亡或通透)细胞占 Venus 阳性、Myc-rtTA3;TRE-shKmt2c 细胞比例的时程分析,这些细胞同时表达针对 Ink4a 和 Arf 的组成型 shRNAs(shCdkn2a)或 Renilla 荧光素酶(shRen)。数值代表平均值 。在感染 shRen 或 shCdkn2a 的两个独立衍生 LPC 株系的每个时间点上,每个基因型的三联孔的平均值
(unpaired two-tailed t-test of final average percentage Draq ). NS, not significant ( ).
(最终平均 Draq 百分比的非配对双尾 t 检验 )。NS,无显著性 ( )。
The online version of this article includes the following source data and figure supplement(s) for figure 5 :
本文的在线版本包括以下源数据和图 5 的补充图:
Figure supplement 1. Kmt2c suppression reduces cell clearance upon enforced Myc expression in vivo.
图 1.抑制 Kmt2c 可减少体内强制表达 Myc 时的细胞清除率。
Figure supplement 2. Endogenous restoration triggers apoptosis and is accompanied by increased expression.
图 2.内源性 的恢复会引发细胞凋亡,并伴随着 表达的增加。
Figure supplement 2—source data 1. Original western blots for Figure 5-figure supplement 2B, F,G.
图 5-图 2-原始数据 1。图 5-图 2B、F、G 的原始 Western 印迹。
presence of MLL3 (i.e. cells infected with Myc-rtTA3 and a dox-inducible shRNA targeting Renilla luciferase) acutely activated INK4A and ARF expression (Figure 5-figure supplement 2B), and these cells could not be maintained in culture. Phenocopying the ability of Myc and Kmt2c suppression to transform liver cells in vivo, combined and sh expression facilitated the persistent growth of cells maintained on Dox (Figure 5-figure supplement 2C,D). By contrast, dox withdrawal induced Kmt2c mRNA expression and H3K4me3 deposition at the Cdkn2a promoters, ultimately leading to elevations in Arf and Ink4a mRNA and protein (Figure 5C and D), reduced colony formation, and increased apoptosis (Figure 5-figure supplement 2D-F). Furthermore, constitutive shRNAmediated knockdown of Arf and Ink4a through targeting of the shared exon 2 (shCdkn2a) significantly rescued colony-forming capacity and prevented cell death following restoration as determined by time-lapse microscopy of cells cultured with a fluorescent dye that stains dead cells (Figure 5E-G, Figure 5-figure supplement 2G). These data support a model whereby a prominent tumor suppressive output of MLL3 in liver cancer involves direct upregulation of Cdkn2a that, when impaired, attenuates the MYC-induced apoptotic program and permits tumor progression.
MLL3的存在(即感染了Myc-rtTA3和针对Renilla荧光素酶的dox诱导性shRNA的细胞)会急性激活INK4A和ARF的表达(图5-图2B),这些细胞无法在培养液中维持生长。与抑制Myc和Kmt2c转化体内肝细胞的能力相似,联合 和sh 的表达促进了Dox维持的细胞的持续生长(图5-图补充2C,D)。相比之下,停用 Dox 会诱导 Kmt2c mRNA 表达和 H3K4me3 在 Cdkn2a 启动子处沉积,最终导致 Arf 和 Ink4a mRNA 和蛋白升高(图 5C 和 D)、集落形成减少和细胞凋亡增加(图 5-图 2D-F)。此外,通过靶向共用的第 2 号外显子(shCdkn2a),组成型 shRNA 介导的 Arf 和 Ink4a 基因敲除显著地挽救了集落形成能力,并防止了 恢复后的细胞死亡(图 5E-G,图 5-补充 2G)。这些数据支持一种模型,即 MLL3 在肝癌中的显著肿瘤抑制作用涉及 Cdkn2a 的直接上调,当 Cdkn2a 受损时,会减弱 MYC 诱导的细胞凋亡程序并允许肿瘤进展。

Wild-type MLL3 野生型 MLL3

Apoptosis and tumor suppression
细胞凋亡和肿瘤抑制

Mutant (loss) MLL3 突变(缺失)MLL3

Tumorigenesis 肿瘤发生
Figure 6. Model of MLL3 as a tumor suppressor in liver cancer. MLL3 restricts MYC-induced liver tumorigenesis by directly activating the Cdkn2a locus to mediate tumor cell apoptosis.
图 6.MLL3 作为肝癌肿瘤抑制因子的模型。MLL3 通过直接激活 Cdkn2a 基因座来介导肿瘤细胞凋亡,从而限制 MYC 诱导的肝脏肿瘤发生。

