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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 binding to the Th proximal promoter. A, chromatin immunoprecipitation assays with human SH-SY5Y cells indicated that hnRNP K directly binds the Th proximal promoter. Three independent ChIP experiments were conducted and the mean relative enrichment of the Th proximal promoter is reported with error bars representing the standard error of the mean. Statistical significance was assessed using the two-tailed Student’s t-test (asterisk indicates p <0.01). Arrows show the position on the Th promoter of the primers used in this assay. B hnRNP K binds single-stranded sequences from the Th promoter. Wild-type and mutant oligonucleotides derived from the GC-R1 and GC-R2 are shown with mutated positions above the wild-type sequence. Highly conserved nucleotides within GC-R1 and GC-R2 are shaded. Western blot analyses showed that wild-type C-rich single strands preferentially pulled down hnRNP K from OB and SN cell line nuclear lysates. Band intensities, shown above the blot, are reported as the mean of duplicate pull-down assays with error bars representing the difference between the mean and the individual trials. Compared to the wild-type strands, mutant C-rich strands significantly reduced the interaction with hnRNP K (asterisk; p <0.01 by Student’s t-test). C, the duplex oligonucleotide containing GC-R1 (show on top with highly conserved nucleotides shaded) was not bound by hnRNP K in electromobility shift assays (lanes 1–4). By contrast, hnRNP K did super-shift this target sequence when CREB was present (lanes 5–8).
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Figure 3: hnRNP K binding to the Th proximal promoter. A, chromatin immunoprecipitation assays with human SH-SY5Y cells indicated that hnRNP K directly binds the Th proximal promoter. Three independent ChIP experiments were conducted and the mean relative enrichment of the Th proximal promoter is reported with error bars representing the standard error of the mean. Statistical significance was assessed using the two-tailed Student’s t-test (asterisk indicates p <0.01). Arrows show the position on the Th promoter of the primers used in this assay. B hnRNP K binds single-stranded sequences from the Th promoter. Wild-type and mutant oligonucleotides derived from the GC-R1 and GC-R2 are shown with mutated positions above the wild-type sequence. Highly conserved nucleotides within GC-R1 and GC-R2 are shaded. Western blot analyses showed that wild-type C-rich single strands preferentially pulled down hnRNP K from OB and SN cell line nuclear lysates. Band intensities, shown above the blot, are reported as the mean of duplicate pull-down assays with error bars representing the difference between the mean and the individual trials. Compared to the wild-type strands, mutant C-rich strands significantly reduced the interaction with hnRNP K (asterisk; p <0.01 by Student’s t-test). C, the duplex oligonucleotide containing GC-R1 (show on top with highly conserved nucleotides shaded) was not bound by hnRNP K in electromobility shift assays (lanes 1–4). By contrast, hnRNP K did super-shift this target sequence when CREB was present (lanes 5–8).

Mentions: To test whether hnRNP K associated with the Th promoter in a cellular context, we performed chromatin immunoprecipitation (ChIP) assays with SH-SY5Y cells. These studies showed that hnRNP K directly bound the G:C-rich regions of the Th promoter (Figure 3A). Previous studies have shown that hnRNP K preferentially binds C-rich single-stranded DNA sequences.14 To establish whether hnRNP K binds single DNA strands from the G:C-rich regions in the Th promoter, pull-down assays used single-stranded biotinylated oligonucleotides incubated with nuclear lysate from either OB or SN cell lines. The target oligonucleotide sequences contained either the wild-type G:C-rich region sequences or mutations overlapping highly conserved positions (Figure 3B). Western blots showed that hnRNP K was preferentially pulled down by the wild-type C-rich strands of both GC-R1 and GC-R2 (Figure 3B; Supplementary Fig. 2).


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 binding to the Th proximal promoter. A, chromatin immunoprecipitation assays with human SH-SY5Y cells indicated that hnRNP K directly binds the Th proximal promoter. Three independent ChIP experiments were conducted and the mean relative enrichment of the Th proximal promoter is reported with error bars representing the standard error of the mean. Statistical significance was assessed using the two-tailed Student’s t-test (asterisk indicates p <0.01). Arrows show the position on the Th promoter of the primers used in this assay. B hnRNP K binds single-stranded sequences from the Th promoter. Wild-type and mutant oligonucleotides derived from the GC-R1 and GC-R2 are shown with mutated positions above the wild-type sequence. Highly conserved nucleotides within GC-R1 and GC-R2 are shaded. Western blot analyses showed that wild-type C-rich single strands preferentially pulled down hnRNP K from OB and SN cell line nuclear lysates. Band intensities, shown above the blot, are reported as the mean of duplicate pull-down assays with error bars representing the difference between the mean and the individual trials. Compared to the wild-type strands, mutant C-rich strands significantly reduced the interaction with hnRNP K (asterisk; p <0.01 by Student’s t-test). C, the duplex oligonucleotide containing GC-R1 (show on top with highly conserved nucleotides shaded) was not bound by hnRNP K in electromobility shift assays (lanes 1–4). By contrast, hnRNP K did super-shift this target sequence when CREB was present (lanes 5–8).
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Figure 3: hnRNP K binding to the Th proximal promoter. A, chromatin immunoprecipitation assays with human SH-SY5Y cells indicated that hnRNP K directly binds the Th proximal promoter. Three independent ChIP experiments were conducted and the mean relative enrichment of the Th proximal promoter is reported with error bars representing the standard error of the mean. Statistical significance was assessed using the two-tailed Student’s t-test (asterisk indicates p <0.01). Arrows show the position on the Th promoter of the primers used in this assay. B hnRNP K binds single-stranded sequences from the Th promoter. Wild-type and mutant oligonucleotides derived from the GC-R1 and GC-R2 are shown with mutated positions above the wild-type sequence. Highly conserved nucleotides within GC-R1 and GC-R2 are shaded. Western blot analyses showed that wild-type C-rich single strands preferentially pulled down hnRNP K from OB and SN cell line nuclear lysates. Band intensities, shown above the blot, are reported as the mean of duplicate pull-down assays with error bars representing the difference between the mean and the individual trials. Compared to the wild-type strands, mutant C-rich strands significantly reduced the interaction with hnRNP K (asterisk; p <0.01 by Student’s t-test). C, the duplex oligonucleotide containing GC-R1 (show on top with highly conserved nucleotides shaded) was not bound by hnRNP K in electromobility shift assays (lanes 1–4). By contrast, hnRNP K did super-shift this target sequence when CREB was present (lanes 5–8).
Mentions: To test whether hnRNP K associated with the Th promoter in a cellular context, we performed chromatin immunoprecipitation (ChIP) assays with SH-SY5Y cells. These studies showed that hnRNP K directly bound the G:C-rich regions of the Th promoter (Figure 3A). Previous studies have shown that hnRNP K preferentially binds C-rich single-stranded DNA sequences.14 To establish whether hnRNP K binds single DNA strands from the G:C-rich regions in the Th promoter, pull-down assays used single-stranded biotinylated oligonucleotides incubated with nuclear lysate from either OB or SN cell lines. The target oligonucleotide sequences contained either the wild-type G:C-rich region sequences or mutations overlapping highly conserved positions (Figure 3B). Western blots showed that hnRNP K was preferentially pulled down by the wild-type C-rich strands of both GC-R1 and GC-R2 (Figure 3B; Supplementary Fig. 2).

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.