Identification and Characterization of Interactions between the Vertebrate Polycomb-Group Protein BMI1 and Human Homologs of Polyhomeotic

Abstract

In Drosophila melanogaster, the Polycomb-group (PcG) genes have been identified as repressors of gene expression. They are part of a cellular memory system that is responsible for the stable transmission of gene activity to progeny cells. PcG proteins form a large multimeric, chromatin-associated protein complex, but the identity of its components is largely unknown. Here, we identify two human proteins, HPH1 and HPH2, that are associated with the vertebrate PcG protein BMI1. HPH1 and HPH2 coimmunoprecipitate and cofractionate with each other and with BMI1. They also colocalize with BMI1 in interphase nuclei of U-2 OS human osteosarcoma and SW480 human colorectal adenocarcinoma cells. HPH1 and HPH2 have little sequence homology with each other, except in two highly conserved domains, designated homology domains I and II. They share these homology domains I and II with the Drosophila PcG protein Polyhomeotic (Ph), and we, therefore, have named the novel proteins HPH1 and HPH2. HPH1, HPH2, and BMI1 show distinct, although overlapping expression patterns in different tissues and cell lines. Two-hybrid analysis shows that homology domain II of HPH1 interacts with both homology domains I and II of HPH2. In contrast, homology domain I of HPH1 interacts only with homology domain II of HPH2, but not with homology domain I of HPH2. Furthermore, BMI1 does not interact with the individual homology domains. Instead, both intact homology domains I and II need to be present for interactions with BMI1. These data demonstrate the involvement of homology domains I and II in protein-protein interactions and indicate that HPH1 and HPH2 are able to heterodimerize.

The Bmi-1 oncoprotein interacts with dinG and MPh2: the role of RING finger domains

“…We found that the mouse homolog of the human Polyhomeotic protein, HPh2, interacts with dinG. Notably, previous studies have shown that Polyhomeotic proteins associate with Bmi-1 (Alkema et al, 1997a;Gunster et al, 1997). Our data further suggest that Bmi-1, dinG, and MPh2 associate to form a stable ternary complex in which each protein contributes to the binding of the others.…”

“…Mammalian PcG genes have been identi®ed in mouse and in man and include bmi-1 and mel18, homologs of Drosophila Psc, and Su(2)Z; M33, HPc1, and HPc2, homologs of Drosophila Pc; Rae28/MPh1/ HPh1 and HPh2, homologs of Drosophila Ph; and eed, a Drosophila esc homolog (Tagawa et al, 1990;Haupt et al, 1991;van Lohuizen et al, 1991a,b;Pearce et al, 1992;Satijn et al, 1997;Alkema et al, 1997a;Gunster et al, 1997;Denisenko and Bomsztyk, 1997). The conserved function of these genes has been demonstrated by the homeotic transformation observed in bmi-1 and mel18 transgenic mice as well as by the ability of the mouse M33 gene to complement Pc mutations in Drosophila (van der Lugt et al, 1994;Alkema et al, 1995;Muller et al, 1995;Akasaka et al, 1996).…”

“…Like their Drosophila counterparts, mammalian PcG proteins appear to associate as a heteromeric complex. Thus, Rae28/MPh1/HPh1 and HPh2 were recently identi®ed as mammalian PcG genes whose products directly interact with Bmi-1 while HPc2 coimmunoprecipitates with Bmi-1 and HPh1 (Alkema et al, 1997a;Gunster et al, 1997;Satijn et al, 1997).…”

“…HPh1 and Rae28/MPh1, like other members of the gene family, encode conserved Polyhomeotic Homology Domains I and II (Alkema et al, 1997a;. Perhaps more important, both human Polyhomeotic proteins as well as mouse MPh1 have recently been shown to interact with Bmi-1 (Alkema et al, 1997a;Gunster et al, 1997).…”

“…The PcG proteins assemble as large multimeric complexes that may repress genes by promoting the formation of heterochromatin (reviewed in Singh, 1994). Recent evidence indicates that Bmi-1 forms a multimeric protein complex with at least two mammalian homologs of PcG proteins, Polycomb (Pc) and, Polyhomeotic (Ph), suggesting that some of the fundamental properties of PcG proteins are conserved from¯ies to mammals (Alkema et al, 1997a;Gunster et al, 1997;Satijn et al, 1997). Furthermore, transgenic mice nullizygous for bmi-1 and overexpressing bmi-1 exhibit homeotic skeletal transformations, thus providing evidence that the function of PcG genes in pattern formation is also conserved through evolution (van der Lugt et al, 1994;Alkema et al, 1995).…”