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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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KLF16 functions as transcription factor involved in the regulation of metabolic and endocrine gene expression in uterine cells.a, selected identifiers for KLF16-regulated genes. Most of these genes (80%) are down-regulated (green) by this transcription factor, which is congruent with the predominant gene-silencing activity of KLF16. Notably, from these genes, 22% participate in estrogen metabolism. b, semantic-based pathway reconstruction algorithms integrate most of the genes found regulated by KLF16 in the PCR microarray into a seamless pathway, showing active interaction among its nodes. KLF16 regulated the expression of several genes connected with metabolism of sex steroid as well as environmental contaminant toxins in endometrial cells. c, chromatin immunoprecipitation was performed on uterine cells transfected with either pCMV/FLAG- or pcDNA3HIS-KLF16 or corresponding empty vectors. Anti-FLAG or anti-HIS antibodies were used for immunoprecipitation, respectively. Shown is a representative sample where FLAG-KLF16 but not empty vector bound a 249-bp CYP1A1 genomic element containing the BTE (−55 to −41) in uterine cells. d, uterine cells were transfected with either FLAG-KLF16 or corresponding empty vector. The cells were treated with 1 nm dioxin to induce CYP1A1 expression. Total RNA was extracted at 48 h, and RT-PCR was done. KLF16 repressed TCDD induction of CYP1A1 mRNA expression by 50% (p < 0.05) compared with the corresponding empty vector. Housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2 microglobulin (B2M) were used as loading controls. e, ChIP was performed for endogenous KLF16 in the presence or absence of 1 nm dioxin. Positive amplification of the CYP1A1 promoter demonstrates that KLF16 binds this genomic element described above both in the presence and absence of dioxin. f, uterine cells were transfected with KLF16 siRNA to knock down endogenous KLF16 levels, which was confirmed by Western blot using anti-KLF16 (upper panel). CYP1A1 mRNA levels were increased 2-fold in cells treated with KLF16 siRNA (lower panel). g, uterine cells were transfected with 7.5 μg of FLAG-KLF16 or corresponding empty vector and 2.5 μg of CYP1A1 promoter-luciferase constructs containing the BTE (−55 to −41). Results of luciferase-reporter assays normalized to protein expression revealed that KLF16 significantly repressed CYP1A1-luciferase activity compared with empty vector control (46%; *, p = 0.03). h, CYP1A1 enzymatic activity was measured using a well characterized CYP substrate-reporter assay. Uterine cells were transfected with either KLF16 (black bars) or empty vector control (white bars) and subsequently treated with either 1 nm TCDD or DMSO control. A luciferin-derived CYP1A1 substrate was added to the medium and enzyme activity determined by reporter activity. CYP1A1 enzymatic activity was significantly inhibited by KLF16 (60%; **, p < 0.001) in TCDD treated cells.
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Figure 2: KLF16 functions as transcription factor involved in the regulation of metabolic and endocrine gene expression in uterine cells.a, selected identifiers for KLF16-regulated genes. Most of these genes (80%) are down-regulated (green) by this transcription factor, which is congruent with the predominant gene-silencing activity of KLF16. Notably, from these genes, 22% participate in estrogen metabolism. b, semantic-based pathway reconstruction algorithms integrate most of the genes found regulated by KLF16 in the PCR microarray into a seamless pathway, showing active interaction among its nodes. KLF16 regulated the expression of several genes connected with metabolism of sex steroid as well as environmental contaminant toxins in endometrial cells. c, chromatin immunoprecipitation was performed on uterine cells transfected with either pCMV/FLAG- or pcDNA3HIS-KLF16 or corresponding empty vectors. Anti-FLAG or anti-HIS antibodies were used for immunoprecipitation, respectively. Shown is a representative sample where FLAG-KLF16 but not empty vector bound a 249-bp CYP1A1 genomic element containing the BTE (−55 to −41) in uterine cells. d, uterine cells were transfected with either FLAG-KLF16 or corresponding empty vector. The cells were treated with 1 nm dioxin to induce CYP1A1 expression. Total RNA was extracted at 48 h, and RT-PCR was done. KLF16 repressed TCDD induction of CYP1A1 mRNA expression by 