A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein α required for prolactin gene transcription

John F. Enwright, Margaret A. Kawecki-Crook, Ty C. Voss, Fred Schaufele, Richard Day

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein α (C/EBPα), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPα with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPα induced PRL gene transcription in pituitary GHFT1-5 cells, whereas the coexpression of Pit-1 and C/EBPα in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPα fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPα from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPα. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPα. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPα, was itself recruited by C/EBPα to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.

Original languageEnglish (US)
Pages (from-to)209-222
Number of pages14
JournalMolecular Endocrinology
Volume17
Issue number2
DOIs
StatePublished - Feb 1 2003
Externally publishedYes

Fingerprint

CCAAT-Enhancer-Binding Proteins
Prolactin
Genes
Heterochromatin
Homeodomain Proteins
HeLa Cells
Point Mutation
Transcriptional Activation
Transcription Factors
Color

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein α required for prolactin gene transcription. / Enwright, John F.; Kawecki-Crook, Margaret A.; Voss, Ty C.; Schaufele, Fred; Day, Richard.

In: Molecular Endocrinology, Vol. 17, No. 2, 01.02.2003, p. 209-222.

Research output: Contribution to journalArticle

Enwright, John F. ; Kawecki-Crook, Margaret A. ; Voss, Ty C. ; Schaufele, Fred ; Day, Richard. / A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein α required for prolactin gene transcription. In: Molecular Endocrinology. 2003 ; Vol. 17, No. 2. pp. 209-222.
@article{d35145ab8fd54361a96097aee8bdaee7,
title = "A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein α required for prolactin gene transcription",
abstract = "The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein α (C/EBPα), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPα with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPα induced PRL gene transcription in pituitary GHFT1-5 cells, whereas the coexpression of Pit-1 and C/EBPα in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPα fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPα from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPα. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPα. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPα, was itself recruited by C/EBPα to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.",
author = "Enwright, {John F.} and Kawecki-Crook, {Margaret A.} and Voss, {Ty C.} and Fred Schaufele and Richard Day",
year = "2003",
month = "2",
day = "1",
doi = "10.1210/me.2001-0222",
language = "English (US)",
volume = "17",
pages = "209--222",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "2",

}

TY - JOUR

T1 - A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein α required for prolactin gene transcription

AU - Enwright, John F.

AU - Kawecki-Crook, Margaret A.

AU - Voss, Ty C.

AU - Schaufele, Fred

AU - Day, Richard

PY - 2003/2/1

Y1 - 2003/2/1

N2 - The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein α (C/EBPα), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPα with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPα induced PRL gene transcription in pituitary GHFT1-5 cells, whereas the coexpression of Pit-1 and C/EBPα in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPα fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPα from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPα. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPα. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPα, was itself recruited by C/EBPα to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.

AB - The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein α (C/EBPα), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPα with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPα induced PRL gene transcription in pituitary GHFT1-5 cells, whereas the coexpression of Pit-1 and C/EBPα in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPα fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPα from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPα. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPα. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPα, was itself recruited by C/EBPα to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.

UR - http://www.scopus.com/inward/record.url?scp=0037317399&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037317399&partnerID=8YFLogxK

U2 - 10.1210/me.2001-0222

DO - 10.1210/me.2001-0222

M3 - Article

C2 - 12554749

AN - SCOPUS:0037317399

VL - 17

SP - 209

EP - 222

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 2

ER -