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

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...

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Autores principales:	Manjula Karpurapu, Ravi Ranjan, Jing Deng, Sangwoon Chung, Yong Gyu Lee, Lei Xiao, Teja Srinivas Nirujogi, Jeffrey R Jacobson, Gye Young Park, John W Christman
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2ed2362a0d03425598f8fb4d2394921e
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spelling	oai:doaj.org-article:2ed2362a0d03425598f8fb4d2394921e2021-11-18T08:25:19ZKrüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.1932-620310.1371/journal.pone.0093362https://doaj.org/article/2ed2362a0d03425598f8fb4d2394921e2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24695324/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Manjula KarpurapuRavi RanjanJing DengSangwoon ChungYong Gyu LeeLei XiaoTeja Srinivas NirujogiJeffrey R JacobsonGye Young ParkJohn W ChristmanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 4, p e93362 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manjula Karpurapu Ravi Ranjan Jing Deng Sangwoon Chung Yong Gyu Lee Lei Xiao Teja Srinivas Nirujogi Jeffrey R Jacobson Gye Young Park John W Christman Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
description	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.
format	article
author	Manjula Karpurapu Ravi Ranjan Jing Deng Sangwoon Chung Yong Gyu Lee Lei Xiao Teja Srinivas Nirujogi Jeffrey R Jacobson Gye Young Park John W Christman
author_facet	Manjula Karpurapu Ravi Ranjan Jing Deng Sangwoon Chung Yong Gyu Lee Lei Xiao Teja Srinivas Nirujogi Jeffrey R Jacobson Gye Young Park John W Christman
author_sort	Manjula Karpurapu
title	Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
title_short	Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
title_full	Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
title_fullStr	Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
title_full_unstemmed	Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
title_sort	krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/2ed2362a0d03425598f8fb4d2394921e
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Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

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