Molecular Determinants for Targeting Heterochromatin Protein 1-Mediated Gene Silencing: Direct Chromoshadow Domain–KAP-1 Corepressor Interaction Is Essential

It is clear that many of the effector domains in eukaryotic transcription factors act as protein-protein interfaces which allow the assembly of macromolecular complexes at various sites in the nucleus. The constellation of transcription factors that are arranged at a gene promoter integrate different complexes via coactivators and corepressors, collectively termed cofactors. Cofactors can act directly upon the process of transcription by regulating components of the RNA polymerase complex or through components of chromatin, often leading to changes in gene expression which are stable through cell division. Core histone modification by histone deacetylases and histone acetyltransferases, which can be targeted, respectively, by corepressors and coactivators, has emerged as a common mechanism for influencing gene expression by altering chromatin (58). Another example is the methylation of CpG islands, which is associated with epigenetically imprinted alleles or repressed genes (8). More recent evidence showing that the methyl-CpG-binding protein is present in a complex with histone deacetylase activity (26, 44) suggests a link between these two chromatin modifications that promote gene silencing. Thus, by acting as links between modifiers of chromatin and site-specific transcription factors, corepressors and coactivators can alter the gene expression profile of a cell in a heritable manner. To investigate the mechanistic steps through which transcription repressors down-regulate gene expression, we have focused on the KRAB (Kruppel-associated box) domain as a model. The KRAB domain is a potent repression domain present in nearly one-third of the members of a family of zinc finger transcription factors for which there are an estimated 300 to 700 human genes (7, 36). The family is characterized by an amino-terminal KRAB repression domain linked to multiple arrays of Cys2His2-type zinc fingers, which are responsible for DNA binding (29). Like many other repression domains, the KRAB repression domain retains repressor activity when transferred to a heterologous DNA binding domain (35, 62, 65). KAP-1 is a universal corepressor for the KRAB domain and is the founding member of a small family of cofactors collectively designated the transcriptional intermediary factor 1 (TIF1) family in humans and mice (61). KAP-1 was isolated by affinity chromatography (19) and subsequently by yeast two-hybrid screening (27, 40). It is a 97-kDa nuclear phosphoprotein and contains a number of domains common to the family but also found in other types of transcriptional regulators (see Fig. ​Fig.1A).1A). At the amino terminus is the RBCC multidomain unit, comprised of a RING finger, two B boxes, and a coiled-coil domain. The RBCC domain is essential for binding to the KRAB domain and participates in multimerization of KAP-1 (19, 48). A plant-like homeodomain (PHD) and a bromodomain are tandemly arranged at the carboxy terminus. The central region of KAP-1 is the least conserved among the family members and is generally rich in prolines, glycines, and serines. KAP-1 itself possesses potent repression activity, and this function is contributed by the carboxy terminus, including the PHD and the bromodomain (19, 40, 61). FIG. 1 Schematic representations of the KAP-1 protein and the HP1BD. (A) The KAP-1 protein is shown in linear format with the conserved domains noted by shaded boxes and with amino acid positions indicated. A RING finger, two B boxes, and a coiled-coil region ... The basis for KAP-1 function in transcription repression is not yet fully understood, but the ability of KAP-1 to bind heterochromatin protein 1 (HP1) is very likely to have a role. The interaction between these families of proteins was first uncovered in a yeast two-hybrid screen using TIF1α (32). There are three different HP1 proteins in humans and mice: HP1α, HP1β, and HP1γ (mouse HP1β and HP1γ are also referred to as M31 or MOD1 and M32 or MOD2, respectively [25]). All the proteins share a basic structure of an amino-terminal chromodomain (CD) and a carboxy-terminal chromoshadow domain (CSD) linked by a hinge region (see Fig. ​Fig.2A).2A). The CD is present in numerous proteins, but the CSD has been found only in the HP1 proteins and is thus considered the signature motif for this family (2, 47, 56). FIG. 2 Schematic representations of the HP1 protein, the CD, and the CSD. (A) Diagram of human HP1α depicting the prototypical structure of the HP1 proteins. The relevant domains are noted; NLS, nuclear localization signal. The CSD polypeptide fragment ... Studies of HP1 in Drosophila melanogaster provide one of the best examples of epigenetic mechanisms of gene regulation. Position effect variegation (PEV) in D. melanogaster refers to the process of silencing euchromatic genes that have integrated adjacent to heterochromatin (63). Gene silencing occurs in only a subset of cells, and this state is faithfully inherited by their progeny, leading to variegated or mosaic patterns of expression. The stochastic nature of PEV is hypothesized to be due to the variable spreading of heterochromatin into adjacent regions (55). Genetic screens for modifiers of PEV led to the discovery of the suppressor allele Su(var)2-5, which encoded HP1, a nonhistone chromosomal protein first identified by its localization to heterochromatin in polytene nuclei (14, 23). D. melanogaster HP1 plays a dose-dependent role in PEV and likely contributes to the formation and/or stabilization of heterochromatin (15). More recent studies showing that there is a correlation between overexpression of the CD protein Swi6 and mat locus imprinting in fission yeast and increased expression of an M31 transgene and changes in variegation gene expression in mice indicate that HP1 function has been strongly conserved (17, 43). Position effects are known to play a role in both human and murine genetic defects (6, 28). In many cases, it appears that positively acting elements are removed and/or that negatively acting (silencing) regions are repositioned near genes. Indeed, boundary elements and locus control regions may function as barriers that prevent inappropriate spreading of heterochromatin or association with heterochromatic regions in nuclei (18, 38, 60). Moreover, enhancer function has been postulated to work by keeping genes out of heterochromatic environs (10). Emerging studies of gene regulation by Ikaros have revealed a colocalization of inactive genes with centromeric heterochromatin and M31 in normal cycling lymphocytes (9). Thus, local compartmentalization of genes in the nucleus is emerging as a common mechanism for repressing transcription in a stable manner. However, the mechanisms responsible for sequence-specific targeting of this repression are unclear. Heterochromatin, as defined classically by cytological appearance, is interspersed throughout chromosomes but is abundant near centromeres and telomeres and is frequently composed of repetitive sequences (64). The mammalian HP1 proteins have distinct euchromatic or heterochromatic staining patterns in nuclei, suggesting that their roles have become specialized or that targeting to chromatin has been regulated uniquely (39, 52). KAP-1 is dynamically associated with the euchromatic and heterochromatic regions, suggesting that it links heterochromatin-mediated gene regulation to localization in a specific chromosomal territory (52). This model proposes that KRAB-zinc finger proteins recruit the KAP-1 corepressor to DNA; this complex, in turn, binds to HP1, which may then nucleate local heterochromatin formation, resulting in gene silencing. In order to understand the interaction between KAP-1 and HP1 in more detail, we have reconstituted the complex using recombinant proteins and have comprehensively defined its biochemical properties. We have found that the CSD in HP1 is required for direct binding to KAP-1 and that a stretch of 15 residues in the middle of KAP-1, the HP1BD, is necessary and sufficient for association. We present evidence that the CSD dimerizes and that it binds the KAP-1 HP1BD in a 2:1 stoichiometry, with an apparent Kd of approximately 60 nM. Mutational analysis with the human HP1α CSD and KAP-1 has pinpointed specific residues that are essential for both binding and transcription repression activity in either protein. We also show that interactions with three other HP1 binding proteins are disrupted by the same CSD mutations as those which disrupt KAP-1 binding; however, these domains are not equally able to repress transcription or inhibit HP1-mediated transcription repression.