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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.


Related in: MedlinePlus

Proposed regulatory mechanism for hnRNP K and DNA secondary structure on the Th proximal promoter. Recruitment of bZIP proteins (in blue) and associated co-activator proteins (in red) to the CRE facilitates assembly of the RNA Polymerase transcription protein complex (in yellow; top panel). Melting of the duplex core promoter DNA by the transcription protein complex generates torsional stress that drives the separation of the duplex DNA in the G:C-rich regions (in green; middle panel). These single DNA strands from the G:C-rich regions either adopt secondary structures, such as G-quadruplexes or i-motifs (bottom left panel), or are bound by proteins such as hnRNP K (bottom right panel). Th transcription is diminished when secondary structures are formed, whereas stabilization of the unfolded single strands in the G:C-rich regions enhances transcription.
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Figure 7: Proposed regulatory mechanism for hnRNP K and DNA secondary structure on the Th proximal promoter. Recruitment of bZIP proteins (in blue) and associated co-activator proteins (in red) to the CRE facilitates assembly of the RNA Polymerase transcription protein complex (in yellow; top panel). Melting of the duplex core promoter DNA by the transcription protein complex generates torsional stress that drives the separation of the duplex DNA in the G:C-rich regions (in green; middle panel). These single DNA strands from the G:C-rich regions either adopt secondary structures, such as G-quadruplexes or i-motifs (bottom left panel), or are bound by proteins such as hnRNP K (bottom right panel). Th transcription is diminished when secondary structures are formed, whereas stabilization of the unfolded single strands in the G:C-rich regions enhances transcription.

Mentions: This study also revealed that individual strands from the conserved G:C-rich regions in the Th promoter adopt either G-quadruplex or i-motif secondary structures. This mirrors previous studies showing that these types of DNA secondary structures form on G:C-rich regions of oncogene promoters also targeted by hnRNP K.17,18 The conserved function of hnRNP K and DNA secondary structure on both Th and oncogene promoters leads us to propose that the Th proximal promoter regulates transcription by a mechanism similar to what has been suggested for the c-myc oncogene17 (Figure 7). On the Th promoter, the recruitment of bZIP proteins and their co-activators to the CRE facilitates assembly of the RNA Polymerase transcription machinery. Melting of core promoter duplex DNA by the RNA Polymerase complex generates negative supercoiling upstream of the complex that can promote separation of duplex DNA strands. The resulting single strands can adopt secondary structures, such as G-quadruplexes and i-motifs, which help dissipate the torsional stress.45 These DNA secondary structures, especially G-quadruplexes, are associated with transcriptional repression through mechanisms that are not established, but may include either disruption of transcription elongation, blocking the binding of transcription activator proteins, or recruitment of transcription repressor proteins.37,38 By contrast, binding of the single DNA strands by proteins such as hnRNP K prevents secondary structure formation and promotes gene transcription by contacting general transcription factors, such as TBP.42,43 The loss of hnRNP K function in vivo would be expected to reduce Th transcription levels due both to the lost interaction with TBP and the increased rate of secondary structure formation. The lack of hnRNP K knock-out mouse strains, however, has precluded direct testing of this prediction. Alternative strategies to knock down hnRNP K by RNA interference in several neural cell lines using either transfection or lentiviral transduction have been impeded by hnRNP K’s regulation of proliferation and survival genes.46


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)

Proposed regulatory mechanism for hnRNP K and DNA secondary structure on the Th proximal promoter. Recruitment of bZIP proteins (in blue) and associated co-activator proteins (in red) to the CRE facilitates assembly of the RNA Polymerase transcription protein complex (in yellow; top panel). Melting of the duplex core promoter DNA by the transcription protein complex generates torsional stress that drives the separation of the duplex DNA in the G:C-rich regions (in green; middle panel). These single DNA strands from the G:C-rich regions either adopt secondary structures, such as G-quadruplexes or i-motifs (bottom left panel), or are bound by proteins such as hnRNP K (bottom right panel). Th transcription is diminished when secondary structures are formed, whereas stabilization of the unfolded single strands in the G:C-rich regions enhances transcription.
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Related In: Results  -  Collection

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Figure 7: Proposed regulatory mechanism for hnRNP K and DNA secondary structure on the Th proximal promoter. Recruitment of bZIP proteins (in blue) and associated co-activator proteins (in red) to the CRE facilitates assembly of the RNA Polymerase transcription protein complex (in yellow; top panel). Melting of the duplex core promoter DNA by the transcription protein complex generates torsional stress that drives the separation of the duplex DNA in the G:C-rich regions (in green; middle panel). These single DNA strands from the G:C-rich regions either adopt secondary structures, such as G-quadruplexes or i-motifs (bottom left panel), or are bound by proteins such as hnRNP K (bottom right panel). Th transcription is diminished when secondary structures are formed, whereas stabilization of the unfolded single strands in the G:C-rich regions enhances transcription.
Mentions: This study also revealed that individual strands from the conserved G:C-rich regions in the Th promoter adopt either G-quadruplex or i-motif secondary structures. This mirrors previous studies showing that these types of DNA secondary structures form on G:C-rich regions of oncogene promoters also targeted by hnRNP K.17,18 The conserved function of hnRNP K and DNA secondary structure on both Th and oncogene promoters leads us to propose that the Th proximal promoter regulates transcription by a mechanism similar to what has been suggested for the c-myc oncogene17 (Figure 7). On the Th promoter, the recruitment of bZIP proteins and their co-activators to the CRE facilitates assembly of the RNA Polymerase transcription machinery. Melting of core promoter duplex DNA by the RNA Polymerase complex generates negative supercoiling upstream of the complex that can promote separation of duplex DNA strands. The resulting single strands can adopt secondary structures, such as G-quadruplexes and i-motifs, which help dissipate the torsional stress.45 These DNA secondary structures, especially G-quadruplexes, are associated with transcriptional repression through mechanisms that are not established, but may include either disruption of transcription elongation, blocking the binding of transcription activator proteins, or recruitment of transcription repressor proteins.37,38 By contrast, binding of the single DNA strands by proteins such as hnRNP K prevents secondary structure formation and promotes gene transcription by contacting general transcription factors, such as TBP.42,43 The loss of hnRNP K function in vivo would be expected to reduce Th transcription levels due both to the lost interaction with TBP and the increased rate of secondary structure formation. The lack of hnRNP K knock-out mouse strains, however, has precluded direct testing of this prediction. Alternative strategies to knock down hnRNP K by RNA interference in several neural cell lines using either transfection or lentiviral transduction have been impeded by hnRNP K’s regulation of proliferation and survival genes.46

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.


Related in: MedlinePlus