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Epigenetic regulation of BMP2 gene in osteoporosis: a DNA methylation study

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Abstract

Osteoporosis is a multifactorial disease in which genetic factors and epigenetic modifications play a major role. DNA methylation is known for gene silencing and its effect on BMP2 promoter has been studied here to understand its regulatory activity in osteoporosis pathogenicity. CpG methylation in the BMP2 promoter was analyzed by performing bisulfite specific PCR on the gDNA samples extracted from whole blood of osteoporotic (n = 24) and healthy (n = 24) individuals. Disproportionate allele frequency of CpG sites was calculated statistically. Differential BMP2 expression was estimated using quantitative RT-PCR technique. Luciferase reporter assay was performed to determine and confirm differential transcriptional activity of BMP2 promoter due to methylation. Total of 14 CpG sites were reporter in the BMP2 promoter of which, CpG site at − 267th position upstream to TSS was found to have disproportionate allele frequency among osteoporotic and healthy individuals and was found to be significantly associated with osteoporosis condition. Functional and gene expression analysis of this methylated site using luciferase reporter vector and Real Time PCR approach, suggested reduced transcriptional activity of BMP2 promoter as well as decreased gene expression in disease condition. BMP2 is being a central signaling molecule, aberrant methylation in the promoter region may result into down regulation of osteoblast markers involved in bone formation.

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Abbreviations

ALP:

Alkaline phosphatase

BMD:

Bone mineral density

BMP2:

Bone morphogenetic protein 2

BMP2K:

BMP2 inducible kinase

BMP4:

Bone morphogenetic protein 4

BMP6:

Bone morphogenetic protein 6

BMP7:

Bone morphogenetic protein 7

cDNA:

Complementary DNA

CI:

Class interval

CpG:

5′—C—Phosphate—G—3

Dlx5:

Distal-less homeobox 5

DNA:

Deoxyribonucleic acid

DXA:

Dual X-Ray absorptiometry

ECACC:

European Collection Of Authenticated Cell Cultures

EDTA:

Ethylenediaminetetraacetic acid

E-MEM:

Eagle’s Minimum Essential Medium

EMP2:

Epithelial membrane protein 2

ERKs:

Extracellular signal-regulated kinases

FC:

Fold change

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

gDNA:

Genomic DNA

ICMR:

Indian Council of Medical Research

ID:

Identity

LA:

Luria agar

LB:

Luria Broth

LD:

Linkage disequilibrium

mRNA:

Messenger RNA

MSA:

Multiple sequence alignment

NCBI:

National Center for Biotechnology Information

OSX:

Osterix

PBMC:

Peripheral blood mononuclear cell

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

qRT-PCR:

Quantitative real time polymerase chain reaction

RefSeq:

Reference sequence database

RLU:

Relative luciferase activity

RNA:

Ribonucleic acid

RR:

Risk ratio

RT-PCR:

Reverse transcriptase polymerase chain reaction

RUNX2:

Runt related transcription factor 2

SD:

Standard deviation

SNP:

Single nucleotide polymorphisms

TGF-Beta:

Activated kinase 1

TF:

Transcription factor

TSS:

Transcription start site

WHO:

World Health Organization

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Authors and Affiliations

  1. Center for Advanced Studies, Department of Zoology, Savitribai Phule Pune University, Pune, 411007, India

    Mehrunnisa M. Raje & Richa Ashma

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  1. Mehrunnisa M. Raje
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  2. Richa Ashma
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Correspondence to Richa Ashma.

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Raje, M.M., Ashma, R. Epigenetic regulation of BMP2 gene in osteoporosis: a DNA methylation study. Mol Biol Rep 46, 1667–1674 (2019). https://doi.org/10.1007/s11033-019-04615-y

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