Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: Genome-wide Binding by MITF (A) Genome browser screenshots derived from ChIP-seq using anti-HA antibody of 501mel cells stably expressing ectopic HA-tagged MITF. (B) Consensus motif for the most significant 900 genome-wide MITF-binding sites predicted from 60-bp regions around peak summits generated by MEME. (C) The proportion of peaks with or without a 5′-TCA(T/C)GTGN-3′ motif at different peak heights. (D) Relationship between motif frequency and peak height as in (C). (E) Sequences associated with a selection of differentiation or non-differentiation-associated MITF target genes. (F) Box and whisker plots of peak height related to motif. Center of notches indicates the median. Green box indicates range of peak heights within which lie a set of well-characterized differentiation-associated genes in addition to many other non-differentiation genes. See also Figure S1 and .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Genome Wide, Binding Assay, Derivative Assay, ChIP-sequencing, Stable Transfection, Expressing, Generated, Selection, Whisker Assay

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: MITF Can Be Acetylated (A) Indicated expression vectors were transfected into Phoenix cells and input and anti-FLAG immunoprecipitates western blotted. (B) Western blot of 501mel cells treated with 200 nM TPA for indicated times. (C) Western blot of extracts from cells transfected with BRAF and/or p300 expression vectors. (D) Western blot of Phoenix cells transfected with indicated vectors and HA-MITF, ±20 μM U0126 immunoprecipitated using anti-HA antibody. (E) Schematic showing the melanocyte-specific MITF-M(+) isoform. The five acetylated lysine residues identified in MITF-M peptides by mass spectrometry are indicated below. ERK, p38, and RSK phosphorylation sites are indicated above with the CBP/p300-binding site. (F) MITF DNA-binding domain-DNA co-crystal structure showing the MITF K243-DNA phosphate-backbone contact. (G) Conservation of K243 between bHLH and bHLH-LZ family members. (H) Peptide array containing indicated residues as 14-amino-acid peptides immobilized on a cellulose membrane probed with rabbit anti-acetyl-K243 antibody. (I) Western blot using anti-acetyl K243 or anti-MITF antibodies of immunoprecipitated GFP-MITF expressed alone or with co-transfected CBP or p300. (J) Western blot using anti-acetyl K243 or anti-MITF antibodies of HIS-tagged MITF purified with nickel beads. All samples were from the same blot. See also Figure S2 .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Mass Spectrometry, Phospho-proteomics, Binding Assay, Peptide Microarray, Membrane, Purification

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: K243 Status Determines MITF DNA-Binding Affinity (A) Comparison of circular dichroism (CD) spectra of bacterially expressed and purified MITF WT and mutant DNA-binding domains. The mean residue ellipticity is plotted in dg × cm 2 × dmol −1 against the wavelength (in nm). CD spectra show the mutations cause no major structural changes. (B) DNA-binding affinity of bacterially expressed and purified MITF WT and mutant DNA-binding domains determined using fluorescence anisotropy. Representative titration curves of each fluorescein-labeled oligonucleotide with MITF WT and mutants. The anisotropy values are the average of triplicate measurements from which the baseline corresponding to the anisotropy of the free fluorescent probe was subtracted. (C) The dissociation constants of MITF WT and mutants on oligonucleotides containing four different recognition sequences determined by fluorescence anisotropy.

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Binding Assay, Comparison, Circular Dichroism, Purification, Mutagenesis, Residue, Fluorescence, Titration, Labeling

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: K243 Controls MITF Function In Vivo (A) Complementation of neural crest MITFa- null nacre zebrafish using MITF WT and K238 (equivalent to K243 in human MITF) mutants (left) and quantification of numbers of melanocytes (right). The dots in the plots represent numbers of melanocytes in each rescued embryo with at least one melanocyte. See also Figure S3 . (B) Western blot of 501mel cells stably expressing HA-MITF WT and mutants (from the same gel). (C) Tumor formation after subcutaneous inoculation of indicated cell lines into athymic nude mice. (D) Example tumors. (E) Tumor size over time using indicated cell lines. Error bars indicate S.E.M.

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: In Vivo, Western Blot, Stable Transfection, Expressing

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: K243 Status Determines MITF Genome-wide Distribution (A) Heatmap of MITF WT and K243 mutant average tag density derived from two biological replicate ChIP-seq experiments of HA-tagged MITF expressed using 0 or 20 ng doxycycline centered on WT occupied regions (20 ng doxycycline). (B) Numbers of ChIP peaks called using HA-tagged MITF WT or mutants induced using 0 or 20 ng doxycycline. See also . (C) Read coverage of two replicates for each of the WT and K243 mutant ChIP-seq experiments expressed using 0 or 20 ng doxycycline centered around peak coordinates of the WT at 5-bp binning intervals. Numbers on the x axis indicate distance from center of the peak (in bp). (D) Genome browser screenshots of indicated loci showing HA-tagged WT and mutant MITF ChIP-seq profiles from iMITF cell lines expressing HA-tagged MITF at 0 or 20 ng doxycycline as indicated. (E) Box and whisker plots showing peak score for two replicate (R1 and R2) ChIP-seq experiments for the WT and two K243 mutants related to the indicated motifs. Expression of HA-MITF WT and mutants induced at 0 or 20 ng doxycycline. Colored line indicates median, and black line indicates mean. See also Figure S4 .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Genome Wide, Mutagenesis, Derivative Assay, ChIP-sequencing, Expressing, Whisker Assay

Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet: Live-Cell Single-Molecule Tracking (SMT) of HALO-Tagged MITF (A) HALO-tagged MITF expression vectors. NLS indicates the nuclear localization sequence. Δbasic lacks residues required for DNA binding. (B) Exemplary frames of SMT movies using WT and mutant HALO-tagged MITF, collected at 100 fps (see also , , , and ). Scale bar, 5 μm. Labeling with 100 pM Halotag JF 594 allows particle densities in the range of a few molecules per frame. (C) SMT movies were tracked to generate a distribution of single-molecule displacements between consecutive frames that was fit with a three-component model (one immobile component and two diffusing components) to provide quantitative estimates for WT MITF and mutants shown in (D) and (E). Cmp, component. (D) Quantitative estimates derived from SMT using WT and mutant HALO-tagged MITF for the fraction of molecules in each state. Error bars indicate SD. (E) Quantitative estimates of the diffusion coefficients of free molecules. For MITF WT, Δ basic, K243Q, and K243R, respectively, N c e l l s = 20 , 6 , 15 , 15 ; N d i s p l a c e m e n t s = 17802 , 2684 , 16422 , 12999 . Error bars indicate SD. (F) Summary derived from the SMT analysis of proportion of MITF calculated to bind high- versus low-affinity sites. (G) Electrophoretic mobility shift assay (EMSA) using bacterially expressed and purified WT and mutant MITF DNA-binding domains, a 30-bp TCACGTGA-motif-containing probe, and competition with 4-fold dilutions of SSD (10 μg to 2.3 fg). Bound DNA is shown. Probe was in excess in all reactions. (H) EMSA as in (G) with competition by indicated competitor oligonucleotides at 3, 10, and 30 ng. Bound DNA is shown. See also and and , , , and .

Article Snippet: GFP-tagged MITF were purified using GFP-trap (Chromotek Cat#gtma-100) from transiently transfected Phoenix-ampho with indicated plasmids (total 6 μg DNA in 6 cm dish) using Fugene 6.

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Labeling, Derivative Assay, Diffusion-based Assay, Electrophoretic Mobility Shift Assay, Purification