Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-B, specificity protein (SP)1-like and GATA-4 binding sites

Emily A. McDonald, Jacqueline E. Smith, Rebecca A. Cederberg, Brett R White

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Background: Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control). Methods: Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA). Results: Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (-1690) within a previously identified upstream enhancer (-1779/-1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-ΚB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-ΚB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-ΚB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P < 0.05). Conclusions: Elevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (-1235), and GATA-4 (-845).

Original languageEnglish (US)
Article number36
JournalReproductive Biology and Endocrinology
Volume14
Issue number1
DOIs
StatePublished - Jun 29 2016

Fingerprint

LHRH Receptors
Genetic Promoter Regions
Gonadotropin-Releasing Hormone
Swine
Binding Sites
Luciferases
Genes
Proteins
Ovulation
Transfection
GATA Transcription Factors
Mutation
Anterior Pituitary Gland
Sequence Deletion
Electrophoretic Mobility Shift Assay
Genetic Markers
Single Nucleotide Polymorphism
Reproduction
Transcription Factors

Keywords

  • Anterior pituitary
  • GATA-4
  • GnRH receptor
  • Gonadotrope
  • NF-ΚB
  • Ovulation rate
  • Porcine
  • SP1-like factor
  • Single nucleotide polymorphism
  • Transcriptional regulation

ASJC Scopus subject areas

  • Reproductive Medicine
  • Endocrinology
  • Obstetrics and Gynecology
  • Developmental Biology

Cite this

@article{a11d8c5cb76145b181d6cc9895409163,
title = "Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-B, specificity protein (SP)1-like and GATA-4 binding sites",
abstract = "Background: Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control). Methods: Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA). Results: Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (-1690) within a previously identified upstream enhancer (-1779/-1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-ΚB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-ΚB binding sites within the context of their native promoters resulted in a 50 and 60 {\%} reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-ΚB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 {\%}, respectively, compared to native sequence (P < 0.05). Conclusions: Elevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (-1235), and GATA-4 (-845).",
keywords = "Anterior pituitary, GATA-4, GnRH receptor, Gonadotrope, NF-ΚB, Ovulation rate, Porcine, SP1-like factor, Single nucleotide polymorphism, Transcriptional regulation",
author = "McDonald, {Emily A.} and Smith, {Jacqueline E.} and Cederberg, {Rebecca A.} and White, {Brett R}",
year = "2016",
month = "6",
day = "29",
doi = "10.1186/s12958-016-0170-0",
language = "English (US)",
volume = "14",
journal = "Reproductive Biology and Endocrinology",
issn = "1477-7827",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-B, specificity protein (SP)1-like and GATA-4 binding sites

AU - McDonald, Emily A.

AU - Smith, Jacqueline E.

AU - Cederberg, Rebecca A.

AU - White, Brett R

PY - 2016/6/29

Y1 - 2016/6/29

N2 - Background: Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control). Methods: Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA). Results: Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (-1690) within a previously identified upstream enhancer (-1779/-1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-ΚB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-ΚB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-ΚB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P < 0.05). Conclusions: Elevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (-1235), and GATA-4 (-845).

AB - Background: Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control). Methods: Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA). Results: Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (-1690) within a previously identified upstream enhancer (-1779/-1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-ΚB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-ΚB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-ΚB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P < 0.05). Conclusions: Elevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (-1235), and GATA-4 (-845).

KW - Anterior pituitary

KW - GATA-4

KW - GnRH receptor

KW - Gonadotrope

KW - NF-ΚB

KW - Ovulation rate

KW - Porcine

KW - SP1-like factor

KW - Single nucleotide polymorphism

KW - Transcriptional regulation

UR - http://www.scopus.com/inward/record.url?scp=84976478570&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84976478570&partnerID=8YFLogxK

U2 - 10.1186/s12958-016-0170-0

DO - 10.1186/s12958-016-0170-0

M3 - Article

VL - 14

JO - Reproductive Biology and Endocrinology

JF - Reproductive Biology and Endocrinology

SN - 1477-7827

IS - 1

M1 - 36

ER -