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 6

ChIP assays show occupancy of the GCMB promoter by GATA3 in parathyroid tumor cells.

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ChIP assays show occupancy of the GCMB promoter by GATA3 in parathyroid ...
(A) Chromatin prepared from parathyroid tumor cells using 3 chromatin sonication conditions. Sonication with 5–10 pulses produced the optimum DNA fragment size range (200–1000 bp) for ChIP reactions. (B) Western blot analysis of the sonicated chromatin using 2 different mouse monoclonal antibodies (HG3-31 and HG3-35) revealed the presence of the intact 50-kDa GATA3 protein. Detection of the intact 70/75-kDa lamin A/C nuclear protein was used as a control. (C) Analysis of chromatin immunoprecipitated with GATA3 antibodies, HG3-31 and HG3-35, and 2 isotype-matched control antibodies, RNApolII and IgG. Purified DNA fragments after ChIP reactions were amplified by PCR with 2 primer pairs specific for the GCMB and GAPDH promoter regions. GAPDH served as a positive control for RNApolII and a negative control for GATA3 antibodies. (D) Quantification of PCR products by SYBR Green quantitative PCR using primers specific for the GCMB and GAPDH promoter regions. The GATA3-independent housekeeping gene GAPDH served as positive control for the RNApolII antibody ChIP, which demonstrated a 7-fold enrichment over IgG ChIP (P < 0.01). (E) Quantification of PCR products by SYBR Green quantitative PCR using primers specific for the GCMB promoter region. This confirmed the enrichment of the GCMB promoter DNA fragments immunoprecipitated with GATA3 antibodies over those immunoprecipitated with IgG and showed a 6.5- and 7.5-fold increase with HG2-31 and HG3-35 antibodies, respectively (*P < 0.01). Results are shown as the mean ± SEM of 3 independent ChIP reactions.