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AIRE-PHD fingers are structural hubs to maintain the integrity of chromatin-associated interactome.

Gaetani M, Matafora V, Saare M, Spiliotopoulos D, Mollica L, Quilici G, Chignola F, Mannella V, Zucchelli C, Peterson P, Bachi A, Musco G - Nucleic Acids Res. (2012)

Bottom Line: In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation.Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level.Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular NMR Laboratory, Center of Translational Genomics and Bioinformatics, Dulbecco Telethon Institute c/o S. Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
Mutations in autoimmune regulator (AIRE) gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of peripheral-tissue antigens to mediate deletional tolerance, thereby preventing self-reactivity. AIRE contains two plant homeodomains (PHDs) which are sites of pathological mutations. AIRE-PHD fingers are important for AIRE transcriptional activity and presumably play a crucial role in the formation of multimeric protein complexes at chromatin level which ultimately control immunological tolerance. As a step forward the understanding of AIRE-PHD fingers in normal and pathological conditions, we investigated their structure and used a proteomic SILAC approach to assess the impact of patient mutations targeting AIRE-PHD fingers. Importantly, both AIRE-PHD fingers are structurally independent and mutually non-interacting domains. In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation. Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level. Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

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(A) Experimental workflow of SILAC combined to mChIP approach. (B) Comparison of H/L ratio of interactors using MAP-SILAC and PAM-SILAC. AIRE interactors ratios are higher in the MAP-SILAC than in the PAM-SILAC experiment, confirming the dynamic nature of the interactions.
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gks933-F3: (A) Experimental workflow of SILAC combined to mChIP approach. (B) Comparison of H/L ratio of interactors using MAP-SILAC and PAM-SILAC. AIRE interactors ratios are higher in the MAP-SILAC than in the PAM-SILAC experiment, confirming the dynamic nature of the interactions.

Mentions: To investigate the role of AIRE-PHD fingers in the assembly of AIRE-mediated macromolecular complexes on chromatin, we compared the chromatin-associated interactome of AIRE wild-type with the one generated by AIRE-PHD finger mutants (D297A, V301M, C446G). To this aim, we applied a SILAC-based immunoprecipitation approach coupled to MS (Figure 3A). mTECs are an extremely rare cell population and their numbers are insufficient for a proteomic approach. The MS screening for AIRE’s partners was therefore performed in HEK293T cell line transfected with AIRE wild-type and mutant constructs. In order to enrich AIRE complexes on chromatin structures, we performed mChIP to favor the identification of chromatin-related interactors. The protocol allows the efficient purification of protein–DNA macromolecules, enabling subsequent protein identification by MS (33) (see Supplementary Data). The enrichment of protein–DNA complexes was confirmed by the increased recovery of both histone H3 and AIRE protein in mChIP extracts when compared with conventional nuclear extracts (Supplementary Figure S4A and B). In two independent SILAC experiments, HEK293T cells cultured in light and heavy SILAC medium were transfected with empty pcDNA3 or with the pcDNA3-AIRE expression vector, respectively. After 24 h, the cells were lysed and their nuclei were isolated as described in (33). The mChIP extracts were immunoprecipitated with anti-AIRE antibody; bound proteins were then mixed 1:1, eluted and separated by SDS–PAGE. Gel pieces were cut, digested with trypsin and analyzed by LC-MS/MS (Figure 3A). Raw data sets of two independent experiments analyzed with MaxQuant led to the identification of 243 proteins. This list was scrutinized according to the following specificity criteria: (i) the ratio (Heavy/Light) H/L 1.3 (confidence interval >80%) was considered as the interactors’ specificity threshold, notably nine-known AIRE interactors fall within this threshold (Supplementary Figure S4C and D; Supplementary Dataset S1); (ii) only proteins with ratio H/L concordant in both biological replicates were considered (Supplementary Figure S5); (iii) for each biological replicate, two technical replicates were analyzed, to increase the robustness of the quantitative data. This filter narrowed the list of putative AIRE-interactors to 24 best candidates (Table 2). Remarkably, most of the interactors could not be detected in conventional nuclear extract immunoprecipitation but were identified only after chromatin enrichment (Supplementary Figure S6).Figure 3.


