Protein Interactions and Modifications Regulate Biological Functions
Cellular responses to external stimuli are mediated by networks of protein interactions. The functions of many proteins are regulated by noncovalent interactions with other proteins and by covalent modifications. Our research is directed toward elucidation of mechanisms whereby protein interactions and modifications control cellular processes.
One biological function that requires interactions between different combinations of proteins in different cell types is the regulation of gene transcription. The independent control of individual transcription units is achieved through combinatorial interactions among multiple transcription factors. The participation of many transcription factors in the regulation of each gene is necessary for the specification of unique sites of transcription initiation within the genome and for integration of the numerous signals that regulate the expression of each gene.
A cornucopia of interactions among transcription regulatory proteins has been identified using genetic screens and in vitro binding assays. Many transcription factors are also covalently modified when isolated from cells or incubated with modifying enzymes in vitro. We investigate transcription factor interactions and modifications in the normal cellular environment.
Bimolecular Fluorescence Complementation Analysis of Protein Interactions
We developed a method for the visualization of protein interactions in living cells. This bimolecular fluorescence complementation (BiFC) assay uses fragments of a fluorescent protein that can form a fluorescent complex if they are brought together by an interaction between proteins fused to the fragments. This approach enables imaging of protein interactions in their normal cellular environment (Figure 1).
The BiFC assay has proved to be generally applicable for the analysis of interactions involving a variety of structurally unrelated proteins. Interactions between many fusion proteins can be detected when they are expressed at levels comparable to their endogenous counterparts. The association between the fluorescent protein fragments traps the complex, enabling detection of rare and transient interactions but preventing real-time analysis of complex dynamics. Since no extrinsic fluorophores or cofactors are required, BiFC analysis can be performed with minimal perturbation and can be used in virtually any cell or organism that can be genetically modified to express fusion proteins.
Competition for Interactions Between Mutually Exclusive Interaction Partners
Most proteins can interact with several alternative interaction partners. Many interactions are mediated by the same contact interface. Such interactions are likely to be mutually exclusive, resulting in competition between alternative interaction partners for complex formation. To investigate the competition between alternative interaction partners in living cells, we developed a multicolor BiFC assay that enables simultaneous visualization of multiple complexes in the same cell (Figure 2).
We examined the competition among bZIP (basic region–leucine zipper) as well as bHLHZIP (basic helix-loop-helix leucine zipper) family proteins for complex formation in living cells. Members of these families can form heterodimers in many combinations in vitro, and all of these combinations tested could also form heterodimers when expressed pairwise in cells. When several alternative interaction partners were expressed in the same cell, however, there were large differences in their relative efficiencies of complex formation. Thus, the selectivity of protein interactions in cells results in part from the competition for contact surfaces among mutually exclusive interaction partners.
Visualization of Novel Functions of Protein Ubiquitination
Many proteins are regulated by covalent modifications that modulate their activities and localization. Ubiquitin family peptide conjugates are the most structurally complex modifications that have been described. This structural complexity is reflected in a diversity of functional roles in the cell. Originally discovered as a signal for proteasomal degradation, ubiquitination has been associated with receptor trafficking, DNA repair, transcription regulation, and other cellular processes.
We have developed a ubiquitin-mediated fluorescence complementation (UbFC) assay for visualization of proteins modified by ubiquitin family peptides in living cells. This approach uses fragments of fluorescent proteins that are fused to the ubiquitin family peptide and to a putative substrate. Covalent conjugation of the ubiquitin family peptide to the substrate brings the fragments of the fluorescent protein together and enables visualization of the conjugate.
We investigated the effects of ubiquitin and SUMO1 (small ubiquitin-related modifier 1) conjugation on the localization and properties of the transcription regulatory protein Jun (Figure 3). We found that ubiquitinated Jun was translocated to lysosomes for degradation. In contrast, SUMO1 modification resulted in Jun localization to subnuclear foci. Ubiquitinated Jun colocalized with the HECT family E3 ligase Itch, and an Itch recognition motif in Jun was required for ubiquitination in living cells. Mutation of the Itch recognition motif or inhibition of lysosomal proteases stabilized Jun. The visualization of ubiquitin family peptide modifications in living cells has revealed new pathways that control the localization and degradation of nuclear proteins.
