GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation

Aysha B. Khalid, Alexandria V. Slayden, Jerusha Kumpati, Chanel D. Perry, Stuart B. Berryhill, Julie A. Crawford, Iram Fatima, Marco Morselli, Matteo Pellegrini, Gustavo Miranda-Carboni, Susan Miranda

Research output: Contribution to journalArticle

Abstract

GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.

Original languageEnglish (US)
Pages (from-to)78-86
Number of pages9
JournalBone
Volume116
DOIs
StatePublished - Nov 1 2018

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Osteoclasts
Osteoblasts
Osteocalcin
Tibia
Histones
Lysine
X-Ray Microtomography
Chromatin Immunoprecipitation
Nucleic Acid Regulatory Sequences
Bone Development
Bone Marrow Cells
Femur
Chromatin
Transcription Factors
Staining and Labeling
Gene Expression
Bone and Bones
Messenger RNA
Genes
Cancellous Bone

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Physiology
  • Histology

Cite this

Khalid, A. B., Slayden, A. V., Kumpati, J., Perry, C. D., Berryhill, S. B., Crawford, J. A., ... Miranda, S. (2018). GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation. Bone, 116, 78-86. https://doi.org/10.1016/j.bone.2018.07.014

GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation. / Khalid, Aysha B.; Slayden, Alexandria V.; Kumpati, Jerusha; Perry, Chanel D.; Berryhill, Stuart B.; Crawford, Julie A.; Fatima, Iram; Morselli, Marco; Pellegrini, Matteo; Miranda-Carboni, Gustavo; Miranda, Susan.

In: Bone, Vol. 116, 01.11.2018, p. 78-86.

Research output: Contribution to journalArticle

Khalid, AB, Slayden, AV, Kumpati, J, Perry, CD, Berryhill, SB, Crawford, JA, Fatima, I, Morselli, M, Pellegrini, M, Miranda-Carboni, G & Miranda, S 2018, 'GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation', Bone, vol. 116, pp. 78-86. https://doi.org/10.1016/j.bone.2018.07.014
Khalid, Aysha B. ; Slayden, Alexandria V. ; Kumpati, Jerusha ; Perry, Chanel D. ; Berryhill, Stuart B. ; Crawford, Julie A. ; Fatima, Iram ; Morselli, Marco ; Pellegrini, Matteo ; Miranda-Carboni, Gustavo ; Miranda, Susan. / GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation. In: Bone. 2018 ; Vol. 116. pp. 78-86.
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abstract = "GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.",
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AU - Khalid, Aysha B.

AU - Slayden, Alexandria V.

AU - Kumpati, Jerusha

AU - Perry, Chanel D.

AU - Berryhill, Stuart B.

AU - Crawford, Julie A.

AU - Fatima, Iram

AU - Morselli, Marco

AU - Pellegrini, Matteo

AU - Miranda-Carboni, Gustavo

AU - Miranda, Susan

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N2 - GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.

AB - GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.

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