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Detailed structural-functional analysis of the Krüppel-like factor 16 (KLF16) transcription factor reveals novel mechanisms for silencing Sp/KLF sites involved in metabolism and endocrinology.

Daftary GS, Lomberk GA, Buttar NS, Allen TW, Grzenda A, Zhang J, Zheng Y, Mathison AJ, Gada RP, Calvo E, Iovanna JL, Billadeau DD, Prendergast FG, Urrutia R - J. Biol. Chem. (2011)

Bottom Line: We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes).Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology.These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota 55905, USA. daftary.gaurang@mayo.edu

ABSTRACT
Krüppel-like factor (KLF) proteins have elicited significant attention due to their emerging key role in metabolic and endocrine diseases. Here, we extend this knowledge through the biochemical characterization of KLF16, unveiling novel mechanisms regulating expression of genes involved in reproductive endocrinology. We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes). KLF16 also regulated the expression of several genes essential for metabolic and endocrine processes in sex steroid-sensitive uterine cells. Mechanistically, we determined that KLF16 possesses an activation domain that couples to histone acetyltransferase-mediated pathways, as well as a repression domain that interacts with the histone deacetylase chromatin-remodeling system via all three Sin3 isoforms, suggesting a higher level of plasticity in chromatin cofactor selection. Molecular modeling combined with molecular dynamic simulations of the Sin3a-KLF16 complex revealed important insights into how this interaction occurs at an atomic resolution level, predicting that phosphorylation of Tyr-10 may modulate KLF16 function. Phosphorylation of KLF16 was confirmed by in vivo (32)P incorporation and controlled by a Y10F site-directed mutant. Inhibition of Src-type tyrosine kinase signaling as well as the nonphosphorylatable Y10F mutation disrupted KLF16-mediated gene silencing, demonstrating that its function is regulatable rather than constitutive. Subcellular localization studies revealed that signal-induced nuclear translocation and euchromatic compartmentalization constitute an additional mechanism for regulating KLF16 function. Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology. These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins.

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Membrane-to-nucleus signaling regulates the accessibility of KLF16 in the nucleus. Localization of KLF16 in endometrial cells was determined by immunofluorescence using anti-KLF16 in uterine cells treated with media supplemented with either 10% (a) or 0% (b) fetal bovine serum. KLF16 demonstrated preferential nuclear localization compared with cytoplasmic localization under normal serum conditions (a, 70.4% versus 27.5%, respectively; *, p = 0.009). In contrast, KLF16 demonstrated preferential cytoplasmic localization under serum-free conditions (b, 24% versus 43.2%, respectively; **, p = 0.04). c, representative cells demonstrating immunolocalization of KLF16 in uterine cells. Cells stained with anti-KLF16 and nuclear (DAPI) as well as cytoplasmic (anti-α-tubulin) markers. Blue, DAPI; green, KLF16; red, α-tubulin.
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Figure 8: Membrane-to-nucleus signaling regulates the accessibility of KLF16 in the nucleus. Localization of KLF16 in endometrial cells was determined by immunofluorescence using anti-KLF16 in uterine cells treated with media supplemented with either 10% (a) or 0% (b) fetal bovine serum. KLF16 demonstrated preferential nuclear localization compared with cytoplasmic localization under normal serum conditions (a, 70.4% versus 27.5%, respectively; *, p = 0.009). In contrast, KLF16 demonstrated preferential cytoplasmic localization under serum-free conditions (b, 24% versus 43.2%, respectively; **, p = 0.04). c, representative cells demonstrating immunolocalization of KLF16 in uterine cells. Cells stained with anti-KLF16 and nuclear (DAPI) as well as cytoplasmic (anti-α-tubulin) markers. Blue, DAPI; green, KLF16; red, α-tubulin.

Mentions: Numerous proteins also respond to signaling pathways via nucleocytoplasmic shuttling. However, information on regulation of KLF proteins by cytoplasm-to-nuclear shuttling as well as their localization within a specific chromatin compartment has remained elusive. Using immunofluorescence, we observed that KLF16 localized to both cytoplasm and nuclei in uterine cells in a serum-responsive manner, used here as a model for activation of multiple signaling cascades (Fig. 8, a–c). In the absence of serum, which keeps signaling quiescently, KLF16 localized to nuclei in 22% of cells (43% cytoplasmic and 35% both; p = 0.04) (Fig. 8b). In contrast, under serum-replete (10%) conditions, nuclear KLF16 significantly increased to 72% (18% cytoplasmic and 10% both; p = 0.009) (Fig. 8a). As cytoplasmic KLF16 cannot regulate gene expression, inhibition of shuttling in quiescent cells suggests that serum growth factors or cytokines regulate nuclear access of KLF16. Therefore, these results strongly support nuclear localization as a key regulatory mechanism of KLF16 in gene expression.


