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.

Publication Type:

Journal Article

Source:

The Journal of biological chemistry, Volume 287, Issue 10, p.7010-25 (2012)

Keywords:

Active Transport, Cell Nucleusdigestive disease, digestive deseases Amino Acid Substitutiondigestive disease, digestive deseases Chromatindigestive disease, digestive deseases Feeder Cellsdigestive disease, digestive deseases Femaledigestive disease, digestive deseases Gene Silencingdigestive disease, digestive deseases Gonadal Steroid Hormonesdigestive disease, digestive deseases Humansdigestive disease, digestive deseases Kruppel-Like Transcription Factorsdigestive disease, digestive deseases Mutation, Missensedigestive disease, digestive deseases Nuclear Localization Signalsdigestive disease, digestive deseases Phosphorylationdigestive disease, digestive deseases Protein Structure, Tertiarydigestive disease, digestive deseases Reproductiondigestive disease, digestive deseases Response Elementsdigestive disease, digestive deseases Sin3 Histone Deacetylase and Corepressor Complexdigestive disease, digestive deseases Structure-Activity Relationshipdigestive disease, digestive deseases Uterus

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.