Discussion 讨论

Our study combined genetic, epigenomic, and animal modeling approaches to identify Cdkn2a as an important regulatory target of MLL3 in both mouse and human liver cancers. Our results support a model whereby oncogenic stress, herein produced by MYC, leads to an increase in the binding of MLL3 to the CDKN2A locus, an event that is associated with the accumulation of histone marks linked to the biochemical activity of MLL3-containing complexes and conducive to gene activation (Figure 6). Accordingly, these events are accompanied by transcriptional upregulation of two key Cdkn2a gene products, Ink4a and Arf. Moreover, suppression of Kmt2c phenocopies the effects of Cdkn2a inactivation in abrogating MYC-induced apoptosis. Conversely, suppression of Cdkn2a diminishes the anti-proliferative effects of restoration. As such, our results establish a conserved epistatic relationship between the chromatin modifier MLL3 and a well-characterized tumor suppressor network.
我们的研究结合了遗传学、表观基因组学和动物模型方法,确定了 Cdkn2a 是 MLL3 在小鼠和人类肝癌中的重要调控靶点。我们的研究结果支持这样一个模型:MYC 在此产生的致癌压力导致 MLL3 与 CDKN2A 基因座的结合增加,这一事件与组蛋白标记的积累有关,组蛋白标记与含 MLL3 复合物的生化活性相关,有利于基因激活(图 6)。因此,这些事件伴随着两个关键的 Cdkn2a 基因产物 Ink4a 和 Arf 的转录上调。此外,Kmt2c 的抑制与 Cdkn2a 失活对 MYC 诱导的细胞凋亡的抑制作用相同。相反,抑制 Cdkn2a 会减弱 恢复的抗增殖作用。因此,我们的研究结果在染色质修饰因子 MLL3 和一个特征明确的肿瘤抑制因子网络之间建立了一种保守的表观关系。
The epistatic relationship described above might be expected to lead to mutual exclusivity of KMT2C and CDKN2A alterations; however, we did not observe significant mutual exclusivity in liver cancer, which is likely due to insufficient samples sizes needed to obtain statistical power. Alternatively, other functionally important components linked to the CDKN2A locus could produce CDKN2Aindependent forces that drive selection for chromosome 9p deletions, including type I interferon genes, CDKN2B, and MTAP (Barriga et al., 2022). Alternatively, mutual exclusivity between KMT2C and CDKN2A alterations would be expected only under circumstances where CDKN2A action is the most dominant MLL3 effector. Indeed, it seems likely that multiple downstream genes, including factors involved in interactions with stromal and immune populations, contribute to MLL3-driven tumor suppression, and their relative importance may vary between cell and tissue types. Such a variable output in cancer-relevant gene regulation has been noted for other chromatin regulators that, at the extreme, serve as pro-oncogenic factors in some contexts and tumor suppressors in others (Fountain et al., 1992; Schmid et al., 2000; Sun et al., 2017; Xia et al., 2021). Furthermore, our observation that Kmt2c deficiency cooperated with MYC but not CTNNB1 to drive HCC highlights such context specificity and is in line with recent findings that chromatin context could favor particular oncogenic alterations over others (Weiss et al., 2022).
上述表观关系可能会导致 KMT2C 和 CDKN2A 基因改变的互斥性;然而,我们在肝癌中没有观察到显著的互斥性,这可能是由于样本量不足,无法获得统计效力。另外,与 CDKN2A 基因位点相关的其他功能上的重要成分也可能产生与 CDKN2A 无关的力量,推动对 9p 染色体缺失的选择,包括 I 型干扰素基因、CDKN2B 和 MTAP(Barriga 等人,2022 年)。或者说,只有在 CDKN2A 作用是最主要的 MLL3 效应体的情况下,KMT2C 和 CDKN2A 的改变才会相互排斥。事实上,多个下游基因,包括参与与基质和免疫群体相互作用的因子,似乎都可能对 MLL3 驱动的肿瘤抑制做出贡献,而且它们的相对重要性可能因细胞和组织类型而异。其他染色质调控因子在癌症相关基因调控中的这种可变输出已被注意到,极端情况下,它们在某些情况下是促癌因子,而在另一些情况下则是肿瘤抑制因子(Fountain 等人,1992 年;Schmid 等人,2000 年;Sun 等人,2017 年;Xia 等人,2021 年)。此外,我们观察到 Kmt2c 缺陷与 MYC 而非 CTNNB1 合作驱动 HCC,这凸显了这种环境特异性,并与最近的研究结果一致,即染色质环境可能有利于特定的致癌改变而非其他改变(Weiss 等人,2022 年)。
