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Promoter-associated proteins of EPAS1 identified by enChIP-MS – A putative role of HDX as a negative regulator

Hamidian, Arash LU ; Vaapil, Marica LU ; von Stedingk, Kristoffer LU ; Fujita, Toshitsugu; Persson, Camilla U. LU ; Eriksson, Pontus LU ; Veerla, Srinivas LU ; De Preter, Katleen; Speleman, Frank and Fujii, Hodaka, et al. (2018) In Biochemical and Biophysical Research Communications 499(2). p.291-298
Abstract

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) – mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and... (More)

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) – mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.

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Contribution to journal
publication status
published
subject
keywords
enChIP, HDX, HIF-2α, Hypoxia, MYCN, Neuroblastoma
in
Biochemical and Biophysical Research Communications
volume
499
issue
2
pages
8 pages
publisher
Elsevier
external identifiers
  • scopus:85044329057
ISSN
0006-291X
DOI
10.1016/j.bbrc.2018.03.150
language
English
LU publication?
yes
id
29bda5f4-e7fc-4397-b745-b1151f59c03f
date added to LUP
2018-04-09 15:14:57
date last changed
2018-07-10 03:00:23
@article{29bda5f4-e7fc-4397-b745-b1151f59c03f,
  abstract     = {<p>Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) – mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.</p>},
  author       = {Hamidian, Arash and Vaapil, Marica and von Stedingk, Kristoffer and Fujita, Toshitsugu and Persson, Camilla U. and Eriksson, Pontus and Veerla, Srinivas and De Preter, Katleen and Speleman, Frank and Fujii, Hodaka and Påhlman, Sven and Mohlin, Sofie},
  issn         = {0006-291X},
  keyword      = {enChIP,HDX,HIF-2α,Hypoxia,MYCN,Neuroblastoma},
  language     = {eng},
  month        = {05},
  number       = {2},
  pages        = {291--298},
  publisher    = {Elsevier},
  series       = {Biochemical and Biophysical Research Communications},
  title        = {Promoter-associated proteins of EPAS1 identified by enChIP-MS – A putative role of HDX as a negative regulator},
  url          = {http://dx.doi.org/10.1016/j.bbrc.2018.03.150},
  volume       = {499},
  year         = {2018},
}