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Identification, Cloning, and Functional Analysis of the TATA-Less Mouse FNDC5 Promoter During Neural Differentiation

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Abstract

FNDC5 (also termed PEP) gene encodes a type I membrane protein which is cleaved and secreted as Irisin hormone. We have identified mouse putative core promoter of FNDC5 and characterized its activity. FNDC5 is located within mouse chromosome 4, spans about 7,534 bp, and consists of 6 exons. The mouse FNDC5 promoter is TATA-less and lacks a consensus initiator sequence. In silico analyses revealed that the core promoter (−561/+101 with respect to translation start site) is located in a GC-rich domain (approximately 70.01 %) with one CpG island as a promoter index and several GC box factors including GC/SP1 which is necessary for transcription of TATA-less promoters. The core promoter showed a lower activity than CMV promoter in CHO and P19 cell lines when located upstream of EGFP CDS in an appropriate expression vector. Data implicated that both exon 1 and intron 1 of the gene are included in the core promoter. Upon treating with retinoic acid, FNDC5 expression was upregulated during embryoid body formation and decreased slowly at final stage of neural differentiation when neurospheres emerged. However, Noggin induction induced up regulation of FNDC5 expression at final stage of neural differentiation. In conclusion, stage dependent expression of FNDC5 is affected by neural induction method used for neural differentiation.

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Abbreviations

aa:

Amino acid

BMP:

Bone morphogenetic protein

DMEM:

Dulbecco’s modified Eagle medium

EGFP:

Enhanced green fluorescent protein

ES-FCS:

Embryonic stem cells qualified-FCS

FCS:

Fetal calf serum

FNDC5:

Fibronectin type III domain containing 5 protein

FNDC5 :

Fibronectin type III domain containing 5 encoding gene

FSC/SSC:

Forward and side light scatter

FRCP2:

Fibronectin type III repeat containing protein 2

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GFP:

Green fluorescent protein

K-DMEM:

Knock out- DMEM

LIF:

Leukemia inhibitory factor

MARRS:

Membrane associated rapid response steroid binding

mESC:

Mouse embryonic stem cell

PEP :

Peroxisomal protein encoding gene

PEP:

Peroxisomal protein

PS:

Penicillin-streptomycin

Q-PCR:

Quantitative real time PCR

RA:

Retinoic acid

SEM:

Standard error of mean

TF:

Transcription factors

TSS:

Translation start site

UTR:

Untranslated region

VDR-RXR:

Vitamin D receptor/retinoic acid X receptor

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Acknowledgments

This study was funded by a Grant-in-aid of research from Royan Institute awarded to Kamran Ghaedi, Ph.D. as the Principal Investigator, and in support of Tahere Seifi for obtaining her M.Sc. degree from the University of Isfahan.

Conflict of interest

None of the authors has any conflict of interest to disclose, and all authors support submission to this journal.

Ethical approval statement

Approval for this study was obtained from the Institutional Review Board of Royan Institute (Tehran, Iran).

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Author notes

  1. Tahere Seifi

    Present address: Department of Biology, Payame Noor University, P.O. Box 19395-4697, Tehran, Iran

Authors and Affiliations

  1. Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran

    Tahere Seifi & Kamran Ghaedi

  2. Department of Cellular Biotechnology at Cell Science Research Center, ACECR, Royan Institute for Biotechnology, 816513-1378, Isfahan, Iran

    Kamran Ghaedi, Sommayeh Tanhaei, Fereshteh Karamali, Abbas Kiani-Esfahani, Maryam Peymani & Mohammad-Hossein Nasr-Esfahani

  3. Department of Stem Cells and Developmental Biology at Cell Science Research Center, ACECR, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran

    Hossein Baharvand

  4. Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran

    Hossein Baharvand

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  1. Tahere Seifi
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Correspondence to Kamran Ghaedi or Mohammad-Hossein Nasr-Esfahani.

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Seifi, T., Ghaedi, K., Tanhaei, S. et al. Identification, Cloning, and Functional Analysis of the TATA-Less Mouse FNDC5 Promoter During Neural Differentiation. Cell Mol Neurobiol 34, 715–725 (2014). https://doi.org/10.1007/s10571-014-0053-x

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  • DOI: https://doi.org/10.1007/s10571-014-0053-x

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