UTX (KDM6A), MLL3 (KMT2C), and MLL4 (KMT2D), the core catalytic components of the COMPASSlike complex, are all considered tumor suppressors, with frequent loss-of-function genomic alterations found in a broad spectrum of human cancers (Revia et al., 2022; Sze and Shilatifard, 2016). While each of these components regulates redundant sets of genes (Hu et al., 2013; Lee et al., 2009), they may exert their tumor suppressive functions through different mechanisms. In liver and pancreas cancer models, UTX can control the expression of negative regulators of mTOR such as DEPTOR, and its disruption prevents their transcription and facilitates tumorigenesis through increased mTORC1 activity (Revia et al., 2022). Additionally, while the mechanisms of MLL4 activity have not been examined in liver cancer, studies suggest that MLL4 suppresses skin carcinogenesis by promoting lineage stability and ferroptosis independently of MLL3 (Egolf et al., 2021). Our study demonstrates that MLL3 is both necessary and sufficient for efficient transcriptional activation of the CDKN2A locus that drives oncogene-induced apoptosis. The molecular basis for this heterogeneity in effector output remains to be determined, but it seems likely that different subsets of target genes are preferentially disabled by haploinsufficiency of individual components and/or subject to compensation by the remaining COMPASS complex activities. Systematic studies comparing the binding, histone modifications, and transcriptional output of cells across a spectrum of allelic configurations of COMPASS complex factors will be needed to achieve a more holistic understanding of their functions and interactions in different contexts.
UTX(KDM6A)、MLL3(KMT2C)和 MLL4(KMT2D)是 COMPASS-like 复合物的核心催化元件,它们都被认为是肿瘤抑制因子,在广泛的人类癌症中经常发现功能缺失的基因组改变(Revia 等人,2022 年;Sze 和 Shilatifard,2016 年)。虽然这些成分中的每一个都调控着冗余的基因集(Hu 等人,2013 年;Lee 等人,2009 年),但它们可能通过不同的机制发挥抑制肿瘤的功能。在肝癌和胰腺癌模型中,UTX 可控制 DEPTOR 等 mTOR 负性调控因子的表达,破坏UTX 可阻止它们的转录,并通过增加 mTORC1 的活性促进肿瘤发生(Revia 等,2022 年)。此外,虽然尚未研究 MLL4 在肝癌中的活性机制,但研究表明,MLL4 可独立于 MLL3,通过促进血统稳定性和铁变态反应抑制皮肤癌的发生(Egolf 等人,2021 年)。我们的研究表明,MLL3 对于 CDKN2A 基因座的有效转录激活既是必要的,也是足够的,而 CDKN2A 基因座的有效转录激活可驱动癌基因诱导的细胞凋亡。效应器输出的这种异质性的分子基础仍有待确定,但不同的靶基因子集很可能因单倍体成分缺陷而优先失效,和/或受到其余 COMPASS 复合物活动的补偿。为了更全面地了解 COMPASS 复合体因子在不同情况下的功能和相互作用,需要进行系统研究,比较不同等位基因配置的 COMPASS 复合体因子的结合、组蛋白修饰和细胞转录输出。
The most well-established role for MLL3/4-UTX-containing complexes is the control of H3K4 monomethylation at enhancers during development (Herz et al., 2010; Hu et al., 2013). While our ChIP-Seq studies also revealed binding of MLL3/4 to enhancers in liver tumor cells, an even larger fraction of genes-including Cdkn2a-showed MLL3/4 chromatin enrichment at gene promoters, and indeed, transcription of this class of genes was most affected by Kmt2c disruption. Interestingly, Kmt2c suppression preferentially limited the MLL3/4 enrichment at promoters and shifted residual complex binding toward intergenic regions. Such dynamic regulation of distinct cis-acting elements by the MLL3/4 complex has also been observed in other contexts (Cheng et al., 2014; Soto-Feliciano et al., 2023), where the non-canonical binding of MLL3/4 at promoters is a recurrent tumor suppressive mechanism in cancer cells. MLL3/4 has also been observed to bind within the exons and introns, which
含 MLL3/4-UTX 复合物最成熟的作用是在发育过程中控制增强子的 H3K4 单甲基化(Herz 等人,2010 年;Hu 等人,2013 年)。虽然我们的 ChIP-Seq 研究也揭示了肝肿瘤细胞中 MLL3/4 与增强子的结合,但包括 Cdkn2a 在内的更大一部分基因在基因启动子处显示出 MLL3/4 染色质富集,而且事实上,这类基因的转录受 Kmt2c 干扰的影响最大。有趣的是,Kmt2c抑制优先限制了启动子上的MLL3/4富集,并将残余的复合体结合转移到基因间区域。MLL3/4 复合物对不同顺式作用元件的这种动态调控在其他情况下也被观察到(Cheng 等人,2014 年;Soto-Feliciano 等人,2023 年),MLL3/4 在启动子上的非规范结合是癌细胞中反复出现的肿瘤抑制机制。还观察到 MLL3/4 在外显子和内含子中结合,这

