The C-terminal transactivation domain of MITF interacts promiscuously with co-activator CBP/p300

Abstract

The microphthalmia-associated transcription factor (MITF) is one of four closely related members of the MiT/TFE family (TFEB, TFE3, TFEC) that regulate a wide range of cellular processes. MITF is a key regulator of melanocyte-associated genes, and essential to proper development of the melanocyte cell lineage. Abnormal MITF activity can contribute to the onset of several diseases including melanoma, where MITF is an amplified oncogene. To enhance transcription, MITF recruits the co-activator CREB-binding protein (CBP) and its homolog p300 to gene promoters, however the molecular determinants of their interaction are not yet fully understood. Here, we characterize the interactions between the C-terminal MITF transactivation domain and CBP/p300. Using NMR spectroscopy, protein pulldown assays, and isothermal titration calorimetry we determine the C-terminal region of MITF is intrinsically disordered and binds with high-affinity to both TAZ1 and TAZ2 of CBP/p300. Mutagenesis studies revealed two conserved motifs within MITF that are necessary for TAZ2 binding and critical for MITF-dependent transcription of a reporter gene. Finally, we observe the transactivation potential of the MITF C-terminal region is reliant on the N-terminal transactivation domain for function. Taken together, our study helps elucidate the molecular details of how MITF interacts with CBP/p300 through multiple redundant interactions that lend insight into MITF function in melanocytes and melanoma.

Figure 1

Schematic of MITF and CBP/p300 domains. MITF contains a basic helix-loop-helix DNA-binding domain (bHLH), N-terminal (NTERM) transactivation domain (TAD) and C-terminal region which contains threonine-rich (Thr), acidic, and serine-rich (Ser) regions. Domain boundaries are numbered based on the MITF-M isoform sequence. CBP/p300 has a catalytic histone acetyltransferase domain (HAT) and several protein-binding domains: nuclear receptor interaction domain (NRID), kinase inducible domain (KIX), IRF-3 binding domain (IBiD), and transcription adapter zinc finger domains (TAZ1 and TAZ2).

Figure 2

(A) ¹H-¹⁵N-HSQC of ¹³C, ¹⁵N-labelled MITF_CTERM, backbone amide resonance peaks are labelled based on their assigned residue. (B) Secondary structure propensity (SSP) values per residue of MITF_CTERM calculated from C⍺ and Cβ chemical shifts.

Figure 3

(A) Coomassie-stained SDS-PAGE analysis of the total amount of KIX, TAZ1, or TAZ2 pulled down by immobilized GB1 or GB1-MITF_CTERM. Lanes 2, 5, and 8 show migration of each isolated CBP/p300 domain and represents 10% of total pulldown input. The migration of immunoglobulin heavy and light chains (IgG H and IgG L), GB1, GB1-MITF_CTERM, KIX, TAZ1, and TAZ2 are denoted. Duplicate uncropped gels are shown in Fig. S3. (B) ITC thermogram following titration of TAZ1 (400 µM) or (C) TAZ2 (400 µM) into MITF_CTERM (30 µM). Data were fit to one-site binding curves, determining dissociation constants (K_d) of 5.0 ± 0.31 µM and 0.76 ± 0.05 µM for the interaction of MITF_CTERM with TAZ1 and TAZ2, respectively).

Figure 4

(A) Chemical shift perturbations (∆δ = [(0.17Δδ_N)² + (Δδ_HN)²]^1/2) of backbone resonances per residue of ¹⁵N-labelled MITF_CTERM (100 µM) upon the addition of TAZ2 (300 μM). Negative values indicate disappearance of a resonance peak, and * denotes a proline or unassigned resonance. Average chemical shift change (0.04 ppm) is shown as dashed line, boxes denote regions chosen for mutation. (B) Average luciferase activation of MITF constructs, reported as fold activation of pcDNA3.1 negative control. Error bars represent standard error, statistical analysis performed using one-way ANOVA followed by Dunnett’s multiple comparison test (***p ≤ 0.001). (C) Coomassie-stained SDS-PAGE analysis of total TAZ2 pulled down by immobilized GB1-MITF_CTERM or GB1-tagged MITF mutant constructs MITF_ΔThr-rich (Δ334–345)_, MITF_ΔAcidic (Δ372–381)_, and MITF_ΔSer-rich (Δ394–411). Lane 2 shows migration of isolated TAZ2 domain and represents 10% total pulldown input. The migration of immunoglobulin heavy and light chains (IgG H and IgG L), GB1, GB1-MITF_CTERM, and TAZ2 are denoted. Uncropped images of duplicate experiments are found in Fig. S4. (D–F) ITC thermograms of MITF_ΔThr-rich (16 µM), MITF_ΔAcidic (17 µM), and MITF_ΔSer-rich (22 µM), following titration with TAZ2 (400 µM) each fit to a one-site binding curve (K_d = 0.41 ± 0.02 µM, 7.24 ± 0.70 µM, and 2.26 ± 0.09 µM, respectively).