Structural And Functional Study Of Oncoprotein DUX4/IGH In Acute B Lymphoblastic Leukemia

DUX4（Double homeobox 4）is a transcription factor that encodes two concatenated homologous domains and is located in the D4Z4 repeat region of subtelomeres 4q and 10q of chromosomes.DUX4plays an important regulatory role in early embryonic development and remains silent or low expressed in normal tissues.The abnormally high expression of DUX4 in myoblasts causes Facioscapulohumeral muscular dystrophy（FSHD）,and the effect of DUX4 on muscle cell differentiation is mainly due to the DNA binding activity of DUX4,which is promoted by the homeobox domain and does not require c-terminal activation domain.In FSHD,the DUX4 copy number decreased to 1-10 and caused chromosomal variation,activating a series of related genes.Gene rearrangement is an important driver of leukemia and an important target of leukemia therapy.B-cell acute lymphocytic leukemia（B-ALL）is a malignant clonal proliferative disease originated from B progenitor cells,accounting for 80%of lymphocytic leukemia.Recent studies have found that DUX4/IGH fusion gene formed by DUX4 and IGH rearrangement plays an important role in promoting the pathogenesis of B-ALL.DUX4/IGH fusion occurred in 7%of B-ALL patients and was associated with abnormally high ERGalt expression.ERG is an Ets transcription factor located on chromosome 21that plays an important role in normal hematopoietic regulation and is associated with the pathogenesis of acute T-lymphocytic leukemia and acute myeloid leukemia.It was found that DUX4/IGH binds to ERG 6intron,prompting ERG to produce the truncated transcript ERGalt.ERGalt induced lymphoblastic leukemia in Arf^-/-knockout mice.Therefore,ERGalt was considered a secondary stroke event in DUX4/IGH-driven leukemia.The ability of DUX4 to bind DNA plays a key role in the occurrence of both FSHD and B-ALL diseases.However,due to the lack of structural information of oncoprotein DUX4/IGH,how DUX4/IGH recognizes binding and transcriptional activation of downstream target genes and the specific mechanism of inducing leukemia is still unclear.In this study,we determined the crystal structure of Apo-DUX4_HD2and DUX4_HD2-DNA_ERG complex,and on the basis of the structure,we verified that the DNA binding ability of DUX4/IGH has an important impact on the occurrence of B-ALL.The protein structure and functional verification together revealed the two-step binding mechanism between DUX4 and ERG of double homeobox genes,and the homeodomain HD1,HD2 and c-terminal domain（CTD）were necessary for DUX4/IGH identification of downstream target genes.Single mutations in the major groove binding interface of HD1 and HD2 significantly disrupt DUX4 binding to ERG and subsequent transcriptional activation.In B cell differentiation experiments,structure-based mutations also significantly destroyed the transcriptional activity of DUX4/IGH and its inhibitory effect on B cell differentiation.At the same time,we systematically analyzed the characteristics of binding elements of DUX4 and DUX4/IGH downstream target genes,and found that DUX4 and DUX4/IGH preferred to bond with TGATXXXATTA reaction elements.These results suggest that DUX4/DUX4-IGH can recognize and bind downstream genes containing TGAT/TAAT or TGAT-TAAT elements and initiate transcriptional activation of these genes.DUX4-ERG recognition and two-step binding mechanism are applicable to other downstream target genes,which are crucial for DUX4/DUX4-IGH driven transcriptional activation and disease occurrence.