may enable chromatin looping of enhancers to activate gene expression (Panigrahi and O'Malley, 2021). Further studies into the action and regulation of MLL3/4 complexes at promoters and gene bodies will be informative and may yield new insights into the actions of the COMPASS-like complex in cancer.
可使染色质环绕增强子激活基因表达(Panigrahi 和 O'Malley,2021 年)。对 MLL3/4 复合物在启动子和基因体上的作用和调控的进一步研究将提供丰富的信息,并可能对 COMPASS-like 复合物在癌症中的作用产生新的认识。
While CDKN2A showed a surprisingly dominant role in mediating the tumor-suppressive effects of the broadly acting MLL3 enzyme, there are precedents for a predominant contribution of a single gene to the functional output of chromatin-complex disruption. Indeed, polycomb repressive complexes (PRCs) broadly repress gene expression in different cell types through the coordinated action of PRC1 and PRC2 complexes that deposit and maintain repressive marks on the enhancers of target genes, including CDKN2A (Bracken et al., 2007; Kotake et al., 2007). Despite these similarly broad effects, CDKN2A is often the most functionally relevant target of PRC-mediated repression, as genetic deletion of either the PRC1 component Bmi1 or the PRC2 component Ezh2, or treatment with small molecule inhibitors of EZH2, can facilitate Cdkn2a induction in normal and tumor cells. This, in turn, triggers anti-proliferative responses that can be rescued by Cdkn2a deletion (Jacobs et al., 1999; Richly et al., 2011). Notably, the COMPASS-like complexes are biochemically and functionally similar to Trithorax complexes in Drosophila, which have an evolutionarily conserved antagonistic relationship with PRC1 and PRC2 that controls epigenetic memory and cell fate during development (Mills, 2010; Piunti and Shilatifard, 2016). Our findings suggest such antagonism extends to tumor suppression in mammalian cells, likely via regulation of Cdkn2a and other tumor suppressor genes (Soto-Feliciano et al., 2023).
虽然 CDKN2A 在介导广泛作用的 MLL3 酶的肿瘤抑制作用方面显示出令人惊讶的主导作用,但染色质复合体破坏的功能输出主要由单个基因贡献的先例也是有的。事实上,多聚核酸抑制复合体(PRCs)通过 PRC1 和 PRC2 复合体的协调作用广泛抑制不同细胞类型中的基因表达,这些复合体沉积并维持靶基因(包括 CDKN2A)增强子上的抑制性 标记(Bracken 等人,2007 年;Kotake 等人,2007 年)。尽管CDKN2A具有类似的广泛作用,但它往往是PRC介导的抑制作用中功能最相关的靶点,因为基因缺失PRC1成分Bmi1或PRC2成分Ezh2,或用EZH2的小分子抑制剂处理,都会促进正常细胞和肿瘤细胞中Cdkn2a的诱导。这反过来又会引发抗增殖反应,而这种反应可以通过 Cdkn2a 的缺失来挽救(Jacobs 等人,1999 年;Richly 等人,2011 年)。值得注意的是,COMPASS 类复合物在生化和功能上与果蝇的 Trithorax 复合物相似,后者与 PRC1 和 PRC2 之间存在进化保守的拮抗关系,在发育过程中控制表观遗传记忆和细胞命运(Mills,2010 年;Piunti 和 Shilatifard,2016 年)。我们的研究结果表明,这种拮抗作用延伸到哺乳动物细胞中的肿瘤抑制,很可能是通过调控 Cdkn2a 和其他肿瘤抑制基因实现的(Soto-Feliciano 等人,2023 年)。

Materials availability statement
材料供应声明

Source files of all original gels and western blots were provided for the following figures:
以下图表提供了所有原始凝胶和 Western 印迹的源文件:
Figure 1-figure supplement ;
图 1-图补充
Figure 4-figure supplement ;
图 4-图补充
Figure 5-figure supplement 2B, F, G.
图 5-图补充 2B、F、G。
RNA sequencing and ChIP-Seq data files that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE85055, as well as in the Dryad digital repository (doi:10.5061/dryad.7pvmcvdwm; doi:10.5061/dryad.f1vhhmhOh). Sequences of sgRNAs, shRNAs, and primers used in this manuscript are included in the Supplementary file 1.
支持本研究结果的 RNA 测序和 ChIP-Seq 数据文件已存入基因表达总库(GSE85055)和 Dryad 数字资源库(doi:10.5061/dryad.7pvmcvdwm; doi:10.5061/dryad.f1vhhmhOh)。本稿件中使用的 sgRNAs、shRNAs 和引物的序列包含在补充文件 1 中。

