Limits...
Cooperation between NRF-2 and YY-1 transcription factors is essential for triggering the expression of the PREPL-C2ORF34 bidirectional gene pair.

Huang CC, Chang WS - BMC Mol. Biol. (2009)

Bottom Line: Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner.The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair.This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan. james@nhri.org.tw

ABSTRACT

Background: Many mammalian genes are organized as bidirectional (head-to-head) gene pairs with the two genes separated only by less than 1 kb. The transcriptional regulation of these bidirectional gene pairs remains largely unclear, but a few studies have suggested that the two closely adjacent genes in divergent orientation can be co-regulated by a single transcription factor binding to a specific regulatory fragment. Here we report an evolutionarily conserved bidirectional gene pair, known as the PREPL-C2ORF34 gene pair, whose transcription relies on the synergic cooperation of two transcription factors binding to an intergenic bidirectional minimal promoter.

Results: While PREPL is present primarily in brain and heart, C2ORF34 is ubiquitously and abundantly expressed in almost all tissues. Genomic analyses revealed that these two non-homologous genes are adjacent in a head-to-head configuration on human chromosome 2p21 and separated by only 405 bp. Within this short intergenic region, a 243-bp GC-rich segment was demonstrated to function as a bidirectional minimal promoter to initiate the transcription of both flanking genes. Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner. The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair.

Conclusion: This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region.

Show MeSH
Determination of the promoter activity of the intergenic region. The directions of gene transcription are indicated by arrows. Here, a 1053-bp fragment (from position -718 to +335) consisting of the entire 405-bp intergenic region and ~300 bp beyond its two boundaries is cloned as the full-length promoter construct. Progressive deletions from this full-length promoter construct were produced and inserted into the promoterless vector in either the sense (relative to PREPL) or antisense (relative to C2ORF34) direction. The resulting luciferase activity of transiently transfected U87MG cells is given adjacent to each construct. Co-transfection of pRL-TK plasmid was performed for normalization of transfection efficiencies and cell viability. Units are relative to SV40 control promoter, defined as 1×. The experiments were performed in triplicate. Data are representative of three independent experiments, and error bars are ± standard deviation. A similar result was also observed in human H4 cells (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2713978&req=5

Figure 3: Determination of the promoter activity of the intergenic region. The directions of gene transcription are indicated by arrows. Here, a 1053-bp fragment (from position -718 to +335) consisting of the entire 405-bp intergenic region and ~300 bp beyond its two boundaries is cloned as the full-length promoter construct. Progressive deletions from this full-length promoter construct were produced and inserted into the promoterless vector in either the sense (relative to PREPL) or antisense (relative to C2ORF34) direction. The resulting luciferase activity of transiently transfected U87MG cells is given adjacent to each construct. Co-transfection of pRL-TK plasmid was performed for normalization of transfection efficiencies and cell viability. Units are relative to SV40 control promoter, defined as 1×. The experiments were performed in triplicate. Data are representative of three independent experiments, and error bars are ± standard deviation. A similar result was also observed in human H4 cells (data not shown).

Mentions: Following characterization of tissue distribution patterns, we attempted to pinpoint the precise transcriptional start sites (TSSs) of the C2ORF34 and PREPL genes to ascertain the length of their intergenic spacer. We first utilized exon-specific primers to amplify genomic DNA fragments encompassing the first exons of both genes. The PCR products were then purified and sequenced to confirm that these two genes are indeed arranged in a head-to-head configuration on human chromosome 2p21 (Figure 2). By performing 5'-RACE with fetal brain-derived cDNA as a template, both genes were identified to contain multiple TSSs including two for C2ORF34 and three for PREPL (Figure 2B). It should be noted that, during the course of 5'-RACE analysis, we unexpectedly detected a PREPL splice variant which uses an alternative, 184-bp untranslated exon 1 (denoted as exon 1a, see Figures 2, Figure 3, and Additional File 1). With this newly found 5'-untranslated exon, the shortest intergenic distance between the closest TSSs for PREPL and C2ORF34 was calculated to be 405 bp (Figure 2). In order to facilitate our subsequent analyses of the promoter activity of this 405-bp intergenic region, here we denoted the positions of nucleotides with respect to -1, the first intergenic nucleotide in front of the PREPL transcriptional start site, and -405, the intergenic nucleotide right before the transcriptional start site of C2ORF34 (see Figure 2).


