Mapping of Tfeb and HDAC5 interaction

To characterize the interaction between Tfeb and HDAC5 in more detail, deletion mutants of both cDNAs were generated and used in Co-IP and luciferase assays. Co-IP experiments indi-cated that the HDAC5/Tfeb interaction was mediated via the N-terminal end of HDAC5 (Fig-ure 22 A and B; constructs in Table 4). Because interaction of HDAC5 and Tfeb was still pre-sent when deletion mutant HDAC5 51-C was used, but disappeared with HDAC5 mutant 100-C, it was assumed that amino acids 51 to 100 of the HDAC5 protein are responsible for inter-action with Tfeb (Figure 22 A and B). These findings are further supported by luciferase as-say analysis. More specifically, deletion of the first 51 amino acids of HDAC5 did not influ-ence its ability to inhibit Tfeb mediated MuRF1-luc induction (Figure 22 C). However, larger deletion of the N-terminal region in HDAC5, using HDAC5 100-C and HDAC5 175-C ex-pression plasmids, abolished Tfeb induced MuRF1 exex-pression (Figure 22 C). In addition, when the HDAC domain in HDAC5 was deleted (using the C-terminal deletion clone HDAC5 1-664) no decrease of Tfeb mediated MuRF1 expression was observed, indicative for the importance of the HDAC domain for its repressive function (Figure 22 C). Interaction of full length Tfeb with HDAC5 1-664 could still be observed (Figure 22 B). These findings implicate a direct interaction of both proteins mediated by amino acids 51 to 100 of HDAC5.

These data also show that HDAC5 mediated inhibition of Tfeb induced MuRF1 expression is mediated by its HDAC domain.

Figure 22: Functional mapping of HDAC5 deletion mutants with full length Tfeb

(A) Schematic illustration of HDAC5 deletion mutant clone design. Binding domains for Tfeb (red) and PKD1 (yellow) as well as nuclear localization signal (NLS, in green) and HDAC family domain (orange) are plotted.

Figure’s information were partially adopted from Zhang et al. 2002 and from own manuscript in preparation.

Interaction of individual deletion clones with full length Tfeb in Co-IP assay, shown in B, is indicated at the right side. (B) HEK293 cells were transfected with expression plasmids encoding full length FLAG-Tfeb and HDAC5-myc full length or deletion clone plasmids as indicated. Input Tfeb and HDAC5 proteins detected by immunoblot (IB) are shown in the top and middle panels. Inputs represent 10% of total lysates. Co-immunoprecipitated eluates representing HDAC5 deletion clones are shown in the bottom panel. (C) HEK293 cells were transfected with expression plasmids encoding FLAG-Tfeb and full length or mutant HDAC5 expres-sion plasmids, as indicated, and M1P (size -543 bp). Values were normalized to expresexpres-sion of pCMV-LacZ and calculated as the fold-activation in luciferase to pCMV-LacZ ratio compared to the reporter alone. Error bars represent SD. * p < 0.05; ** p < 0.005; *** p < 0.001; n = 3. (Ref.: Figure data are included in manuscript in preparation/submission)

Next step was the determination of the region in Tfeb which mediates its interaction with HDAC5 (Figure 23 A, constructs in Table 4). Tfeb deletion mutants were designed according to the known domain structure of mouse Tfeb; this included the bHLH and the LZ domain.

The N-terminal deletion clone, lacking the first 127 amino acids (Tfeb 128-C), which includes a glycine rich region with unknown function, did not interact with full length HDAC5 in

Co-IP experiments (Figure 23 B). This clone showed a reduced effectiveness in M1P construct (size -543 bp) induction, and was less sensitive to HDAC5 in luciferase assays (Figure 23 C).

Similar results were observed for the Tfeb deletion mutant Δ129-237, showing no interaction with HDAC5 and comparable induction of the MuRF1-luc activity (Figure 23 B and C). The other two internal deletion clones Tfeb Δ238-400 and Tfeb Δ299-352 were amongst others designed to confirm Tfebs’ MuRF1 induction via the bHLH DNA binding domain. Tfeb Δ238-400, which comprised the bHLH and the LZ domain, showed stable interaction with HDAC5 in Co-IP experiments (Figure 23 B). As expected, this deletion mutant was unable to induce MuRF1 expression (Figure 23 C). Tfeb Δ299-352, missing the amino acids encoding for the bHLH domain, retained its interaction capacity with HDAC5 (Figure 23 B) and was also unable to increase MuRF1 expression (Figure 23 C). In addition, this expression plasmid did not induce MuRF1 at protein level when transfected in C2C12 myoblasts compared to wild type full-length Tfeb (Figure 23 D).

Figure 23: Functional mapping of Tfeb deletion mutants with full length HDAC5

(A) Schematic illustration of full length mouse Tfeb isoform A (Q6P203) and deletion mutants used in this study. Deletion-clone nomenclature is shown on left side. Indicated domains and regions: GR = Glycine-rich, violet box; bHLH = basic Helix-Loop-Helix domain, green box; LZ = Leucine Zipper domain, blue box. (B) HEK293 cells were transfected with expression plasmids encoding full length HDAC5-myc and full length or mutant FLAG-Tfeb proteins as indicated. Input HDAC5 and full length and mutant Tfeb proteins detected by immunoblot (IB) are shown in the top and middle panels. Inputs represent 10% of total lysates. Co-immunoprecipitated eluates representing HDAC5 are shown in the bottom panel. (C) HEK293 cells were trans-fected with expression plasmids encoding full length or mutant Tfeb or full length histone deacetylase (HDAC) 5 at equal amounts and the M1P reporter construct (size -543 bp). Values were normalized to expression of pCMV-LacZ and calculated as the fold-activation in luciferase to pCMV-LacZ ratio compared to the reporter

alone. Error bars represent SD. n.s. = not significant; * p < 0.05; *** p < 0.001; n = 3. (Ref.: Figure data are included in manuscript in preparation/submission)

Subcellular localization of the N-terminal Tfeb deletion clone 128-C and Δ129-237 did not change when over-expressed in C2C12 myoblasts and compared with the full-length FLAG-tagged Tfeb isoform (Figure 24). Both clones and wild type Tfeb were localized to nucleus, cytosol and vesicular structures according to previous publications (Sardiello et al., 2009a;

Settembre et al., 2012a). In contrast, Tfeb Δ238-400 and Tfeb Δ299-352, both lacking the bHLH domain (Figure 23 A), were localized to the cytoplasm and not found in the nucleus of C2C12 myoblasts (Figure 24).

Figure 24: Subcellular localization of Tfeb deletion mutants in C2C12 myoblasts

Fluorescence images showing the subcellular localization of full length FLAG-Tfeb and indicated deletion mu-tants of Tfeb in transfected C2C12 myoblasts, detected by immunofluorescence (24 h after transfection). Left panel shows immunostaining against FLAG tag and secondary antibody Alexa 488, right panel shows merged pictures with DAPI channel. Scale bars represent 20 µm. (Ref.: Figure data are included in manuscript in prepa-ration/submission)

In summary, Tfeb and HDAC5 interact with each other at their individual N-termini respec-tively. HDAC5 inhibits Tfeb induced MuRF1 expression, most likely via its HDAC domain.

Tfeb induced MuRF1 expression requires Tfebs’ bHLH DNA-binding domain.