Abstract
The response of the human acute myeloid leukemia cell line THP-1 to phorbol esters has been widely studied to test candidate leukemia therapies and as a model of cell cycle arrest and monocyte-macrophage differentiation. Here we have employed Cap Analysis of Gene Expression (CAGE) to analyze a dense time course of transcriptional regulation in THP-1 cells treated with phorbol myristate acetate (PMA) over 96 h. PMA treatment greatly reduced the numbers of cells entering S phase and also blocked cells exiting G2/M. The PMA-treated cells became adherent and expression of mature macrophage-specific genes increased progressively over the duration of the time course. Within 1–2 h PMA induced known targets of tumor protein p53 (TP53), notably CDKN1A, followed by gradual down-regulation of cell-cycle associated genes. Also within the first 2 h, PMA induced immediate early genes including transcription factor genes encoding proteins implicated in macrophage differentiation (EGR2, JUN, MAFB) and down-regulated genes for transcription factors involved in immature myeloid cell proliferation (MYB, IRF8, GFI1). The dense time course revealed that the response to PMA was not linear and progressive. Rather, network-based clustering of the time course data highlighted a sequential cascade of transient up- and down-regulated expression of genes encoding feedback regulators, as well as transcription factors associated with macrophage differentiation and their inferred target genes. CAGE also identified known and candidate novel enhancers expressed in THP-1 cells and many novel inducible genes that currently lack functional annotation and/or had no previously known function in macrophages. The time course is available on the ZENBU platform allowing comparison to FANTOM4 and FANTOM5 data.
Highlights
The KMT2A/MLL gene encodes an epigenetic modifier, histone lysine methyl-transferase 2A (Hess, 2004)
In order to study the process of differentiation in a population in which the majority of cells respond synchronously, the FANTOM4 consortium cloned THP-1 cells obtained from ATCC by limiting dilution and chose one subclone in which >90% of cells became adherent within 48 h of addition of phorbol myristate acetate (PMA) (Suzuki et al, 2009)
Since the hallmark of leukemia is dysregulated proliferation we have examined in detail the coordinated expression of these cell cycle genes in THP-1 cells during PMA-induced growth inhibition
Summary
The KMT2A/MLL gene encodes an epigenetic modifier, histone lysine methyl-transferase 2A (Hess, 2004). Genome-wide analysis of MLL-AF9 binding in THP1 cells revealed a substantial overlap with enhancers bound by RUNX1, a transcription factor that regulates myeloid differentiation and is itself commonly involved in leukemogenic translocations (Prange et al, 2017). These studies identified a novel target of MLL-AF9, the transcription factor ZNF521. The consortium used CAP Analysis of Gene Expression (CAGE) to identify regulated promoters across a time course of differentiation These studies identified a cohort of transcription factor genes rapidly down-regulated following PMA addition. In the macrophage time course, and in several other systems studied (Arner et al, 2015) a transient pulse of eRNA from transcribed enhancers was detected prior to the detection of promoter activity of inducible genes
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