B細胞慢性淋巴性白血病中CD22基因的轉錄調控

Abstract

CD22 是一種唾液酸結合免疫球蛋白樣凝集素， 摘要 是B 細胞受體 (BCR) 訊號傳 導的抑 制性輔助受體。CD22受到激化後，會招募 SHP-1 磷酸酶並抑制 BCR 參與引起的鈣流入。先前的研究指出CD22 在慢性淋巴球白血病 (CLL) 患者的 B 細胞中減少。慢性淋巴球白血 病是一種 B 細胞起源的白血病， 其特徵是細胞死 亡的累積血液、骨髓和次級淋巴組織中的抗藥性 B 細胞。一個公開的CLL 患者轉錄組顯示，CD22的低表達與患者預後不良 有關。在CLL 中，一些異常表 達轉錄因子會導致促進癌症的基因異常表達細胞存活、增殖和 對治療的抵抗力。 例如，IRF4已被鑑定作為一種 CLL 抑制基因，在小鼠體內剔 除該基因會導致 CLL 的發生。在這個計畫中，我們探討了 B 細胞中 CD22 的轉錄調節 因子， 以探究CLL B 細胞中 CD22可能失調的機制。雖然之前的研究已鑑定了 CD22 基因的啟動子區域，然而有關其轉錄的知識有限。我們首先證實了B細胞上CD22蛋白的表現量在台灣CLL患者的比例低於健康的人。我們測試了 CD22 mRNA CLL 和非 CLL B 細胞系中的蛋白質表現量，證實了CLL 細胞株的 CD22的表達較低。 透過使用一系列截短 的 CD22 啟動子區域分析，我們鑑定出 CD22基因轉錄起始 位點下游的〜90個鹼基對區域 是最小啟動子。這個最小區 域包含保守的轉錄 因子結合位點（GC 盒和 E 盒），這些位點對於CD22 的轉錄可能是必需的調節 區域。為了確定控制 CD22 表現的主要轉錄因子， 我們使用合成誘餌DNA 進行 DNA 親和蛋白質體學，鎖定最小啟動子 區域和GC盒/E盒 突變體。使用合成的 誘餌DNA從 CLL 和非 CLL 細胞株的 核提取物中釣出幾個有顯著差異 轉錄因子， 並透過染色質免疫沉澱、定量 PCR 和EMSA交叉驗證。這些調節因子中，B 細胞系株中的基因分析顯示 SPIB 和 PU.1 是正向調節因子，而IRF4則是CD22的潛 在轉錄 抑制因子。這項研究揭示了 人類 CD22基因轉錄調控的基本機制，以及在 CLL的 B 細胞中失調的可能機制。 本次研究中所獲得的知識將有利於患者分類策 略和發展針對慢性淋巴球白血病 CD22 的標靶治療。

CD22 is a sialic acid-binding immunoglobulin-like lectin serving as an inhibitory coreceptor for B-cell receptor (BCR) signaling. It recruits SHP-1 phosphatase upon activation and suppresses the calcium influx elicited by BCR engagement. Previous studies reported that CD22 is downregulated in the B cells of chronic lymphocytic leukemia (CLL) patients. CLL is a type of leukemia of B cell origins and is characterized by the accumulation of cell death-resistant B cell clones in blood, bone marrow, and secondary lymphoid tissues. A publicly available transcriptomic dataset for CLL patients indicated that low CD22 expression level is associated with a poor prognosis of patients. In CLL, aberrant expression of some transcription factors is known to lead to abnormal expression of genes that promote cancer cell survival, proliferation, and resistance to treatment. For example, IRF4 has been identified as a CLL suppressor gene, whose deletion in mice leads to the development of CLL. In this project, we explored the transcription regulator of CD22 in B-cells to decipher possible mechanisms by which CD22 is dysregulated in CLL B cells. Although a previous study identified the promoter region of the CD22 gene, the knowledge regarding its transcriptional regulators is limited. We first confirmed that CD22 protein expression levels on the B cells of Taiwanese CLL patients are lower than those of healthy donors. We then tested CD22 mRNA and protein levels in CLL and non-CLL B cell lines, confirming the lower expression of CD22 in CLL cell lines. The minimal promoter region of the CD22 gene was identified through reporter assays using a series of truncated CD22 promoter regions, revealing that a ~90-base pair region located downstream of the previously reported transcription start site is the minimal promoter. This minimal region contains conserved transcription factor binding sites (GC box and E box), indicating that these sites could be essential for transcriptional regulation of CD22. To identify the major transcription factors controlling CD22 expression, we conducted DNA-affinity proteomics analyses using synthetic bait DNA corresponding to the minimal promoter region and GC box/E box mutants. Several transcription factors were significantly enriched from the nuclear extracts of CLL and non-CLL cell lines with the bait DNA, and further characterized by functional reporter assays, such as chromatin immunoprecipitation with quantitative PCR and electrophoretic mobility shift assay. Among those regulators, genetic manipulation in B cell lines demonstrated that SPIB and PU.1 are positive regulators and IRF4 is a potential transcriptional repressor of CD22. This study revealed the basic mechanism of transcriptional regulation of the human CD22 gene and a possible mechanism of its dysregulation in CLL B cells. The knowledge gained in this study may benefit the development of patient stratification strategies and CD22-targeting therapies for CLL.