Detailed structural-functional analysis of the Krüppel-like factor 16 (KLF16) transcription factor reveals novel mechanisms for silencing Sp/KLF sites involved in metabolism and endocrinology.

Daftary GS, Lomberk GA, Buttar NS, Allen TW, Grzenda A, Zhang J, Zheng Y, Mathison AJ, Gada RP, Calvo E, Iovanna JL, Billadeau DD, Prendergast FG, Urrutia R - J. Biol. Chem. (2011)

Membrane-to-nucleus signaling regulates the accessibility of KLF16 in the nucleus. Localization of KLF16 in endometrial cells was determined by immunofluorescence using anti-KLF16 in uterine cells treated with media supplemented with either 10% (a) or 0% (b) fetal bovine serum. KLF16 demonstrated preferential nuclear localization compared with cytoplasmic localization under normal serum conditions (a, 70.4% versus 27.5%, respectively; *, p = 0.009). In contrast, KLF16 demonstrated preferential cytoplasmic localization under serum-free conditions (b, 24% versus 43.2%, respectively; **, p = 0.04). c, representative cells demonstrating immunolocalization of KLF16 in uterine cells. Cells stained with anti-KLF16 and nuclear (DAPI) as well as cytoplasmic (anti-α-tubulin) markers. Blue, DAPI; green, KLF16; red, α-tubulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293586&req=5

Figure 8: Membrane-to-nucleus signaling regulates the accessibility of KLF16 in the nucleus. Localization of KLF16 in endometrial cells was determined by immunofluorescence using anti-KLF16 in uterine cells treated with media supplemented with either 10% (a) or 0% (b) fetal bovine serum. KLF16 demonstrated preferential nuclear localization compared with cytoplasmic localization under normal serum conditions (a, 70.4% versus 27.5%, respectively; *, p = 0.009). In contrast, KLF16 demonstrated preferential cytoplasmic localization under serum-free conditions (b, 24% versus 43.2%, respectively; **, p = 0.04). c, representative cells demonstrating immunolocalization of KLF16 in uterine cells. Cells stained with anti-KLF16 and nuclear (DAPI) as well as cytoplasmic (anti-α-tubulin) markers. Blue, DAPI; green, KLF16; red, α-tubulin.
Mentions: Numerous proteins also respond to signaling pathways via nucleocytoplasmic shuttling. However, information on regulation of KLF proteins by cytoplasm-to-nuclear shuttling as well as their localization within a specific chromatin compartment has remained elusive. Using immunofluorescence, we observed that KLF16 localized to both cytoplasm and nuclei in uterine cells in a serum-responsive manner, used here as a model for activation of multiple signaling cascades (Fig. 8, a–c). In the absence of serum, which keeps signaling quiescently, KLF16 localized to nuclei in 22% of cells (43% cytoplasmic and 35% both; p = 0.04) (Fig. 8b). In contrast, under serum-replete (10%) conditions, nuclear KLF16 significantly increased to 72% (18% cytoplasmic and 10% both; p = 0.009) (Fig. 8a). As cytoplasmic KLF16 cannot regulate gene expression, inhibition of shuttling in quiescent cells suggests that serum growth factors or cytokines regulate nuclear access of KLF16. Therefore, these results strongly support nuclear localization as a key regulatory mechanism of KLF16 in gene expression.

Bottom Line: We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes).Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology.These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota 55905, USA. daftary.gaurang@mayo.edu

ABSTRACT
Krüppel-like factor (KLF) proteins have elicited significant attention due to their emerging key role in metabolic and endocrine diseases. Here, we extend this knowledge through the biochemical characterization of KLF16, unveiling novel mechanisms regulating expression of genes involved in reproductive endocrinology. We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes). KLF16 also regulated the expression of several genes essential for metabolic and endocrine processes in sex steroid-sensitive uterine cells. Mechanistically, we determined that KLF16 possesses an activation domain that couples to histone acetyltransferase-mediated pathways, as well as a repression domain that interacts with the histone deacetylase chromatin-remodeling system via all three Sin3 isoforms, suggesting a higher level of plasticity in chromatin cofactor selection. Molecular modeling combined with molecular dynamic simulations of the Sin3a-KLF16 complex revealed important insights into how this interaction occurs at an atomic resolution level, predicting that phosphorylation of Tyr-10 may modulate KLF16 function. Phosphorylation of KLF16 was confirmed by in vivo (32)P incorporation and controlled by a Y10F site-directed mutant. Inhibition of Src-type tyrosine kinase signaling as well as the nonphosphorylatable Y10F mutation disrupted KLF16-mediated gene silencing, demonstrating that its function is regulatable rather than constitutive. Subcellular localization studies revealed that signal-induced nuclear translocation and euchromatic compartmentalization constitute an additional mechanism for regulating KLF16 function. Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology. These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins.

Show MeSH
Related in: MedlinePlus