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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 compartmentalization to euchromatin with chromatin cofactors in cultured endometrial cells. For coimmunolocalization of KLF16 in uterine cells, representative images are shown. All cells were stained with DAPI (blue, column 3, panels C, H, M, R, W, and BB) to visualize the DAPI-light euchromatic and DAPI-intense heterochromatic regions. All cells were also stained with anti-KLF16 (green, column 2, panels B, G, L, Q, V, and AA). For chromatin colocalization, cells were additionally stained with specific monoclonal antibodies (red, column 1, panels A, F, K, P, U, and Z) to either euchromatic markers trimethyl H3K4 or dimethyl H3K4 (panels A–E and F–J, respectively) or a heterochromatic marker trimethyl H3K9 (panels K–O). Overlay of corresponding KLF16 and individual chromatin markers (column 4, panels D, I, N, S, X, and CC) and of KLF16, individual chromatin markers and DAPI (column 5, panels E, J, O, T, Y, and DD), respectively, is shown in the figure, in columns 4 and 5 are labeled merge 1,2 and merge 1, 2, 3, respectively. KLF16 preferentially colocalized with euchromatin markers (A–J) compared with the heterochromatic marker (K–O). To evaluate colocalization with the Sin3a corepressor complex, cells were stained with monoclonal antibodies to Sin3a, HDAC1, and HDAC2 (panels P–T, U–Y, and Z–DD, respectively). KLF16 extensively colocalized with Sin3a, HDAC1, and HDAC2 (P–DD).
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Figure 9: KLF16 compartmentalization to euchromatin with chromatin cofactors in cultured endometrial cells. For coimmunolocalization of KLF16 in uterine cells, representative images are shown. All cells were stained with DAPI (blue, column 3, panels C, H, M, R, W, and BB) to visualize the DAPI-light euchromatic and DAPI-intense heterochromatic regions. All cells were also stained with anti-KLF16 (green, column 2, panels B, G, L, Q, V, and AA). For chromatin colocalization, cells were additionally stained with specific monoclonal antibodies (red, column 1, panels A, F, K, P, U, and Z) to either euchromatic markers trimethyl H3K4 or dimethyl H3K4 (panels A–E and F–J, respectively) or a heterochromatic marker trimethyl H3K9 (panels K–O). Overlay of corresponding KLF16 and individual chromatin markers (column 4, panels D, I, N, S, X, and CC) and of KLF16, individual chromatin markers and DAPI (column 5, panels E, J, O, T, Y, and DD), respectively, is shown in the figure, in columns 4 and 5 are labeled merge 1,2 and merge 1, 2, 3, respectively. KLF16 preferentially colocalized with euchromatin markers (A–J) compared with the heterochromatic marker (K–O). To evaluate colocalization with the Sin3a corepressor complex, cells were stained with monoclonal antibodies to Sin3a, HDAC1, and HDAC2 (panels P–T, U–Y, and Z–DD, respectively). KLF16 extensively colocalized with Sin3a, HDAC1, and HDAC2 (P–DD).

Mentions: Subsequent to nuclear translocation, KLF16 predominantly localized to the euchromatic compartment. Euchromatin is enriched in cofactors used by KLF16 to regulate gene expression. Selective localization of KLF16 to euchromatin was supported by colocalization data demonstrating endogenous KLF16 alongside specific markers for nuclear domains (Fig. 9). These markers included trimethyl histone H3 K4 (Fig. 9, A–E) and dimethyl histone H3 K4 (Fig. 9, F–J) for euchromatin, as well as trimethyl histone H3 K9 for heterochromatin (Fig. 9, K–O). Additionally, we also evaluated colocalization of endogenous KLF16 with its chromatin regulators Sin3a (Fig. 9, P–T), HDAC1 (Fig. 9, U–Y), and HDAC2 (Fig. 9, Z–DD). Colocalization of KLF16 with Sin3a and HDAC1–2 within euchromatin is consistent with our biochemical data from Fig. 3d. Fig. 9 also demonstrates that KLF16 was largely excluded from heterochromatin. These data describe for the first time nuclear translocation as an important regulatory mechanism. Additionally, we show that KLF16 is a euchromatic protein that colocalized with short term gene silencing complexes, Sin3-histone-deacetylases. Collectively, these results demonstrate that nuclear translocation in response to signaling as well as euchromatic targeting is a defining functional feature of KLF16, revealing a second important mechanism for regulation of this KLF protein.


