Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.
Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.
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截短SOX 9变异是基因组稳定的结直肠癌中的杂合等位基因。
DOI：
10.1016/j.gastha.2022.04.011
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Sethi, N S
中科院分区：
文献类型：
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作者：
Duronio, G N;Liang, X;Hebbar, P;Islam, M;Spisak, S;Sethi, N S
Duronio等人，p3通过利用患者来源的分子信息的综合分析，我们最近定义了结直肠癌（CRC）的基因组稳定亚型，这是一种以前未被识别的亚组，缺乏显著的非整倍性和突变密度升高1。这一新类别的一个显著分子特征是在发育转录因子和WNT途径靶点SOX 92中存在高度复发的突变。然而，这些改变在CRC中的功能意义仍然知之甚少。先前的研究基于人类CRC病例的基因组分析假设突变体SOX 9的功能获得作用3，4。然而，到目前为止，截短的SOX 9蛋白的作用的直接的，功能分析尚未进行。在这封研究信中，我们注释了CRC中的SOX 9突变，描述了它们的转录和表观基因组后果，并假设为什么它们被选择用于基因组稳定的CRC。在CRC中，SOX 9改变主要是无义/移码突变，优先聚集在基因的c-末端一半内发现的三个功能域中（图1A-B）。我们证实了截短形式的SOX 9在携带内源性突变的CRC细胞系亚组中表达（补充图1A-B），通常以高于内源性野生型SOX 9的水平表达。来自癌症基因组图谱（TCGA）和癌细胞系百科全书（CCLE）的人CRC的基因组分析表明，大多数S 0X 9突变是杂合的，保留了基因的野生型（WT）拷贝（图1C）。这一结果提出了关于突变截短的SOX 9蛋白的功能的几种可能性：（1）它们通过调节不同的转录程序而携带功能获得特性，（2）它们通过抑制WT SOX 9功能而发挥显性负活性，或（3）它们表现为导致杂合状态的无效等位基因。为了研究突变体SOX 9的转录和表观基因组后果以及区分这些可能性，我们对突变体和WT SOX 9进行了全面的全基因组分子分析。我们在HT-115 CRC细胞中条件性过表达了四种具有N-末端V5蛋白标签的SOX 9蛋白构建体。这些包括一个WT构建体和三个突变体SOX 9等位基因，其c-末端结构域连续丢失，代表了在患者中观察到的突变谱（图2A-B）。截短的S 0X 9突变体以比WT S 0X 9更高的水平表达，这可能反映了对截短变体的表达升高的更大耐受性。V5蛋白标签确保了条件性过表达的WT和突变体S 0X 9的特异性评估，而不涉及细胞中表达的内源性S 0X 9（图2C）。使用染色质免疫沉淀，随后使用抗V5抗体进行DNA测序（ChIP-seq），测定突变体和WT S 0X 9的全基因组结合。突变体和WT S 0X 9结合至整个基因组中的相同位置（~ 1750个位点，图2D）;这些位点的基序分析显示天然S 0X 9结合序列的最大富集，如通过SeqPos确定的（p= lxl 0 -336）。
Duronio et al., p3 Through an integrative analysis leveraging patient-derived molecular information, we recently defined the genome stable subtype of colorectal cancer (CRC), a previously unrecognized subgroup that lacks significant aneuploidy and elevated mutational density1. A striking molecular feature of this new class is the presence of highly recurrent mutations in the developmental transcription factor and WNT pathway target SOX92. However, the functional significance of these alterations in CRC remains poorly understood. Prior studies hypothesized a gain-of-function role for mutant SOX9 based on genomic analyses of human CRC cases3, 4. However, to date a direct, functional analysis of the role of truncated SOX9 proteins has yet to be performed. In this Research Letter, we annotate SOX9 mutations in CRC, describe their transcriptional and epigenomic consequences, and postulate as to why they are selected for in genome stable CRC.In CRC, SOX9 alterations are predominantly nonsense/frameshift mutations that preferentially cluster in three functional domains found within the c-terminal half of the gene (Figure 1A-B). We confirmed that truncated forms of SOX9 are expressed in a subset of CRC cell-lines harboring endogenous mutations (Supplementary Figure 1A-B), often at higher levels than endogenous wildtype SOX9. Genomic analyses of human CRC from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) indicated that the majority of SOX9 mutations are heterozygous, preserving a wildtype (WT) copy of the gene (Figure 1C). This result raised a few possibilities as to the function of mutant truncated SOX9 proteins:(1) they carry gain-of-function properties by regulating a distinct transcriptional program,(2) they exert dominant-negative activity by inhibiting WT SOX9 function, or (3) they behave as a null allele leading to a heterozygous state. To investigate the transcriptional and epigenomic consequence of mutant SOX9 as well as distinguish between these possibilities, we pursued a comprehensive genome-wide molecular analysis of mutant and WT SOX9. We conditionally overexpressed four SOX9 proteins constructs with N-terminal V5 protein tags in HT-115 CRC cells. These included one WT construct and three mutant SOX9 alleles with sequential loss of its c-terminal domains, representing the spectrum of mutations observed in patients (Figure 2A-B). Truncated SOX9 mutants were expressed at higher levels than WT SOX9, which may reflect greater tolerance to elevated expression of truncated variants. The V5 protein tag ensured the specific assessment of the conditionally overexpressed WT and mutant SOX9 without engaging endogenous SOX9 expressed in the cells (Figure 2C). Genomewide binding of mutant and WT SOX9 was determined using chromatin-immunoprecipitation followed by DNA sequencing (ChIP-seq) using an anti-V5 antibody. Mutant and WT SOX9 bound to identical locations throughout the genome (~ 1750 sites, Figure 2D); the motif analysis of these sites showed greatest enrichment for the native SOX9 binding sequence as determined by SeqPos (p= 1x10-336).