Cooperation between NRF-2 and YY-1 transcription factors is essential for triggering the expression of the PREPL-C2ORF34 bidirectional gene pair.

Huang CC, Chang WS - BMC Mol. Biol. (2009)

Determination of the promoter activity of the intergenic region. The directions of gene transcription are indicated by arrows. Here, a 1053-bp fragment (from position -718 to +335) consisting of the entire 405-bp intergenic region and ~300 bp beyond its two boundaries is cloned as the full-length promoter construct. Progressive deletions from this full-length promoter construct were produced and inserted into the promoterless vector in either the sense (relative to PREPL) or antisense (relative to C2ORF34) direction. The resulting luciferase activity of transiently transfected U87MG cells is given adjacent to each construct. Co-transfection of pRL-TK plasmid was performed for normalization of transfection efficiencies and cell viability. Units are relative to SV40 control promoter, defined as 1×. The experiments were performed in triplicate. Data are representative of three independent experiments, and error bars are ± standard deviation. A similar result was also observed in human H4 cells (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713978&req=5

Figure 3: Determination of the promoter activity of the intergenic region. The directions of gene transcription are indicated by arrows. Here, a 1053-bp fragment (from position -718 to +335) consisting of the entire 405-bp intergenic region and ~300 bp beyond its two boundaries is cloned as the full-length promoter construct. Progressive deletions from this full-length promoter construct were produced and inserted into the promoterless vector in either the sense (relative to PREPL) or antisense (relative to C2ORF34) direction. The resulting luciferase activity of transiently transfected U87MG cells is given adjacent to each construct. Co-transfection of pRL-TK plasmid was performed for normalization of transfection efficiencies and cell viability. Units are relative to SV40 control promoter, defined as 1×. The experiments were performed in triplicate. Data are representative of three independent experiments, and error bars are ± standard deviation. A similar result was also observed in human H4 cells (data not shown).
Mentions: Following characterization of tissue distribution patterns, we attempted to pinpoint the precise transcriptional start sites (TSSs) of the C2ORF34 and PREPL genes to ascertain the length of their intergenic spacer. We first utilized exon-specific primers to amplify genomic DNA fragments encompassing the first exons of both genes. The PCR products were then purified and sequenced to confirm that these two genes are indeed arranged in a head-to-head configuration on human chromosome 2p21 (Figure 2). By performing 5'-RACE with fetal brain-derived cDNA as a template, both genes were identified to contain multiple TSSs including two for C2ORF34 and three for PREPL (Figure 2B). It should be noted that, during the course of 5'-RACE analysis, we unexpectedly detected a PREPL splice variant which uses an alternative, 184-bp untranslated exon 1 (denoted as exon 1a, see Figures 2, Figure 3, and Additional File 1). With this newly found 5'-untranslated exon, the shortest intergenic distance between the closest TSSs for PREPL and C2ORF34 was calculated to be 405 bp (Figure 2). In order to facilitate our subsequent analyses of the promoter activity of this 405-bp intergenic region, here we denoted the positions of nucleotides with respect to -1, the first intergenic nucleotide in front of the PREPL transcriptional start site, and -405, the intergenic nucleotide right before the transcriptional start site of C2ORF34 (see Figure 2).

Bottom Line: Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner.The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair.This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan. james@nhri.org.tw

ABSTRACT

Background: Many mammalian genes are organized as bidirectional (head-to-head) gene pairs with the two genes separated only by less than 1 kb. The transcriptional regulation of these bidirectional gene pairs remains largely unclear, but a few studies have suggested that the two closely adjacent genes in divergent orientation can be co-regulated by a single transcription factor binding to a specific regulatory fragment. Here we report an evolutionarily conserved bidirectional gene pair, known as the PREPL-C2ORF34 gene pair, whose transcription relies on the synergic cooperation of two transcription factors binding to an intergenic bidirectional minimal promoter.

Results: While PREPL is present primarily in brain and heart, C2ORF34 is ubiquitously and abundantly expressed in almost all tissues. Genomic analyses revealed that these two non-homologous genes are adjacent in a head-to-head configuration on human chromosome 2p21 and separated by only 405 bp. Within this short intergenic region, a 243-bp GC-rich segment was demonstrated to function as a bidirectional minimal promoter to initiate the transcription of both flanking genes. Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner. The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair.

Conclusion: This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region.

Show MeSH