50% (p < 0.05) compared with the corresponding empty vector. Housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2 microglobulin (B2M) were used as loading controls. e, ChIP was performed for endogenous KLF16 in the presence or absence of 1 nm dioxin. Positive amplification of the CYP1A1 promoter demonstrates that KLF16 binds this genomic element described above both in the presence and absence of dioxin. f, uterine cells were transfected with KLF16 siRNA to knock down endogenous KLF16 levels, which was confirmed by Western blot using anti-KLF16 (upper panel). CYP1A1 mRNA levels were increased 2-fold in cells treated with KLF16 siRNA (lower panel). g, uterine cells were transfected with 7.5 μg of FLAG-KLF16 or corresponding empty vector and 2.5 μg of CYP1A1 promoter-luciferase constructs containing the BTE (−55 to −41). Results of luciferase-reporter assays normalized to protein expression revealed that KLF16 significantly repressed CYP1A1-luciferase activity compared with empty vector control (46%; *, p = 0.03). h, CYP1A1 enzymatic activity was measured using a well characterized CYP substrate-reporter assay. Uterine cells were transfected with either KLF16 (black bars) or empty vector control (white bars) and subsequently treated with either 1 nm TCDD or DMSO control. A luciferin-derived CYP1A1 substrate was added to the medium and enzyme activity determined by reporter activity. CYP1A1 enzymatic activity was significantly inhibited by KLF16 (60%; **, p < 0.001) in TCDD treated cells.

Mentions: To identify bona fide KLF16 gene targets that participate in metabolic and endocrine functions in endometrial cells, we utilized several complementary approaches as follows: pathway-specific microarray analysis with pathway reconstruction, ChIP, promoter assays, and EMSA. Overexpression of KLF16 in uterine epithelial cells combined with microarray analysis revealed that KLF16 regulates genes comprising key nodes within the metabolic-endocrine biosynthetic pathways such as NRR2 and RARB, nuclear hormone receptors ESRRA and ESRRB, and the estrogen-metabolizing enzyme CYP1A1 (Fig. 2, a and b). Therefore, these results demonstrate that KLF16 is at least as important as KLF9 and -13 in regulating endometrial genes, most notably those implicated in metabolic and endocrine homeostasis.


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)

KLF16 functions as transcription factor involved in the regulation of metabolic and endocrine gene expression in uterine cells.a, selected identifiers for KLF16-regulated genes. Most of these genes (80%) are down-regulated (green) by this transcription factor, which is congruent with the predominant gene-silencing activity of KLF16. Notably, from these genes, 22% participate in estrogen metabolism. b, semantic-based pathway reconstruction algorithms integrate most of the genes found regulated by KLF16 in the PCR microarray into a seamless pathway, showing active interaction among its nodes. KLF16 regulated the expression of several genes connected with metabolism of sex steroid as well as environmental contaminant toxins in endometrial cells. c, chromatin immunoprecipitation was performed on uterine cells transfected with either pCMV/FLAG- or pcDNA3HIS-KLF16 or corresponding empty vectors. Anti-FLAG or anti-HIS antibodies were used for immunoprecipitation, respectively. Shown is a representative sample where FLAG-KLF16 but not empty vector bound a 249-bp CYP1A1 genomic element containing the BTE (−55 to −41) in uterine cells. d, uterine cells were transfected with either FLAG-KLF16 or corresponding empty vector. The cells were treated with 1 nm dioxin to induce CYP1A1 expression. Total RNA was extracted at 48 h, and RT-PCR was done. KLF16 repressed TCDD induction of CYP1A1 mRNA expression by 50% (p < 0.05) compared with the corresponding empty vector. Housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2 microglobulin (B2M) were used as loading controls. e, ChIP was performed for endogenous KLF16 in the presence or absence of 1 nm dioxin. Positive amplification of the CYP1A1 promoter demonstrates that KLF16 binds this genomic element described above both in the presence and absence of dioxin. f, uterine cells were transfected with KLF16 siRNA to knock down endogenous KLF16 levels, which was confirmed by Western blot using anti-KLF16 (upper panel). CYP1A1 mRNA levels were increased 2-fold in cells treated with KLF16 siRNA (lower panel). g, uterine cells were transfected with 7.5 μg of FLAG-KLF16 or corresponding empty vector and 2.5 μg of CYP1A1 promoter-luciferase