AIRE-PHD fingers are structural hubs to maintain the integrity of chromatin-associated interactome.

Gaetani M, Matafora V, Saare M, Spiliotopoulos D, Mollica L, Quilici G, Chignola F, Mannella V, Zucchelli C, Peterson P, Bachi A, Musco G - Nucleic Acids Res. (2012)

(A) Experimental workflow of SILAC combined to mChIP approach. (B) Comparison of H/L ratio of interactors using MAP-SILAC and PAM-SILAC. AIRE interactors ratios are higher in the MAP-SILAC than in the PAM-SILAC experiment, confirming the dynamic nature of the interactions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526288&req=5

gks933-F3: (A) Experimental workflow of SILAC combined to mChIP approach. (B) Comparison of H/L ratio of interactors using MAP-SILAC and PAM-SILAC. AIRE interactors ratios are higher in the MAP-SILAC than in the PAM-SILAC experiment, confirming the dynamic nature of the interactions.
Mentions: To investigate the role of AIRE-PHD fingers in the assembly of AIRE-mediated macromolecular complexes on chromatin, we compared the chromatin-associated interactome of AIRE wild-type with the one generated by AIRE-PHD finger mutants (D297A, V301M, C446G). To this aim, we applied a SILAC-based immunoprecipitation approach coupled to MS (Figure 3A). mTECs are an extremely rare cell population and their numbers are insufficient for a proteomic approach. The MS screening for AIRE’s partners was therefore performed in HEK293T cell line transfected with AIRE wild-type and mutant constructs. In order to enrich AIRE complexes on chromatin structures, we performed mChIP to favor the identification of chromatin-related interactors. The protocol allows the efficient purification of protein–DNA macromolecules, enabling subsequent protein identification by MS (33) (see Supplementary Data). The enrichment of protein–DNA complexes was confirmed by the increased recovery of both histone H3 and AIRE protein in mChIP extracts when compared with conventional nuclear extracts (Supplementary Figure S4A and B). In two independent SILAC experiments, HEK293T cells cultured in light and heavy SILAC medium were transfected with empty pcDNA3 or with the pcDNA3-AIRE expression vector, respectively. After 24 h, the cells were lysed and their nuclei were isolated as described in (33). The mChIP extracts were immunoprecipitated with anti-AIRE antibody; bound proteins were then mixed 1:1, eluted and separated by SDS–PAGE. Gel pieces were cut, digested with trypsin and analyzed by LC-MS/MS (Figure 3A). Raw data sets of two independent experiments analyzed with MaxQuant led to the identification of 243 proteins. This list was scrutinized according to the following specificity criteria: (i) the ratio (Heavy/Light) H/L 1.3 (confidence interval >80%) was considered as the interactors’ specificity threshold, notably nine-known AIRE interactors fall within this threshold (Supplementary Figure S4C and D; Supplementary Dataset S1); (ii) only proteins with ratio H/L concordant in both biological replicates were considered (Supplementary Figure S5); (iii) for each biological replicate, two technical replicates were analyzed, to increase the robustness of the quantitative data. This filter narrowed the list of putative AIRE-interactors to 24 best candidates (Table 2). Remarkably, most of the interactors could not be detected in conventional nuclear extract immunoprecipitation but were identified only after chromatin enrichment (Supplementary Figure S6).Figure 3.

Bottom Line: In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation.Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level.Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular NMR Laboratory, Center of Translational Genomics and Bioinformatics, Dulbecco Telethon Institute c/o S. Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
Mutations in autoimmune regulator (AIRE) gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of peripheral-tissue antigens to mediate deletional tolerance, thereby preventing self-reactivity. AIRE contains two plant homeodomains (PHDs) which are sites of pathological mutations. AIRE-PHD fingers are important for AIRE transcriptional activity and presumably play a crucial role in the formation of multimeric protein complexes at chromatin level which ultimately control immunological tolerance. As a step forward the understanding of AIRE-PHD fingers in normal and pathological conditions, we investigated their structure and used a proteomic SILAC approach to assess the impact of patient mutations targeting AIRE-PHD fingers. Importantly, both AIRE-PHD fingers are structurally independent and mutually non-interacting domains. In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation. Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level. Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

Show MeSH
Related in: MedlinePlus