MITF and TFEB cross-regulation in melanoma cells

Abstract

The MITF, TFEB, TFE3 and TFEC (MiT-TFE) proteins belong to the basic helix-loop-helix family of leucine zipper transcription factors. MITF is crucial for melanocyte development and differentiation, and has been termed a lineage-specific oncogene in melanoma. The three related proteins MITF, TFEB and TFE3 have been shown to be involved in the biogenesis and function of lysosomes and autophagosomes, regulating cellular clearance pathways. Here we investigated the cross-regulatory relationship of MITF and TFEB in melanoma cells. Like MITF, the TFEB and TFE3 genes are expressed in melanoma cells as well as in melanoma tumors, albeit at lower levels. We show that the MITF and TFEB proteins, but not TFE3, directly affect each other's mRNA and protein expression. In addition, the subcellular localization of MITF and TFEB is subject to regulation by the mTOR signaling pathway, which impacts their cross-regulatory relationship at the transcriptional level. Our work shows that the relationship between MITF and TFEB is multifaceted and that the cross-regulatory interactions of these factors need to be taken into account when considering pathways regulated by these proteins.

Fig 1. MITF binds to the MITF and TFEB genes in melanoma cells.

MITF ChIP-seq data in 501Mel and Colo829 melanoma cells show peaks for MITF in the MITF gene (A) and a peak in the intron 1 of TFEB containing a CAGCTG regulatory element (B). No binding sites were observed within the TFE3 gene (C) nor within 20 kb upstream or downstream (10.6084/m9.figshare.12568646).

Fig 2. MITF and TFEB modulate each other’s expression upon overexpression in 501Mel cells.

(A-B) The expression of MITF, TFEB and TFE3 as determined by RT-qPCR after overexpression of MITF(-) (A) or TFEB (B) in 501mel cells compared to empty vector (EV). Bars represent SEM. * indicates significance at p<0.05. (C-D) Western blot analysis of MITF (C) and TFEB (D) proteins, using specific antibodies, after overexpression of TFEB (C) and MITF (C, D) in 501mel cells. Shown is a representative figure for at least three independent experiments. (E) Quantification of the Western blots presented in C-D. Bars represent SEM. * indicates significance at p<0.05. (F) Western blot analysis of MITF and TFEB proteins, using FLAG (MITF) or TFEB-specific antibodies, upon dose-dependent doxycycline-inducible overexpression of FLAG-tagged MITF in 501mel cells. Shown is a representative figure for three independent experiments. (G) Quantification of the Western blot presented in F. Bars represent SEM. * indicates significance at p<0.05.

Fig 3. MITF and TFEB modulate each other’s expression upon knockdown in 501Mel and Skmel28 cells.

(A) The expression of MITF and TFEB as determined by RT-qPCR after siRNA-mediated knockdown of each factor compared to control siRNA in 501Mel cells. Bars represent SEM. * indicates significance at p<0.05. (B) Western blot analysis of MITF and TFEB proteins after siRNA-mediated knockdown of MITF or TFEB in 501 Mel cells. Shown is a representative figure and quantification of three independent experiments (C). Bars represent SEM. * indicates significance at p<0.05. (D) The expression of MITF and TFEB as determined by RT-qPCR after doxycycline-induced (1μg/ml) miR-MITF knockdown, compared to miR-NTC control in Skmel28 cells; three independent experiments are shown. Bars represent SEM. * indicates significance at p<0.05.

Fig 4. MITF activates TFEB expression through a CAGCTG motif in intron 1.

(A) Schematic representation of the luciferase reporter constructs generated for the MITF binding sites observed in the TFEB gene. (B) HEK293T cells were transiently co-transfected with a p3XFLAG-CMV-14 construct with or without MITF-M (empty vector, EV) and reporter constructs and assayed for luciferase activity after 24h. Luminescence signal is expressed as fold change over an empty luciferase reporter for Tyrosinase and for a luciferase reporter containing the fragment from TFEB intron 1 and a mutated version thereof in front of the minimal SV40 promoter. Error bars represent the SEM of three experiments. * indicates significance at p<0.05. (C) EMSA showing binding of in vitro translated MITF protein to oligos containing the CACGTG and CAGCTG sequences. Supershifts with the C5 MITF specific antibody are indicated, confirming the specificity of the gel shifts. (D) A total of 11,173 sequences under the MITF ChIP-seq peaks were called with p<0.05. Graphs show the number of peaks among all the peaks called with any given number of matches for each motif. Bottom table shows the total number of matches for each motif in all the peaks.

Fig 5. mTOR signaling affects the subcellular localization and cross-regulation of MITF and TFEB.

(A) Immunofluorescence images of human 501Mel cells after treatment with vehicle (DMSO) or an mTOR inhibitor (Torin-1, 1 μM, 3 hours) showing endogenous TFEB and MITF proteins in green. (B) Human 501Mel cells expressing doxycycline-inducible GFP-tagged MITF-M or TFEB were treated with vehicle (DMSO) or Torin-1, then fixed for GFP imaging. (C) The expression of MITF as determined by RT-qPCR upon overexpression of MITF(-) or TFEB with or without mTOR inhibition for 3 hours (Torin-1, 1 μM). (D) The expression of TFEB as determined by RT-qPCR upon overexpression of MITF(-) with or without mTOR inhibition (Torin-1, 1 μM, 3 hours) compared to vehicle (DMSO). Error bars represent SEM. * indicates significance at p<0.05. “ns” indicates no significance at p>0.05.

Fig 6. Model of the cross-regulatory relationship between MITF and TFEB in melanoma.

MITF can inhibit its own gene expression and increase that of TFEB. TFEB can inhibit the gene expression of MITF. The nuclear localization of TFEB is increased upon mTOR inhibition, which enhances its inhibitory effects on MITF gene expression. In contrast, the MITF-M isoform expressed in melanoma cells is predominantly nuclear under basal conditions and its activity is less affected by mTOR regulation.