Krüppel-like factors are effectors of nuclear receptor signaling

Highlights

• Krüppel-like factors (KLFs) are evolutionarily conserved transcription factors.

• KLFs function in nuclear receptor signaling.

• Several KLF genes are regulated by nuclear receptors.

• Some KLFs act as accessory transcription factors for nuclear receptor action.

• Networks of KLFs integrate cellular hormone responses.

Abstract

Binding of steroid and thyroid hormones to their cognate nuclear receptors (NRs) impacts virtually every aspect of postembryonic development, physiology and behavior, and inappropriate signaling by NRs may contribute to disease. While NRs regulate genes by direct binding to hormone response elements in the genome, their actions may depend on the activity of other transcription factors (TFs) that may or may not bind DNA. The Krüppel-like family of transcription factors (KLF) is an evolutionarily conserved class of DNA-binding proteins that influence many aspects of development and physiology. Several members of this family have been shown to play diverse roles in NR signaling. For example, KLFs (1) act as accessory transcription factors for NR actions, (2) regulate expression of NR genes, and (3) as gene products of primary NR response genes function as key players in NR-dependent transcriptional networks. In mouse models, deletion of different KLFs leads to aberrant transcriptional and physiological responses to hormones, underscoring the importance of these proteins in the regulation of hormonal signaling. Understanding the functional relationships between NRs and KLFs will yield important insights into mechanisms of NR signaling. In this review we present a conceptual framework for understanding how KLFs participate in NR signaling, and we provide examples of how these proteins function to effect hormone action.

Introduction

Almost three decades after the first nuclear receptor (NR) genes were cloned, many questions remain about how these proteins activate or repress target genes to mediate the actions of hormones. In particular, the role of other transcription factors (TFs) in NR signaling is increasingly recognized as an essential but poorly understood aspect of NR function. Krüppel-like factors (KLFs) have emerged as key players in NR signaling. The KLFs are zinc-finger TFs with diverse functions in cell proliferation, differentiation and tumorigenic transformation (McConnell and Yang, 2010). Krüppel-like factors bind to GC/GT-rich regions in the genome and activate or repress target genes in concert with chromatin-modifying enzymes. Several KLFs have been found to function in NR signaling by different mechanisms. Some are direct NR target genes whose protein products regulate secondary response genes to mediate hormone action. Others act as accessory TFs to cooperatively activate or repress NR target genes. Still others regulate the expression of genes coding for NRs, influencing NR protein expression and therefore cellular sensitivity to hormone. Here we review the current state of knowledge of the diverse roles that KLFs play in hormone action. We focus on NR signaling in vertebrates, discuss gaps in our current knowledge of KLF functions, and propose experiments to better understand KLF/NR interactions.

Krüppel-like factors comprise a family of transcription factors that bind GC/GT rich sequences in the genome (McConnell and Yang, 2010). They have C-terminal DNA binding domains (DBDs) consisting of three Cys2–His2 zinc fingers that are highly conserved among KLFs within and among vertebrate species, and that also exhibit sequence similarity with DBDs of members of the Specificity Protein (Sp) family (Kaczynski et al., 2003). By contrast to the DBDs, the N-terminal regions are highly variable, which likely explain the divergent functions of KLFs despite their similar DNA-binding capabilities.

There are seventeen KLF genes in mammals that group into three major subfamilies based on similarities in their N-terminal domains (Fig. 1) (Moore et al., 2011). Krüppel-like factors 3, 8, and 12 (Group 1) typically repress gene transcription by binding to the C-terminal binding proteins (CtBPs), a family of transcriptional repressors that recruit chromatin-modifying enzymes that add repressive marks to histones (Turner and Crossley, 1998). Krüppel-like factors 1, 2, 4, 6 and 7 (Group 2) share acidic activation domains and thus typically act as transcriptional activators. Krüppel-like factors 9, 10, 11, 13, 14 and 16 (Group 3) share a Sin3a-interacting domain (SID), an α-helical motif that interacts with the repressor protein Sin3a (Zhang et al., 2001). These factors can repress transcription of target genes, but they are also capable of acting as transactivators (Imataka et al., 1992); whether a Group 3 KLF functions in tranrepression or transactivation may depend on the stage of cellular differentiation that it is expressed. For example, KLF9 activates the Fgfr1 promoter in proliferating myoblasts but represses it in differentiated myotubes via the same DNA binding site (Mitchell and DiMario, 2010). Krüppel-like factors 5, 15 and 17 do not group into any of the other families based on the presence of identifiable protein–protein interaction motifs, although their primary amino acid sequences place KLFs 15 and 17 closer to Group 3, and KLF5 closer to Group 1 (McConnell and Yang, 2010, Moore et al., 2011). All three can act as activators or repressors of transcription (Gumireddy et al., 2009, Oishi et al., 2008, van Vliet et al., 2006) (and see Section 3.2), In some cases, KLFs of the same subfamily have overlapping or redundant functions and can compensate for each other if one is lost or deleted (Heard et al., 2012, Veldman et al., 2007). Members of all three subfamilies have been found to function in NR signaling.

