RESUMO
BACKGROUND: Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. RESULTS: To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. CONCLUSIONS: We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Expressão Gênica , Humanos , Células K562 , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas/antagonistas & inibidores , RNA Polimerase II/genética , Sítio de Iniciação de TranscriçãoRESUMO
TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial-mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation and EMT, we found TWIST1 to be upregulated during EMT and downregulated early in carcinogenesis. The TWIST1 3'UTR contains putative regulatory elements, including miRNA target sites and two cytoplasmic polyadenylation elements (CPE). We found that miR-580, CPEB1, and CPEB2 act as negative regulators of TWIST1 expression in a sequence-specific and additive/cooperative manner.
