Nuclear matrix association of the human beta-globin locus utilizing a novel approach to quantitative real-time PCR.
The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.
Ostermeier
G Charles
GC
Department of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA.
Liu
Zhandong
Z
Martins
Rui Pires
RP
Bharadwaj
Rikki R
RR
Ellis
James
J
Draghici
Sorin
S
Krawetz
Stephen A
SA
D002460:Cell Line; D005914:Globins / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D019901:Locus Control Region; D008957:Models, Genetic; D015530:Nuclear Matrix / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid; D012984:Software; D013997:Time Factors
D005914:Globins