Materials and methods 材料和方法

Animal experiments 动物实验

8- to 10-week-old female C57BL/6 animals were purchased from Envigo (formerly Harlan). Each experiment was performed in mice from the same order. Arf-null animals (C57BL/6 background), originally provided by Dr. Charles Sherr, St. Jude Children's Research Hospital, were maintained in our breeding colony. For HTVI, a sterile solution/plasmid mix was prepared containing oncogene transposons (5 g DNA of pT3-Myc or pT3-Ctnnb1 N90) with either of pX330 expressing the indicated sgRNAs or of pT3-EF1a-GFP-miRE plasmid together with CMV-SB13 Transposase 1:5 ratio). Mice were randomly assigned to experimental groups and injected with the solution/plasmid mix into the lateral tail vein with a total volume corresponding to of body weight in 5-7 s as described before (Largaespada, 2009; Moon et alo, 2019; Tschaharganeh et alo, 2014; Xue et al., 2014). Injected mice were monitored for tumor formation by abdominal palpation. All animal experiments were approved by the Memorial Sloan Kettering Cancer Center (MSK) Institutional Animal Care and Use Committee (protocol 11-06-011). Animals were monitored for signs of ill health by veterinary staff at the Research Animal Resource Center at MSK, and efforts were made to minimize suffering.
8至10周大的雌性C57BL/6小鼠购自Envigo公司(前身为Harlan公司)。每次实验都在同一订单的小鼠中进行。Arf-null小鼠(C57BL/6背景)最初由圣裘德儿童研究医院的Charles Sherr博士提供,在我们的繁殖群中饲养。对于HTVI,制备了无菌 溶液/质粒混合物,其中含有癌基因转座子(5 g DNA的pT3-Myc或 pT3-Ctnnb1 N90)和 表达所述sgRNA的pX330或 pT3-EF1a-GFP-miRE质粒以及CMV-SB13转座酶(1:5的比例)。小鼠被随机分配到实验组,按照之前的描述(Largaespada,2009;Moon et alo,2019;Tschaharganeh et alo,2014;Xue et alo,2014),在5-7秒内将 溶液/质粒混合物注入小鼠尾侧静脉,总体积相当于 体重。注射小鼠通过腹部触诊监测肿瘤的形成。所有动物实验均已获得纪念斯隆-凯特琳癌症中心(MSK)机构动物护理和使用委员会的批准(协议 11-06-011)。MSK研究动物资源中心的兽医人员负责监测动物的健康状况,并努力将动物的痛苦降到最低。

Vector constructs 向量结构

The pT3-Myc vector Addgene (#92046) and pT3-EF1a-GFP-miRE plasmid were described before (Huang et al., 2014). The pT3-Ctnnb1 N90 vector (Tward et al., 2007) was obtained from Addgene (#31785). For CRISPR/Cas9-mediated genome editing, sgRNAs were subcloned into pX330 (Addgene, #42230; Hsu et al., 2013). All shRNA and sgRNA sequences are listed in Supplementary file 1.
pT3-Myc载体Addgene(#92046)和pT3-EF1a-GFP-miRE质粒之前已有描述(Huang等人,2014)。pT3-Ctnnb1 N90 载体(Tward 等人,2007 年)来自 Addgene(#31785)。对于 CRISPR/Cas9 介导的基因组编辑,sgRNA 被亚克隆到 pX330(Addgene,#42230;Hsu 等人,2013 年)中。所有 shRNA 和 sgRNA 序列见补充文件 1。