constructs containing the BTE (−55 to −41). Results of luciferase-reporter assays normalized to protein expression revealed that KLF16 significantly repressed CYP1A1-luciferase activity compared with empty vector control (46%; *, p = 0.03). h, CYP1A1 enzymatic activity was measured using a well characterized CYP substrate-reporter assay. Uterine cells were transfected with either KLF16 (black bars) or empty vector control (white bars) and subsequently treated with either 1 nm TCDD or DMSO control. A luciferin-derived CYP1A1 substrate was added to the medium and enzyme activity determined by reporter activity. CYP1A1 enzymatic activity was significantly inhibited by KLF16 (60%; **, p < 0.001) in TCDD treated cells.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: KLF16 functions as transcription factor involved in the regulation of metabolic and endocrine gene expression in uterine cells.a, selected identifiers for KLF16-regulated genes. Most of these genes (80%) are down-regulated (green) by this transcription factor, which is congruent with the predominant gene-silencing activity of KLF16. Notably, from these genes, 22% participate in estrogen metabolism. b, semantic-based pathway reconstruction algorithms integrate most of the genes found regulated by KLF16 in the PCR microarray into a seamless pathway, showing active interaction among its nodes. KLF16 regulated the expression of several genes connected with metabolism of sex steroid as well as environmental contaminant toxins in endometrial cells. c, chromatin immunoprecipitation was performed on uterine cells transfected with either pCMV/FLAG- or pcDNA3HIS-KLF16 or corresponding empty vectors. Anti-FLAG or anti-HIS antibodies were used for immunoprecipitation, respectively. Shown is a representative sample where FLAG-KLF16 but not empty vector bound a 249-bp CYP1A1 genomic element containing the BTE (−55 to −41) in uterine cells. d, uterine cells were transfected with either FLAG-KLF16 or corresponding empty vector. The cells were treated with 1 nm dioxin to induce CYP1A1 expression. Total RNA was extracted at 48 h, and RT-PCR was done. KLF16 repressed TCDD induction of CYP1A1 mRNA expression by 50% (p < 0.05) compared with the corresponding empty vector. Housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2 microglobulin (B2M) were used as loading controls. e, ChIP was performed for endogenous KLF16 in the presence or absence of 1 nm dioxin. Positive amplification of the CYP1A1 promoter demonstrates that KLF16 binds this genomic element described above both in the presence and absence of dioxin. f, uterine cells were transfected with KLF16 siRNA to knock down endogenous KLF16 levels, which was confirmed by Western blot using anti-KLF16 (upper panel). CYP1A1 mRNA levels were increased 2-fold in cells treated with KLF16 siRNA (lower panel). g, uterine cells were transfected with 7.5 μg of FLAG-KLF16 or corresponding empty vector and 2.5 μg of CYP1A1 promoter-luciferase constructs containing the BTE (−55 to −41). Results of luciferase-reporter assays normalized to protein expression revealed that KLF16 significantly repressed CYP1A1-luciferase activity compared with empty vector control (46%; *, p = 0.03). h, CYP1A1 enzymatic activity was measured using a well characterized CYP substrate-reporter assay. Uterine cells were transfected with either KLF16 (black bars) or empty vector control (white bars) and subsequently treated with either 1 nm TCDD or DMSO control. A luciferin-derived CYP1A1 substrate was added to the medium and enzyme activity determined by reporter activity. CYP1A1 enzymatic activity was significantly inhibited by KLF16 (60%; **, p < 0.001) in TCDD treated cells.
Mentions: To identify bona fide KLF16 gene targets that participate in metabolic and endocrine functions in endometrial cells, we utilized several complementary approaches as follows: pathway-specific microarray analysis with pathway reconstruction, ChIP, promoter assays, and EMSA. Overexpression of KLF16 in uterine epithelial cells combined with microarray analysis revealed that KLF16 regulates genes comprising key nodes within the metabolic-endocrine biosynthetic pathways such as NRR2 and RARB, nuclear hormone receptors ESRRA and ESRRB, and the estrogen-metabolizing enzyme CYP1A1 (Fig. 2, a and b). Therefore, these results demonstrate that KLF16 is at least as important as KLF9 and -13 in regulating endometrial genes, most notably those implicated in metabolic and endocrine homeostasis.

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