Nuclear receptors comprise an ancient family of transcription factors that mediate signaling by hormones, vitamins A and D, metabolic intermediates and products, and xenobiotics; some NRs are orphan receptors for which a ligand has not yet been discovered, or that may function in a ligand-independent manner (Germain et al., 2006). All NRs share three key functional protein domains. The ligand binding domain (LBD) located at the C-terminus binds hormone (or metabolic intermediate or xenobiotic), and also contains an activator function domain (AF-2) that binds coactivators or co-repressors in a ligand-dependent manner. The DNA binding domain (DBD) located in the center of the molecule binds to DNA, allowing for activation or repression of direct NR target genes. The N-terminus has a domain with weak, ligand-independent activation function (AF-1) (Mangelsdorf et al., 1995). Molecular phylogenetic analysis divides the human NR family into six clades that differ in size. The NRs that mediate hormone action fall into two functional groups (type I and type II) based on subcellular distribution in the unliganded state and mechanisms of action (Germain et al., 2006). The type I receptors (e.g., steroid hormone receptors) reside in the cytoplasm in the absence of hormone where they are bound by heat shock and other proteins that facilitate folding of the receptor into a ligand-binding state (the receptor is ‘activated’). Upon ligand binding the NR and heat shock protein complex dissociate, transforming the NR into a DNA binding conformation. The NR then translocates to the nucleus where it binds to hormone response elements as a homodimer and activates or represses gene transcription. By contrast, type II receptors (e.g., thyroid – TR, and retinoid receptors – RAR/RXR) are bound to DNA in the absence of hormone, often as heterodimers. Unliganded type II receptors can function as powerful transcriptional repressors when resident in chromatin; whereas, in the liganded state they may activate or repress transcription. This switch in function is due to conformational changes that lead to changes in protein–protein interactions; unliganded receptors resident in chromatin recruit corepressors such as Nuclear Receptor Corepressor (NCoR) and Silencing Mediator of Retinoid and Thyroid receptors (SMRT) that in turn recruit histone deacetylases (HDACs). By contrast, liganded receptors recruit coactivators, some with histone acetyltransferase (HAT) activity such as CBP/p300 that catalyze acetylation of lysine residues on histone tails (Cheng et al., 2010). Type II receptors often bind DNA as heterodimers (e.g., TR-RAR or TR-RXR), which may allow for crosstalk between hormone and retinoid signaling pathways (Bagamasbad and Denver, 2011, Lee and Privalsky, 2005).

In this review we focus on KLF interactions with nuclear hormone receptors because these have been the best studied, but there are some reports of KLFs influencing the expression or function of other NR types. For example, KLFs 8 and 9 were shown to regulate the expression of peroxisome proliferator activated receptor γ in differentiating adipocytes (Lee et al., 2012, Pei et al., 2011a). In mouse liver KLF9 was found to work cooperatively with hepatocyte nuclear factor 4α on the promoter of the Deiodinase 1 gene (Ohguchi et al., 2008).

Before discussing the roles that KLFs play in NR signaling it is important to establish a conceptual framework for understanding which types of associations with NRs are most relevant for KLFs to act as mediators/integrators of NR function. Here we will discuss three ways in which KLFs can influence NR signaling (summarized in Fig. 2):

• KLF genes are direct transcriptional targets of NRs, and therefore mediate the cellular actions of NRs by regulating secondary hormone response genes (up or down-regulation). Also, KLFs may regulate transcription of genes that code for proteins that influence NR function in chromatin, which can enhance or suppress ongoing or subsequent NR signaling. Several KLF genes may be targeted by multiple NRs. Krüppel-like factor genes may be convergence points for different intracellular signaling pathways, thus serving as integrators of cellular endocrine responses.

• Some KLFs act as cofactors/accessory TFs for NR function, thereby enhancing or enabling transcriptional activation or repression at NR target genes. This can involve direct DNA binding of the KLF to the NR regulated gene, or protein–protein interactions among the KLF and the NR. Modulation of NR signaling by accessory TFs is an important mechanism for imparting specificity to hormonal responses.

• KLFs can regulate expression of NR genes, which would impact cellular sensitivity to hormones.

As we discuss below, there are examples for each of these mechanisms of action for KLFs on NR signaling. Furthermore, some KLFs function in multiple NR signaling pathways and/or perform different functions within a single pathway. In this review we focus on roles for KLFs in thyroid hormone, corticosteroid and sex steroid signaling via their cognate NRs.