Gata3-deficient mice develop parathyroid abnormalities due to dysregulation of the parathyroid-specific transcription factor Gcm2
Irina V. Grigorieva, … , Frank Grosveld, Rajesh V. Thakker
Irina V. Grigorieva, … , Frank Grosveld, Rajesh V. Thakker
Published May 17, 2010
Citation Information: J Clin Invest. 2010;120(6):2144-2155. https://doi.org/10.1172/JCI42021.
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Research Article Endocrinology

Gata3-deficient mice develop parathyroid abnormalities due to dysregulation of the parathyroid-specific transcription factor Gcm2

Abstract

Heterozygous mutations of GATA3, which encodes a dual zinc-finger transcription factor, cause hypoparathyroidism with sensorineural deafness and renal dysplasia. Here, we have investigated the role of GATA3 in parathyroid function by challenging Gata3+/– mice with a diet low in calcium and vitamin D so as to expose any defects in parathyroid function. This led to a higher mortality among Gata3+/– mice compared with Gata3+/+ mice. Compared with their wild-type littermates, Gata3+/– mice had lower plasma concentrations of calcium and parathyroid hormone (PTH) and smaller parathyroid glands with a reduced Ki-67 proliferation rate. At E11.5, Gata3+/– embryos had smaller parathyroid-thymus primordia with fewer cells expressing the parathyroid-specific gene glial cells missing 2 (Gcm2), the homolog of human GCMB. In contrast, E11.5 Gata3–/– embryos had no Gcm2 expression and by E12.5 had gross defects in the third and fourth pharyngeal pouches, including absent parathyroid-thymus primordia. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays showed that GATA3 binds specifically to a functional double-GATA motif within the GCMB promoter. Thus, GATA3 is critical for the differentiation and survival of parathyroid progenitor cells and, with GCM2/B, forms part of a transcriptional cascade in parathyroid development and function.

Authors

Irina V. Grigorieva, Samantha Mirczuk, Katherine U. Gaynor, M. Andrew Nesbit, Elena F. Grigorieva, Qiaozhi Wei, Asif Ali, Rebecca J. Fairclough, Joanna M. Stacey, Michael J. Stechman, Radu Mihai, Dorota Kurek, William D. Fraser, Tertius Hough, Brian G. Condie, Nancy Manley, Frank Grosveld, Rajesh V. Thakker

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Figure 4

Analysis of DNA binding by GATA3 protein to putative GATA motifs in upstream sequence of GCMB.

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Analysis of DNA binding by GATA3 protein to putative GATA motifs in upst...
(A) Diagram showing location of 3 putative GATA sites (A to C) in the 1400-bp sequence upstream of the transcription site (+1) of the GCMB gene. The direction of GATA motifs and their positions on the forward or reverse DNA strands are shown (arrows). Bioinformatic analysis of the 1400-bp sequence predicted only site C as a putative binding site of GATA3 (marked +); sites A and B and motifs i and ii were not predicted by this analysis (marked –). Binding by GATA3 protein to each site or motif assessed by EMSAs (B) is indicated (+++, strong; +, weak; –, absent). (B) EMSAs using nuclear extracts from COS-7 cells, transfected with the GATA3 expression vector. Binding reactions utilized a radiolabeled (32P) double-stranded (ds) oligonucleotide containing putative GATA site (A–C). Control binding reactions using untransfected (UT) cells and probes containing the consensus GATA binding site (+) or GATA motifs (i and ii) in the reverse orientation were performed. Densitometry revealed that more than 90% of ds oligonucleotide C was bound, but less than 10% of the ds oligonucleotides A and B were bound, and 0% of the oligonucleotides i and ii were bound. (C) Use of an anti-GATA3 antibody in the binding reaction (+) with ds oligonucleotide containing GATA site C revealed a supershift, indicating that the complex was specific for the GATA3 protein.