We mapped amino acid residues that are required for Jun ubiquitination and lysosomal localization. Jun was polyubiquitinated by ubiquitin containing a single lysine residue at position 27, suggesting that the polyubiquitin chain has a novel topology. Both the δ region of Jun and isoleucine-44 of ubiquitin are required for lysosomal localization, suggesting that the specificity of translocation is established by combinatorial recognition of determinants in both Jun and ubiquitin. The HRS and TSG101 ubiquitin-binding proteins are required for lysosomal translocation of Jun and affect the rate of Jun turnover. Among members of the Fos and Jun families, lysosomal translocation is unique to ubiquitinated Jun since ubiquitin conjugates formed by other members of these families have distinct distributions. The lysosomal translocation of ubiquitinated Jun is mediated by a selective pathway that requires specific recognition of a novel conjugate structure.
Visualization of the Association of Polycomb Group Repressive Complexes with Native Chromatin
Epigenetic regulatory proteins maintain cellular states by propagating the pattern of gene expression across cell generations. Polycomb group (PcG) proteins sustain the pluripotency of stem cells and control the switch to differentiation by silencing the expression of numerous genes in a cell-type-specific manner. We used several independent approaches to investigate chromatin association by CBX family PcG proteins in live embryonic stem (ES) cells. Fluorescence photobleaching demonstrated that the majority of CBX proteins have high mobilities, but a small subpopulation is immobile. BiFC-based analysis of nucleosome binding demonstrated that the distribution of each chromatin-associated CBX protein is distinct from the total population, consistent with the interpretation that the bulk of each CBX protein is not bound to nucleosomes. The fraction of CBX proteins that is stably bound to chromatin increased during ES cell differentiation, consistent with recruitment of mobile CBX protein complexes to new target genes during ES cell differentiation.
The mechanisms that specify the genes to be silenced by PcG proteins are unknown. All PcG proteins whose binding specificities have been investigated recognize the same target genes. All CBX proteins contain conserved chromodomain regions that can bind histones in vitro. We compared the binding specificities of CBX proteins in living cells by imaging the distributions of chromatin-associated CBX proteins in interphase and on metaphase chromosomes. Different CBX proteins bound to nucleosomes in distinct subnuclear regions and in distinct chromosomal banding patterns (Figure 4). Deletion of the conserved regions of CBX proteins did not prevent chromatin association, suggesting that the association of each CBX protein with a different set of genomic targets is mediated by unique sequences in each protein. Deletion of the chromodomains also had no effect on the mobilities of CBX proteins, suggesting that chromatin association in cells is mediated by other regions of the proteins or their interaction partners. CBX proteins are recruited to distinct target genes through interactions involving nonconserved sequences in each protein.
Many histone modifications correlate with gene expression, and most genes that are occupied by PcG proteins are enriched in H3 lysine-27 trimethylation. Lysine-27 trimethylation is catalyzed by polycomb repressive complex 2 (PRC2), and the chromodomains of CBX proteins that associate with polycomb repressive complex 1 (PRC1) can bind lysine-27 trimethylated H3 in vitro. These observations have engendered the model that H3 lysine-27 trimethylation is required for PRC1 recruitment. Since the chromodomains of CBX proteins are dispensable for chromatin association by CBX proteins, we examined the effect of elimination of H3 lysine-27 trimethylation on CBX protein binding to nucleosomes by using cells containing a mutation in the EED subunit of PRC2. CBX proteins bound to nucleosomes with identical efficiencies in EED-null ES cells that had no detectable H3 lysine-27 trimethylation, as in wild-type ES cells. The chromatin-associated CBX proteins did not colocalize with H3 lysine-27 trimethylation in wild-type cells, with the exception of the inactive X. H3 lysine-27 trimethylation therefore does notaffect CBX protein recruitment to chromatin in ES cells.
Comprehensive identification of the modified histones in nucleosomes that are bound by a specific regulatory protein has been difficult. To identify modified histones associated with CBX proteins, we developed a strategy based on the identification of BiFC stabilized complexes (iBiSC). We purified histones from BiFC complexes formed by CBX2 and compared the levels of modified histones purified from the BiFC complexes with their levels in total chromatin. H3 associated with CBX2 was enriched in K27 trimethylation, K4 dimethylation, and K9 acetylation. Some of these modifications could affect CBX2 recruitment, but others may be produced through the recruitment of histone-modifying complexes subsequent to CBX2 binding. The iBiSC strategy is a novel approach for the analysis of chromatin-associated regulatory protein complexes.