HMG20B stabilizes interaction of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block [ChIP-seq I]

Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukaemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we made use of mass spectrometry approaches. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is colocated on chromatin genome-wide with GFI1 and LSD1; the strongest HMG20B binding colocates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukaemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing the interaction on chromatin of LSD1 with GFI1. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukaemia cell differentiation block. Human THP1 AML cells were infected with lentivirus to induce doxycycline-induced expression of Flag-tagged full HMG20B, or a fusion protein containing the GFI1 DNA binding domain (GFI1-ZNF) fused with the full HMG20B. Cells were grown in the presence or absence of doxycycline for 48 hr. and subsequently ChIP-sequencing for LSD1, GFI1, Flag-HMG20B/GFI1-ZNF-HMG20B or H3K27ac was done.

LSD1（赖氨酸特异性去甲基化酶1）的药物抑制，可诱导携带MLL基因易位的急性髓系白血病（AML）患者体内的白血病原始细胞发生分子与形态学分化。除具备去甲基化酶活性外，LSD1在SNAG结构域转录抑制因子GFI1所占据的基因组位点上，发挥关键的支架功能。重要的是，LSD1抑制剂可同时阻断其酶促活性与支架功能，其中对支架功能的阻断通过破坏GFI1与LSD1之间的蛋白质-蛋白质相互作用实现。为探究LSD1抑制对LSD1蛋白质复合物的更广泛影响，我们采用了质谱分析方法。我们发现，LSD1抑制同样会破坏HMG盒蛋白HMG20B与LSD1的相互作用。后续研究显示，HMG20B在全基因组染色质上与GFI1和LSD1共定位；HMG20B结合最强的区域，与GFI1和LSD1结合最强的区域完全重合。功能实验证实，HMG20B敲低可诱导白血病细胞分化；进一步研究表明，HMG20B通过稳定LSD1与GFI1在染色质上的相互作用，对GFI1的转录抑制活性起到必需作用。HMG20B与LSD1的相互作用通过其卷曲螺旋结构域介导。综上，HMG20B是GFI1:LSD1转录抑制复合物的关键组成部分，该复合物参与白血病细胞的分化阻滞过程。我们使用慢病毒感染人THP1 AML细胞，以诱导强力霉素诱导表达的Flag标签标记的全长HMG20B，或诱导表达包含GFI1 DNA结合结构域（GFI1-ZNF）与全长HMG20B融合的融合蛋白。将细胞在含或不含强力霉素的培养基中培养48小时，随后分别针对LSD1、GFI1、Flag-HMG20B/GFI1-ZNF-HMG20B或组蛋白H3赖氨酸27乙酰化（H3K27ac）进行染色质免疫沉淀测序（ChIP-seq）。