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 compartmentalization to euchromatin with chromatin cofactors in cultured endometrial cells. For coimmunolocalization of KLF16 in uterine cells, representative images are shown. All cells were stained with DAPI (blue, column 3, panels C, H, M, R, W, and BB) to visualize the DAPI-light euchromatic and DAPI-intense heterochromatic regions. All cells were also stained with anti-KLF16 (green, column 2, panels B, G, L, Q, V, and AA). For chromatin colocalization, cells were additionally stained with specific monoclonal antibodies (red, column 1, panels A, F, K, P, U, and Z) to either euchromatic markers trimethyl H3K4 or dimethyl H3K4 (panels A–E and F–J, respectively) or a heterochromatic marker trimethyl H3K9 (panels K–O). Overlay of corresponding KLF16 and individual chromatin markers (column 4, panels D, I, N, S, X, and CC) and of KLF16, individual chromatin markers and DAPI (column 5, panels E, J, O, T, Y, and DD), respectively, is shown in the figure, in columns 4 and 5 are labeled merge 1,2 and merge 1, 2, 3, respectively. KLF16 preferentially colocalized with euchromatin markers (A–J) compared with the heterochromatic marker (K–O). To evaluate colocalization with the Sin3a corepressor complex, cells were stained with monoclonal antibodies to Sin3a, HDAC1, and HDAC2 (panels P–T, U–Y, and Z–DD, respectively). KLF16 extensively colocalized with Sin3a, HDAC1, and HDAC2 (P–DD).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: KLF16 compartmentalization to euchromatin with chromatin cofactors in cultured endometrial cells. For coimmunolocalization of KLF16 in uterine cells, representative images are shown. All cells were stained with DAPI (blue, column 3, panels C, H, M, R, W, and BB) to visualize the DAPI-light euchromatic and DAPI-intense heterochromatic regions. All cells were also stained with anti-KLF16 (green, column 2, panels B, G, L, Q, V, and AA). For chromatin colocalization, cells were additionally stained with specific monoclonal antibodies (red, column 1, panels A, F, K, P, U, and Z) to either euchromatic markers trimethyl H3K4 or dimethyl H3K4 (panels A–E and F–J, respectively) or a heterochromatic marker trimethyl H3K9 (panels K–O). Overlay of corresponding KLF16 and individual chromatin markers (column 4, panels D, I, N, S, X, and CC) and of KLF16, individual chromatin markers and DAPI (column 5, panels E, J, O, T, Y, and DD), respectively, is shown in the figure, in columns 4 and 5 are labeled merge 1,2 and merge 1, 2, 3, respectively. KLF16 preferentially colocalized with euchromatin markers (A–J) compared with the heterochromatic marker (K–O). To evaluate colocalization with the Sin3a corepressor complex, cells were stained with monoclonal antibodies to Sin3a, HDAC1, and HDAC2 (panels P–T, U–Y, and Z–DD, respectively). KLF16 extensively colocalized with Sin3a, HDAC1, and HDAC2 (P–DD).
Mentions: Subsequent to nuclear translocation, KLF16 predominantly localized to the euchromatic compartment. Euchromatin is enriched in cofactors used by KLF16 to regulate gene expression. Selective localization of KLF16 to euchromatin was supported by colocalization data demonstrating endogenous KLF16 alongside specific markers for nuclear domains (Fig. 9). These markers included trimethyl histone H3 K4 (Fig. 9, A–E) and dimethyl histone H3 K4 (Fig. 9, F–J) for euchromatin, as well as trimethyl histone H3 K9 for heterochromatin (Fig. 9, K–O). Additionally, we also evaluated colocalization of endogenous KLF16 with its chromatin regulators Sin3a (Fig. 9, P–T), HDAC1 (Fig. 9, U–Y), and HDAC2 (Fig. 9, Z–DD). Colocalization of KLF16 with Sin3a and HDAC1–2 within euchromatin is consistent with our biochemical data from Fig. 3d. Fig. 9 also demonstrates that KLF16 was largely excluded from heterochromatin. These data describe for the first time nuclear translocation as an important regulatory mechanism. Additionally, we show that KLF16 is a euchromatic protein that colocalized with short term gene silencing complexes, Sin3-histone-deacetylases. Collectively, these results demonstrate that nuclear translocation in response to signaling as well as euchromatic targeting is a defining functional feature of KLF16, revealing a second important mechanism for regulation of this KLF protein.

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