Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

Abstract

The role of different lineage specific transcription factors in directing hematopoietic cell fate towards myeloid lineage is well established but the status of epigenetic modifications has not been defined during this important developmental process. We used non proliferating, PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages to study the PU.1 dependent KLF4 transcriptional regulation and its promoter demethylation during monocyte/macrophage differentiation. Expression of KLF4 was regulated by active demethylation of its promoter and PU.1 specifically bound to KLF4 promoter oligo harboring the PU.1 consensus sequence. Methylation specific quantitative PCR and Bisulfite sequencing indicated demethylation of CpG residues most proximal to the transcription start site of KLF4 promoter. Cloned KLF4 promoter in pGL3 Luciferase and CpG free pcpgf-bas vectors showed accentuated reporter activity when co-transfected with the PU.1 expression vector. In vitro methylation of both KLF4 promoter oligo and cloned KLF4 promoter vectors showed attenuated in vitro DNA binding activity and Luciferase/mouse Alkaline phosphotase reporter activity indicating the negative influence of KLF4 promoter methylation on PU.1 binding. The Cytosine deaminase, Activation Induced Cytidine Deaminase (AICDA) was found to be critical for KLF4 promoter demethylation. More importantly, knock down of AICDA resulted in blockade of KLF4 promoter demethylation, decreased F4/80 expression and other phenotypic characters of macrophage differentiation. Our data proves that AICDA mediated active demethylation of the KLF4 promoter is necessary for transcriptional regulation of KLF4 by PU.1 during monocyte/macrophage differentiation.

Activation-induced cytidine deaminase and active cytidine demethylation

“…These observations suggest that AID is involved in the epigenetic changes that establish the EMT transcriptional program. Karpurapu et al reached a similar conclusion, finding that decreased AID expression blocked myeloid progenitor cell maturation, as judged by their morphology, and that AID is essential for the demethylation of Klf4, a gene involved in inflammatory monocyte differentiation [39]. Furthermore, Ratnu et al, investigating primary cultures of post-mitotic cortical neurons, reported that AID is involved in the demethylation of Bdnf, an activity-dependent gene necessary for neuronal survival [40].…”

“…Evidence for a role of AID in genome-wide demethylation is also limited, either because the effect is subtle, as in the PGC study [29], or because the introduction of artificial constructs might not be physiologically relevant, as in the zebrafish study [25] or the GAL4/UAS system [24]. However, AID knockdown led to the loss of neurons in zebrafish [25] and clear cellular and molecular phenotypes in three independent systems [38][39][40], suggesting that AID may be involved in key gene-specific demethylation events linked to differentiation of non-lymphoid cells. These findings beg the question: is AID-dependent demethylation also observed in B cells?…”

“…Knockdowns for AID shRNAs against Aicda RNA block the epithelialmesenchymal transition, which is partially rescued by AID [38] PU/ER(T) cell line AID encoding plasmid was electroporated AID interacts with GADD45a and TDG shRNAs against Aicda RNA block the demethylation of the Klf4 promoter and interfere with macrophage differentiation [39] Primary cortical neuron culture…”

The DNA methylation profile of activated human natural killer cells

“…DNA methylation is thought to provide a chemically stable mark that serves to initiate and reinforce these cell fate decisions. 2 Large-scale methylation studies of specific lineage pathways have been performed on differentiating erythroid, 3,4 T lymphocyte, 5,6 B lymphocyte, 7 and myeloid 4 cells. These important developmental studies reveal insights into the regulation of cell fate decisions but also serve as a potential source of biomarkers that help characterize different immune compartments during the immune response.…”

Epigenetic dynamics in immunity and autoimmunity

“…Furthermore, dynamic changes were also observed in the expression of the TET2, DNMT1, DNMT3A and DNMT3B genes coupled with temporal locus-specific demethylation of SRC, AHRR, CYP1B1 and PPARG, providing possible mechanisms accounting for the loss in DNA methylation (Zhang, Ulm, 2014). In addition, recent findings showed that Activation Induced Cytidine Deaminase (AICDA) mediated active demethylation of the KLF4 promoter, which is necessary for PU.1-mediated transcriptional regulation during monocyte/macrophage differentiation (Karpurapu et al, 2014).…”