Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

Manjula Karpurapu,Ravi Ranjan,Gye Young Park,Sangwoon Chung,John W Christman,Teja Srinivas Nirujogi,Jing Deng,Yong Gyu Lee,Jeffrey R Jacobson,Lei Xiao,Chi Zhang

doi:10.1371/journal.pone.0093362

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.

Highlights

Myeloid cell differentiation is controlled by a complex circuitry of lineage specific transcription factors and the role of the Ets family transcription factor PU.1 in myeloid lineage specification is well documented [1,2]
PU.1 binds to Kruppel like factor 4 (KLF4) promoter We used PU/ER(T) cell line generated from fetal liver cells of PU.1-/- mice that shows conditional PU.1 dependent macrophage differentiation [30,34]
5) Activation Induced Cytidine Deaminase (AICDA) interacts with GADD45a and Thymine DNA Glycosylase (TDG) but GADD45a was found to be dispensable for KLF4 promoter demethylation. 6) shRNA knock down of AICDA blocked demethylation of KLF4 promoter, F4/80 expression and macrophage differentiation

Summary

Introduction

Myeloid cell differentiation is controlled by a complex circuitry of lineage specific transcription factors and the role of the Ets family transcription factor PU. in myeloid lineage specification is well documented [1,2]. PU. expression levels critically determine the specification of myeloid and common lymphoid progenitors [3] and knock down of PU. in mice results in defective development of macrophages and granulocytes [4,5]. X-ray crystal studies of KLF4 protein revealed that the deletion of the two C-terminal zinc fingers lead to deficiency of KLF4 expression resulting in macrophage self renewal and defective differentiation [12]. Recent genome wide studies on epigenetic and transcription factor profiling in different cell types correlated the lineage specific transcription factor binding with changes in epigenetic marks like histone acetylation/methylation and DNA methylation at the target gene promoters that potentially affect the cell fate

Objectives

Methods

Results

Conclusion