Advertisement

Analysis of Transcriptional Regulation in Bone Cells

  • Huilin JinEmail author
  • Antonia Sophocleous
  • Asim Azfer
  • Stuart H. Ralston
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1914)

Abstract

Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay, five commonly used methods for studying and altering gene transcription.

Key words

Transcription Chromatin Immunoprecipitation Chromosome conformation capture (3C) technology ChIP EMSA CRISPR-Cas9 Promoter Gene 

References

  1. 1.
    Strachan T, Read AP (2011) Human molecular genetics. Garland Science, New YorkGoogle Scholar
  2. 2.
    Sati S, Cavalli G (2017) Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma 126:33–44CrossRefGoogle Scholar
  3. 3.
    Das PM, Ramachandran K, vanWert J, Singal R (2004) Chromatin immunoprecipitation assay. BioTechniques 37:961–969CrossRefGoogle Scholar
  4. 4.
    Banerjee B, Sherwood RI (2017) A CRISPR view of gene regulation. Curr Opin Syst Biol 1:1–8CrossRefGoogle Scholar
  5. 5.
    Mann V, Hobson EE, Li B, Stewart TL, Grant SF, Robins SP, Aspden RM, Ralston SH (2001) A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest 107:899–907CrossRefGoogle Scholar
  6. 6.
    Jin H, van't Hof RJ, Albagha OM, Ralston SH (2009) Promoter and intron 1 polymorphisms of COL1A1 interact to regulate transcription and susceptibility to osteoporosis. Hum Mol Genet 18:2729–2738CrossRefGoogle Scholar
  7. 7.
    Garcia-Giralt N, Enjuanes A, Bustamante M, Mellibovsky L, Nogues X, Carreras R, ez-Perez A, Grinberg D, Balcells S (2005) In vitro functional assay of alleles and haplotypes of two COL1A1-promoter SNPs. Bone 36:902–908CrossRefGoogle Scholar
  8. 8.
    Arai H, Miyamoto KI, Yoshida M, Yamamoto H, Taketani Y, Morita K, Kubota M, Yoshida S, Ikeda M, Watabe F, Kanemasa Y, Takeda E (2001) The polymorphism in the caudal-related homeodomain protein Cdx-2 binding element in the human vitamin D receptor gene. J Bone Miner Res 16:1256–1264CrossRefGoogle Scholar
  9. 9.
    Chen X, Zhu D, Yang M, Hu W, Duan Y, Lu B, Rong Y, Dong S, Hao R, Chen J, Chen Y, Yao S, Thynn HN, Guo Y, Yang T (2018) An osteoporosis risk SNP at 1p36.12 acts as an allele-specific enhancer to modulate LINC00339 expression via long-range loop formation. Am J Hum Genet 102:776–793CrossRefGoogle Scholar
  10. 10.
    Zhu D, Chen X, Hu W, Dong S, Lu B, Rong Y, Chen Y, Chen H, Thynn HN, Wang N, Guo Y, Yang T (2018) Multiple functional variants at 13q14 risk locus for osteoporosis regulate RANKL expression through long-range super-enhancer. J Bone Miner Res. https://doi.org/10.1002/jbmr.3419 [Epub ahead of print]CrossRefGoogle Scholar
  11. 11.
    Xiao SM, Kung AW, Gao Y, Lau KS, Ma A, Zhang ZL, Liu JM, Xia W, He JW, Zhao L, Nie M, Fu WZ, Zhang MJ, Sun J, Kwan JSH, Tso GHW, Dai ZJ, Cheung CL, Bow CH, Leung AYHL, Tan KCB, Sham PC (2012) Post-genome wide association studies and functional analyses identify association of MPP7 gene variants with site-specific bone mineral density. Hum Mol Genet 21:1648–1657CrossRefGoogle Scholar
  12. 12.
    Keupp K, Beleggia F, Kayserili H, Barnes AM, Steiner M, Semler O, Fischer B, Yigit G, Janda CY, Becker J, Breer S, Altunoglu U, Grunhagen J, Krawitz P, Hecht J, Schinke T, Makareeva E, Lausch E, Cankaya T, Caparros-Martın JA, Lapunzina P, Temtamy S, Aglan M, Zabel B, Eysel P, Koerber F, Leikin S, Garcia KC, Netzer C, Schonau E, Ruiz-Perez VL, Mundlos S, Amling M, Kornak U, Marini J, Wollnik B (2013) Mutations in WNT1 cause different forms of bone fragility. Am J Hum Genet 92:565–574CrossRefGoogle Scholar
  13. 13.
    Hakim O, Misteli T (2012) SnapShot: chromosome conformation capture. Cell 148:1068–1068CrossRefGoogle Scholar
  14. 14.
    Didovyk A, Borek B, Tsimring L, Hasty J (2016) Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Curr Opin Biotechnol 40:177–184CrossRefGoogle Scholar
  15. 15.
    Naughton C, MacLeod K, Kuske B, Clarke R, Cameron DA, Langdon SP (2007) Progressive loss of estrogen receptor alpha cofactor recruitment in endocrine resistance. Mol Endocrinol 21:2615–2626CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Huilin Jin
    • 1
    • 2
    Email author
  • Antonia Sophocleous
    • 3
  • Asim Azfer
    • 4
  • Stuart H. Ralston
    • 4
  1. 1.South West Thames Regional Genetics LaboratorySt George’s HospitalLondonUK
  2. 2.Kennedy Institute of RheumatologyLondonUK
  3. 3.Department of Life Sciences, School of SciencesEuropean University CyprusNicosiaCyprus
  4. 4.Rheumatic Diseases Unit, Centre for Genomics and Experimental MedicineMRC IGMM, Western General Hospital, University of EdinburghEdinburghUK

Personalised recommendations