Derivation of primary liver tumor cell lines
原发性肝肿瘤细胞系的培育

Liver tumors were resected with sterile instruments, and 10-50 mg of tumor tissue was minced and washed in sterile PBS, incubated in a mix of collagenase IV and dispase (dissolved in sterile, serum-free DMEM(Dulbecco's Modified Eagle Medium)) with gentle shaking, washed with PBS, incubated for in (w/v) trypsin, and washed and plated in complete DMEM (10% FBS(fetal bovine serum), penicillin/streptomycin) on collagen-coated plates (PurCol, Advanced Biomatrix). Primary cultures were passaged until visibly free from fibroblasts. Cell lines were authenticated on a routine basis using short tandem repeat profiling, as well as tested for mycoplasma contamination and immediately discarded upon a positive test.
用无菌器械切除肝脏肿瘤,将 10-50 毫克肿瘤组织切碎并用无菌 PBS 冲洗,在 胶原酶 IV 和 分散酶(溶于无菌、无血清 DMEM(Dulbecco's Modified Eagle Medium,杜氏改良鹰培养基))的混合液中轻轻振荡孵育,用 PBS 冲洗,在 胰蛋白酶中孵育 。(w/v) 胰蛋白酶中孵育 ,然后在完全 DMEM(10% FBS(胎牛血清), 青霉素/链霉素)中洗涤并移栽到涂有胶原蛋白的平板(PurCol,Advanced Biomatrix)上。对原代培养物进行传代培养,直至其明显脱离成纤维细胞。使用短串联重复图谱对细胞系进行常规鉴定,并进行支原体污染检测,检测结果呈阳性时立即丢弃。

Analysis of CRISPR-directed mutations
分析 CRISPR 引导的突变

CRISPR-mediated insertions and deletions were detected by surveyor assay as directed by the manufacturer (Transgenomic/IDT). Briefly, after overnight lysis of primary tumors and cell lines at in buffer containing proteinase K, Tris, EDTA, and 0.5% SDS, pH 8.0, genomic DNA was extracted by isopropanol precipitation. bp regions flanking predicted CRISPR cleavage sites were PCR amplified with Herculase II taq polymerase, column purified (Qiagen), heated to , and slowly cooled to promote annealing of heteroduplexes. Following treatment with Surveyor nuclease, products were analyzed by electrophoresis on a polyacrylamide gel. Primers used for surveyor assay are listed in Supplementary file 1. Amplified PCR products were separately gel purified and ligated into blunt-end digested pBlueScript (Stratagene). DNA from 48 transformed colonies was analyzed by Sanger sequencing using a T7 primer.
按照生产商(Transgenomic/IDT)的指示,通过测量器检测 CRISPR 介导的插入和缺失。简而言之,在含有 蛋白酶 K、{{2Tris, {{3}EDTA 和 0.5% SDS(pH 值为 8.0)的缓冲液中过夜裂解原代肿瘤和细胞株,然后用异丙醇沉淀法提取基因组 DNA。{用 Herculase II taq 聚合酶进行 PCR 扩增,柱纯化(Qiagen),加热至 。然后缓慢冷却以促进异源双链体退火。用 Surveyor 核酸酶处理后,在 聚丙烯酰胺凝胶上电泳分析产物。用于 Surveyor 检测的引物列于补充文件 1。扩增的 PCR 产物分别进行凝胶纯化,并连接到钝端消化的 pBlueScript(Stratagene)中。使用 T7 引物对 48 个转化菌落的 DNA 进行桑格测序分析。

CRISPR activation CRISPR 激活

Human HCC cell line HLE, purchased from JCRB Cell Bank (JCRB0404), was transduced by the lentivirus expressing nuclease-dead Cas9 (dCas9) fused with VPR (Chavez et al., 2015) and sgRNAs against KMT2C (sequence in Supplementary file 1) to generate stable MLL3 CRISPRa HLE line by puromycin selection.
人类 HCC 细胞系 HLE 购自 JCRB 细胞库(JCRB0404),通过表达融合了 VPR 的核酸酶死 Cas9(dCas9)(Chavez 等,2015 年)和针对 KMT2C 的 sgRNA(序列见补充文件 1)的慢病毒转导,经嘌呤霉素选择产生稳定的 MLL3 CRISPRa HLE 系。

