Limits...
Identification and Characterization of LHX8 DNA Binding Elements.

Park M, Jeon S, Jeong JH, Park M, Lee DR, Yoon TK, Choi DH, Choi Y - (2012)

Bottom Line: We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE).In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element.These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biomedical Science, CHA University, Seoul 135-081, Korea.

ABSTRACT
Lhx8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian folliculogenesis. However, Lhx8 DNA binding sequences are not characterized yet. We aimed to identify and characterize a cis-acting sequence of germ-cell specific transcriptional factor, Lhx8. To identify Lhx8 DNA binding element, Cyclic Amplification of Sequence Target (CAST) Analysis was performed. Electrophoretic Mobility Shift Assay (EMSA) was processed for the binding specificity of Lhx8. Luciferase assay was for the transcriptional activity of Lhx8 through identified DNA binding site. We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE). In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element. These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

No MeSH data available.


Transactivation of reporter genes through the LBE. The pCMV Tag5 or pCMV Tag5-Lhx8 was co-transfected with 3xLBE-pGL4 luciferase reporter vector into HEK293 cells. Cell extracts were collected after 48h of transfection and analyzed for luciferase activity. The black box indicates the artificial 3xLBE (TGATTG). (* P<0.01)
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Figure 4: Transactivation of reporter genes through the LBE. The pCMV Tag5 or pCMV Tag5-Lhx8 was co-transfected with 3xLBE-pGL4 luciferase reporter vector into HEK293 cells. Cell extracts were collected after 48h of transfection and analyzed for luciferase activity. The black box indicates the artificial 3xLBE (TGATTG). (* P<0.01)

Mentions: To examine whether Lhx8 can regulate the expression of LBE driven reporter gene in vivo, we constructed a luciferase reporter vector containing three LBEs (TGATTG), 3xLBE-pGL4 upstream of the luciferase reporter gene. This reporter vector was co-transfected with either empty vector (pCMV-Tag5) or Lhx8 over-expression vector (pCMV-Tag5) into HEK293 cells. The relative luciferase activity of reporter construct containing three copies of TGATTG was increased by 2.3-fold with the overexpression of Lhx8 (Fig. 4). This suggests that Lhx8 can activate the transcription of target genes through the LBE.


Identification and Characterization of LHX8 DNA Binding Elements.

Park M, Jeon S, Jeong JH, Park M, Lee DR, Yoon TK, Choi DH, Choi Y - (2012)

Transactivation of reporter genes through the LBE. The pCMV Tag5 or pCMV Tag5-Lhx8 was co-transfected with 3xLBE-pGL4 luciferase reporter vector into HEK293 cells. Cell extracts were collected after 48h of transfection and analyzed for luciferase activity. The black box indicates the artificial 3xLBE (TGATTG). (* P<0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282238&req=5

Figure 4: Transactivation of reporter genes through the LBE. The pCMV Tag5 or pCMV Tag5-Lhx8 was co-transfected with 3xLBE-pGL4 luciferase reporter vector into HEK293 cells. Cell extracts were collected after 48h of transfection and analyzed for luciferase activity. The black box indicates the artificial 3xLBE (TGATTG). (* P<0.01)
Mentions: To examine whether Lhx8 can regulate the expression of LBE driven reporter gene in vivo, we constructed a luciferase reporter vector containing three LBEs (TGATTG), 3xLBE-pGL4 upstream of the luciferase reporter gene. This reporter vector was co-transfected with either empty vector (pCMV-Tag5) or Lhx8 over-expression vector (pCMV-Tag5) into HEK293 cells. The relative luciferase activity of reporter construct containing three copies of TGATTG was increased by 2.3-fold with the overexpression of Lhx8 (Fig. 4). This suggests that Lhx8 can activate the transcription of target genes through the LBE.

Bottom Line: We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE).In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element.These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biomedical Science, CHA University, Seoul 135-081, Korea.

ABSTRACT
Lhx8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian folliculogenesis. However, Lhx8 DNA binding sequences are not characterized yet. We aimed to identify and characterize a cis-acting sequence of germ-cell specific transcriptional factor, Lhx8. To identify Lhx8 DNA binding element, Cyclic Amplification of Sequence Target (CAST) Analysis was performed. Electrophoretic Mobility Shift Assay (EMSA) was processed for the binding specificity of Lhx8. Luciferase assay was for the transcriptional activity of Lhx8 through identified DNA binding site. We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE). In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element. These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

No MeSH data available.