RESUMO
BACKGROUND: Blood typing is essential for safe transfusions and is performed serologically or genetically. Genotyping predominantly focuses on coding regions, but non-coding variants may affect gene regulation, as demonstrated in the ABO, FY and XG systems. To uncover regulatory loci, we expanded a recently developed bioinformatics pipeline for discovery of non-coding variants by including additional epigenetic datasets. METHODS: Multiple datasets including ChIP-seq with erythroid transcription factors (TFs), histone modifications (H3K27ac, H3K4me1), and chromatin accessibility (ATAC-seq) were analyzed. Candidate regulatory regions were investigated for activity (luciferase assays) and TF binding (electrophoretic mobility shift assay, EMSA, and mass spectrometry, MS). RESULTS: In total, 814 potential regulatory sites in 47 blood-group-related genes were identified where one or more erythroid TFs bound. Enhancer candidates in CR1, EMP3, ABCB6, and ABCC4 indicated by ATAC-seq, histone markers, and co-occupancy of 4 TFs (GATA1/KLF1/RUNX1/NFE2) were investigated but only CR1 and ABCC4 showed increased transcription. Co-occupancy of GATA1 and KLF1 was observed in the KEL promoter, previously reported to contain GATA1 and Sp1 sites. TF binding energy scores decreased when three naturally occurring variants were introduced into GATA1 and KLF1 motifs. Two of three GATA1 sites and the KLF1 site were confirmed functionally. EMSA and MS demonstrated increased GATA1 and KLF1 binding to the wild-type compared to variant motifs. DISCUSSION: This combined bioinformatics and experimental approach revealed multiple candidate regulatory regions and predicted TF co-occupancy sites. The KEL promoter was characterized in detail, indicating that two adjacent GATA1 and KLF1 motifs are most crucial for transcription.
Assuntos
Antígenos de Grupos Sanguíneos , Epigênese Genética , Humanos , Antígenos de Grupos Sanguíneos/genética , Fator de Transcrição GATA1/genética , Fatores de Transcrição Kruppel-Like/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Infant and adult MLL1/KMT2A-rearranged (MLLr) leukemia represents a disease with a dismal prognosis. Here, we present a functional and proteomic characterization of in utero-initiated and adult-onset MLLr leukemia. We reveal that fetal MLL::ENL-expressing lymphomyeloid multipotent progenitors (LMPPs) are intrinsically programmed towards a lymphoid fate but give rise to myeloid leukemia in vivo, highlighting a complex interplay of intra- and extracellular factors in determining disease subtype. We characterize early proteomic events of MLL::ENL-mediated transformation in fetal and adult blood progenitors and reveal that whereas adult pre-leukemic cells are mainly characterized by retained myeloid features and downregulation of ribosomal and metabolic proteins, expression of MLL::ENL in fetal LMPPs leads to enrichment of translation-associated and histone deacetylases signaling proteins, and decreased expression of inflammation and myeloid differentiation proteins. Integrating the proteome of pre-leukemic cells with their secretome and the proteomic composition of the extracellular environment of normal progenitors highlights differential regulation of Igf2 bioavailability, as well as of VLA-4 dimer and its ligandome, upon initiation of fetal- and adult-origin leukemia, with implications for human MLLr leukemia cells' ability to communicate with their environment through granule proteins. Our study has uncovered opportunities for targeting ontogeny-specific proteomic vulnerabilities in in utero-initiated and adult-onset MLLr leukemia.