Generation and modification of primary cells
原始细胞的生成和改造

LPCs from E13.5-15.5 C57BL/6 embryos were isolated and grown in hepatocyte growth media (HGM) as previously described (Zender et alo, 2005). To simultaneously overexpress Myc and conditionally suppress , LPCs were co-infected with a retroviral construct constitutively expressing both Myc and a reverse tet-transactivator (rtTA) (MSCV-Myc-IRES-rtTA) along with retroviral TRMPV vectors (MSCV-TRE-dsRed-miR30/shRNA-PGK-Venus-IRES-NeoR) (Zuber et al., 2011) expressing ds-Red linked, teint-responsive shRNAs targeting Kmt2c cloned into an optimized mir-30 context ('mir-E,' TRPMVe; Zuber et al., 2011). For selection of infected cells and sustained shKmt2c expression, cells were maintained in HGM with neomycin ( and dox ( starting 2 days after infection. To introduce constitutively expressed shRNAs in the setting of inducible shKmt2c, retroviral MLPe vectors (MSCV-LTR-mir-E-PGK-Puro-IRES-GFP; Dickins et al., 2005). GFP-linked shRNAs targeting either Cdkn2a or Renilla luciferase (as control) were co-infected with MSCV-Myc-IRES-rtTa and TRMPVe-shKmt2c. Triple-infected cells were maintained in media with neomycin, puromycin , and dox 2 days post infection. Infected continuously proliferating cells were transitioned to growth in complete DMEM and maintained on collagen-coated plates.
按照之前的描述(Zender et alo, 2005),从 E13.5-15.5 C57BL/6 胚胎中分离并在肝细胞生长培养基(HGM)中培养 LPCs。为了同时过表达 Myc 和有条件地抑制 ,LPCs 被同时感染。为了同时过表达 Myc 和有条件抑制 ,LPCs 被同时表达 Myc 和反向 tet-transactivator (rtTA) 的逆转录病毒构建体(MSCV-Myc-IRES-rtTA)以及逆转录病毒 TRMPV 载体(MSCV-TRE-dsRed-miR30/shRNA-PGK-Venus-IRES-NeoR)共同感染(Zuber 等人,2011 年)、MSCV-TRE-dsR-miR30/shRNA-PGK-Venus-IRES-NeoR)(Zuber 等人,2011 年)。为了选择感染细胞并维持 shKmt2c 的表达,细胞在感染 2 天后开始用新霉素( )和多克隆( )维持在 HGM 中。为了在诱导性 shKmt2c 的环境中引入组成型表达的 shRNA,使用了逆转录病毒 MLPe 载体(MSCV-LTR-mir-E-PGK-Puro-IRES-GFP;Dickins 等人,2005 年)。MSCV-Myc-IRES-rtTa 和 TRMPVe-shKmt2c 共同感染了靶向 Cdkn2a 或 Renilla 荧光素酶(作为对照)的 GFP 链接 shRNA。三重感染的细胞在感染后 2 天用新霉素、嘌呤霉素 和多抗霉素培养基培养。和 dox 的培养基中。感染后持续增殖的细胞转入完全 DMEM 培养基中生长,并保存在涂有胶原蛋白的平板上。

Colony assays 菌落测定

For measurement of cell proliferation, 5000 transduced and selected LPCs or MEFs were plated in triplicate in 6-well plates. Tetracycline-inducible shKmt2c-expressing LPCs were grown in the presence or absence of dox, and after 5 days, cells were fixed with formalin and methanol and stained with crystal violet. MEFs were fixed after 6 days with formalin and methanol and stained with crystal violet.
为了测量细胞增殖,将 5000 个转导和筛选过的 LPC 或 MEF 一式三份培养在 6 孔板中。四环素诱导的表达 shKmt2c 的 LPCs 在有或没有 dox 的情况下生长,5 天后用福尔马林和甲醇固定细胞,并用 结晶紫染色。6 天后用福尔马林和甲醇固定 MEF,并用 水晶紫染色。

Apoptosis assays 细胞凋亡测定

Apoptosis was measured in LPCs via Annexin V staining according to the manufacturer's instructions (eBiosciences, Annexin-V APC). 25,000 cells were grown with and without dox for 3 days, trypsinized,
根据生产商的说明(eBiosciences,Annexin-V APC),通过Annexin V染色测量LPC细胞的凋亡情况。25,000 个细胞在含有或不含有多克斯的情况下生长 3 天,胰蛋白酶灭活、

washed with Annexin-V binding buffer, and cells were incubated with Annexin-V APC and analyzed on an LSRII flow cytometer (BD).
用 Annexin-V 结合缓冲液清洗细胞, 细胞与 Annexin-V APC 一起孵育,并在 LSRII 流式细胞仪(BD)上进行分析。

Live imaging 实时成像

Imaging was performed on LPCs immortalized by linked overexpression of Myc and two independent, inducible Kmt2c shRNAs constitutively expressing shRNAs targeting Renilla luciferase or Cdkn2a (generated as detailed above). 1000 cells were plated on collagen-coated, 96 well, clear bottom imaging plates in media supplemented with Draq7 (Invitrogen) with and without dox, in triplicate by genotype. after plating cells, Venus (marking all plated cells) and Draq7 fluorescence was collected in two, fields of each well every for using an automated, high content microscope (InCell 6000, General Electric).
通过连接过表达 Myc 和两个独立的诱导型 Kmt2c shRNA,构成表达针对雷尼拉荧光素酶或 Cdkn2a 的 shRNA(生成方法详见上文),对永生的 LPCs 进行成像。将 1000 个细胞培养在涂有胶原蛋白的 96 孔透明底成像板上,培养基中添加 Draq7(Invrogen)。Draq7(Invitrogen 公司)的培养基中,加入或不加入 dox,按基因型一式三份。细胞培养 后,使用自动高内涵显微镜(InCell 6000,通用电气公司)每 在{{4}每孔的两个{{2}视野中收集维纳斯(标记所有培养细胞)和 Draq7 荧光。)

