Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia [4C]. Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia [4C]

CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops. In addition to insulation of euchromatin from heterochromatin, CTCF is an important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCF inactivating events have been found in human cancer, resulting in deregulation of global gene expression by altered methylated genomic states. In contrast to these studies, we here describe that inactivation of CTCF can drives subtle and local genomic effects that elevates oncogene expression levels from driving chromosomal rearrangements. For T cell acute lymphoblastic leukemia (T-ALL), heterozygous CTCF deletions or inactivating mutations are predominantly found in nearly 50 percent of t(5;14)(q35;q32.2) rearranged patients that couples the TLX3 oncogene in the vicinity of the BCL11B enhancer. This unique entity has been associated with γδ-lineage development. Functional CTCF loss results in diminished expression of the αβ-lineage commitment factor BCL11B from the non-rearranged allele, but unexpectedly drives higher levels of the TLX3 oncogene from the translocated allele. In line, Ctcf conditional knockout mice have reduced numbers of αβ T cells but increased numbers of γδ T cells, a phenotype identical to that of Bcl11b knockout mice and implying that CTCF is directly involved in the regulation of the BCL11B enhancer. We demonstrate that most TLX3-rearranged patients with heterozygous CTCF aberrations preserved single intervening CTCF bindings sites in the translocation breakpoint areas located in between the BCL11B enhancer and the TLX3 oncogene. These intervening CTCF sites insulate TLX3 from the enhancer by forming competitive loops with TLX3 regulatory sequences. Using reverse genetics, we provide evidence that heterozygous inactivation of CTCF diminishes competitive loop formation in favor of high-affinity TLX3 promoter loops to BCL11B enhancer sequences formed among multiple convergent CTCF binding sites. This boosts oncogene expression levels and leukemia burden in T-ALL patients. Overall design: 4C experiment where interacting-loops of BCL11B and TLX3 has been studied in bulk and single cell clones from HPB-ALL have been studied

CCCTC结合因子（CCCTC-binding factor, CTCF）通过介导染色体环（chromosomal loops）的形成，调控三维染色质结构（3D chromatin architecture）。除了介导常染色质与异染色质的绝缘分隔之外，CCCTC结合因子还是一类重要的转录因子，同时参与抗原受体与T细胞受体（T cell receptor）的重组事件（recombination events）调控。人类癌症中已发现CCCTC结合因子的失活突变，这类事件会通过改变基因组甲基化状态，引发全局基因表达失调。与此前的研究结论不同，本研究证实CCCTC结合因子的失活可引发细微且局限的基因组效应，通过驱动染色体重排提升致癌基因（oncogene）的表达水平。针对T细胞急性淋巴细胞白血病（T cell acute lymphoblastic leukemia, T-ALL），杂合性CCCTC结合因子缺失或失活突变主要见于近50%的t(5;14)(q35;q32.2)重排患者，这类重排会将TLX3致癌基因（TLX3 oncogene）置于BCL11B增强子（BCL11B enhancer）的邻近区域。该独特白血病亚型与γδ谱系发育（γδ-lineage development）密切相关。功能性CCCTC结合因子缺失会导致非易位等位基因上的αβ谱系定向因子BCL11B的表达水平下调，但意外地会使易位等位基因上的TLX3致癌基因表达水平升高。与此一致，Ctcf条件性敲除小鼠（Ctcf conditional knockout mice）的αβ T细胞数量减少，而γδ T细胞数量增加，该表型（phenotype）与Bcl11b敲除小鼠完全一致，提示CCCTC结合因子直接参与BCL11B增强子的调控过程。本研究证实，大多数携带有杂合性CCCTC结合因子异常的TLX3重排患者，其位于BCL11B增强子与TLX3致癌基因之间的易位断点区域（breakpoint areas）内，仍保留有单个间隔的CCCTC结合因子结合位点。这些间隔的CCCTC结合因子位点通过与TLX3调控序列（regulatory sequences）形成竞争性环，将TLX3与BCL11B增强子分隔开。通过反向遗传学（reverse genetics）手段，本研究证明CCCTC结合因子的杂合性失活会削弱竞争性环的形成，转而促进由多个相向结合的CCCTC结合位点介导的、与BCL11B增强子序列结合的高亲和力（high-affinity）TLX3启动子环（promoter loops）的形成，进而提升T-ALL患者体内的致癌基因表达水平与白血病负荷（leukemia burden）。实验设计总览：本研究针对HPB-ALL细胞系的整体克隆与单细胞克隆（single cell clones）开展4C实验（4C experiment），以解析BCL11B与TLX3之间的相互作用环（interacting-loops）。