Limits...
Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure.

Banerjee K, Wang M, Cai E, Fujiwara N, Baker H, Cave JW - Nat Commun (2014)

Bottom Line: It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds to C-rich single-stranded DNA within these conserved regions and also associates with double-stranded sequences when proteins, such as CRE-binding protein, are bound to an adjacent cis-regulatory element.The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures.These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, New York 10605, USA.

ABSTRACT
Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds to C-rich single-stranded DNA within these conserved regions and also associates with double-stranded sequences when proteins, such as CRE-binding protein, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype.

No MeSH data available.


hnRNP K interacts with the conserved G:C-rich regions in the Th proximal promoter and is co-expressed with TH in vivo. A, protein pull-down assays with nuclear lysates from mouse Th-expressing cells derived from the mouse olfactory bulb (OB) and substantia nigra (SN). Forward strand of wild-type and mutant oligonucleotide duplexes derived from GC-R1 are shown with mutant positions above the wild-type sequence. Proteins pulled down with either the wild-type or mutant duplex probes were revealed by coomassie stained SDS-polyacrylamide gels. Mass spectrometry identified the differentially isolated protein (indicated by arrow) as hnRNP K. B, transcription assays in human SH-SY5Y cells showed over-expression of hnRNP K could up-regulate Th promoter activity. Luciferase activities are reported as the mean of three independent transfection experiments with error bars representing the standard error of the mean. Pairwise comparisons of luciferase activity levels used the Student’s t-test (single asterisk indicates p <0.01; double asterisk indicates p<0.001). C–R, TH and hnRNP K co-expression in mice and humans. In both the SN (C–F and K–N) and OB (G–J and O–R), most TH-containing cells co-express hnRNP K. Bar equals 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4264680&req=5

Figure 2: hnRNP K interacts with the conserved G:C-rich regions in the Th proximal promoter and is co-expressed with TH in vivo. A, protein pull-down assays with nuclear lysates from mouse Th-expressing cells derived from the mouse olfactory bulb (OB) and substantia nigra (SN). Forward strand of wild-type and mutant oligonucleotide duplexes derived from GC-R1 are shown with mutant positions above the wild-type sequence. Proteins pulled down with either the wild-type or mutant duplex probes were revealed by coomassie stained SDS-polyacrylamide gels. Mass spectrometry identified the differentially isolated protein (indicated by arrow) as hnRNP K. B, transcription assays in human SH-SY5Y cells showed over-expression of hnRNP K could up-regulate Th promoter activity. Luciferase activities are reported as the mean of three independent transfection experiments with error bars representing the standard error of the mean. Pairwise comparisons of luciferase activity levels used the Student’s t-test (single asterisk indicates p <0.01; double asterisk indicates p<0.001). C–R, TH and hnRNP K co-expression in mice and humans. In both the SN (C–F and K–N) and OB (G–J and O–R), most TH-containing cells co-express hnRNP K. Bar equals 50 μm.

Mentions: To identify proteins that bind to the G:C-rich regions, pull-down experiments were performed using biotinylated double-stranded oligonucleotides containing either the wild-type or mutated sequences. Initial studies focused on the 5′-GGTGG-3′ sequence in GC-R1 (Figure 2A) since this motif was strongly conserved in vertebrates (Figure 1A and 1B). The target oligonucleotides were incubated with nuclear lysate from Th-expressing cell lines derived from either the substantia nigra (SN) or the OB. Coomassie-stained gels showed a band that was differentially pulled down in both cell lines (Figure 2A). Mass spectrometry established that this band corresponded to hnRNP K. Transcription assays in human SH-SY5Y cells showed that luciferase activity under the control of the 4.5kb upstream rat Th promoter was up-regulated when hnRNP K was over-expressed (Figure 2B). Human Th promoter activity in SH-SY5Y cells was also up-regulated in SH-SY5Y cells (Supplementary Fig. 1).


Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure.

Banerjee K, Wang M, Cai E, Fujiwara N, Baker H, Cave JW - Nat Commun (2014)

hnRNP K interacts with the conserved G:C-rich regions in the Th proximal promoter and is co-expressed with TH in vivo. A, protein pull-down assays with nuclear lysates from mouse Th-expressing cells derived from the mouse olfactory bulb (OB) and substantia nigra (SN). Forward strand of wild-type and mutant oligonucleotide duplexes derived from GC-R1 are shown with mutant positions above the wild-type sequence. Proteins pulled down with either the wild-type or mutant duplex probes were revealed by coomassie stained SDS-polyacrylamide gels. Mass spectrometry identified the differentially isolated protein (indicated by arrow) as hnRNP K. B, transcription assays in human SH-SY5Y cells showed over-expression of hnRNP K could up-regulate Th promoter activity. Luciferase activities are reported as the mean of three independent transfection experiments with error bars representing the standard error of the mean. Pairwise comparisons of luciferase activity levels used the Student’s t-test (single asterisk indicates p <0.01; double asterisk indicates p<0.001). C–R, TH and hnRNP K co-expression in mice and humans. In both the SN (C–F and K–N) and OB (G–J and O–R), most TH-containing cells co-express hnRNP K. Bar equals 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264680&req=5

Figure 2: hnRNP K interacts with the conserved G:C-rich regions in the Th proximal promoter and is co-expressed with TH in vivo. A, protein pull-down assays with nuclear lysates from mouse Th-expressing cells derived from the mouse olfactory bulb (OB) and substantia nigra (SN). Forward strand of wild-type and mutant oligonucleotide duplexes derived from GC-R1 are shown with mutant positions above the wild-type sequence. Proteins pulled down with either the wild-type or mutant duplex probes were revealed by coomassie stained SDS-polyacrylamide gels. Mass spectrometry identified the differentially isolated protein (indicated by arrow) as hnRNP K. B, transcription assays in human SH-SY5Y cells showed over-expression of hnRNP K could up-regulate Th promoter activity. Luciferase activities are reported as the mean of three independent transfection experiments with error bars representing the standard error of the mean. Pairwise comparisons of luciferase activity levels used the Student’s t-test (single asterisk indicates p <0.01; double asterisk indicates p<0.001). C–R, TH and hnRNP K co-expression in mice and humans. In both the SN (C–F and K–N) and OB (G–J and O–R), most TH-containing cells co-express hnRNP K. Bar equals 50 μm.
Mentions: To identify proteins that bind to the G:C-rich regions, pull-down experiments were performed using biotinylated double-stranded oligonucleotides containing either the wild-type or mutated sequences. Initial studies focused on the 5′-GGTGG-3′ sequence in GC-R1 (Figure 2A) since this motif was strongly conserved in vertebrates (Figure 1A and 1B). The target oligonucleotides were incubated with nuclear lysate from Th-expressing cell lines derived from either the substantia nigra (SN) or the OB. Coomassie-stained gels showed a band that was differentially pulled down in both cell lines (Figure 2A). Mass spectrometry established that this band corresponded to hnRNP K. Transcription assays in human SH-SY5Y cells showed that luciferase activity under the control of the 4.5kb upstream rat Th promoter was up-regulated when hnRNP K was over-expressed (Figure 2B). Human Th promoter activity in SH-SY5Y cells was also up-regulated in SH-SY5Y cells (Supplementary Fig. 1).

Bottom Line: It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds to C-rich single-stranded DNA within these conserved regions and also associates with double-stranded sequences when proteins, such as CRE-binding protein, are bound to an adjacent cis-regulatory element.The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures.These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, New York 10605, USA.

ABSTRACT
Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds to C-rich single-stranded DNA within these conserved regions and also associates with double-stranded sequences when proteins, such as CRE-binding protein, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype.

No MeSH data available.