Chromatin immunoprecipitation
染色质免疫沉淀

Histone ChIP was performed as previously described (Lee et al., 2006). Briefly, cell samples were cross-linked in formaldehyde for , and the reaction was stopped by addition of glycine to final concentration. Fixed cells were lysed in SDS lysis buffer, and the chromatin was fragmented by sonication (Covaris). Sheared chromatin was incubated with antibodies (final concentration ) against H3K4me3 (Abcam, ab8580, Lot:GR164706-1), H3K27ac (Abcam, ab4729, Lot:GR200563-1), or H3K4me1 (Abcam; ab8895, Lot:GR114265-2) or with normal rabbit lgG (Abcam, ab46540) at for overnight. Antibodies were recovered by binding to protein A/G agarose (Millipore), and the eluted DNA fragments were used directly for qPCR or subjected to high-throughput sequencing (ChIP-Seq) using a HiSeq 2000 platform (Illumina). High-throughput reads were aligned to mouse genome assembly NCBI37/mm9 as previously described (Barradas et al., 2009). Reads that aligned to multiple loci in the mouse genome were discarded. The ChIP-Seq signal for each gene was quantified as total number of reads per million in the region upstream to downstream of the transcription start site (TSS). Primers used for ChIP-qPCR of mouse Cdkn2a promoter (Barradas et al., 2009) are listed in Table S1.
组蛋白 ChIP 按照之前的描述进行(Lee 等人,2006 年)。简而言之,细胞样本在 甲醛中交联 ,然后加入甘氨酸至 终浓度停止反应。然后加入 终浓度的甘氨酸停止反应。固定的细胞在 SDS 裂解缓冲液中裂解,染色质通过超声(Covaris)破碎。剪切后的染色质与针对 H3K4me3(Abcam,ab8580,Lot:GR164706-1)、H3K27ac(Abcam,ab4729,Lot:GR200563-1)或 H3K4me1(Abcam;ab8895,Lot:GR114265-2)的抗体(最终浓度 )或正常兔 lgG(Abcam,ab46540)在 孵育过夜。抗体与蛋白 A/G 琼脂糖(Millipore)结合回收,洗脱的 DNA 片段直接用于 qPCR 或使用 HiSeq 2000 平台(Illumina)进行高通量测序(ChIP-Seq)。根据之前的描述(Barradas 等人,2009 年),高通量读数与小鼠基因组组装 NCBI37/mm9 进行了比对。舍弃了与小鼠基因组中多个位点对齐的读数。每个基因的 ChIP-Seq 信号被量化为转录起始位点(TSS)上游 至下游 区域的每百万读数总数。表 S1 列出了用于小鼠 Cdkn2a 启动子 ChIP-qPCR 的引物(Barradas 等人,2009 年)。
The complete dataset is available at NCBI Gene Expression Omnibus (GSE85055), as well as the Dryad digital repository (doi:10.5061/dryad.7pvmcvdwm).
完整的数据集可在 NCBI 基因表达总库(GSE85055)和 Dryad 数字资源库(doi:10.5061/dryad.7pvmcvdwm)上查阅。
For the MLL3 ChIP-Seq, the following protocol was used. Cross-linking ChIP in mouse and human cells was performed with cells per immunoprecipitation. Cells were collected, washed once with ice-cold PBS, and flash-frozen. Cells were resuspended in ice-cold PBS and cross-linked using paraformaldehyde (PFA; Electron Microscopy Sciences) for at room temperature with gentle rotation. Unreacted PFA was quenched with glycine (final concentration ) for at room temperature with gentle rotation. Cells were washed once with ice-cold PBS and pelleted by centrifugation ( for ). To obtain a soluble chromatin extract, cells were resuspended in of LB1 (50 mM HEPES pH 7.5, EDTA, glycerol, 0.5% NP-40, 0.25% Triton X-100, and complete protease inhibitor cocktail) and incubated at for 10 min while rotating. Samples were centrifuged (1400 for , resuspended in of LB2 (10 Tris- pH 8.0, EDTA, EGTA, and complete protease inhibitor cocktail), and incubated at for while rotating. Finally, samples were centrifuged ( for ) and resuspended in of LB3 (10 mM Tris-HCl pH 8.0, EDTA, EGTA, sodium deoxycholate, -lauroylsarcosine, and complete protease inhibitor cocktail). Samples were homogenized by passing seven to eight times through a 28-gauge needle, then Triton X-100 was added to a final concentration of