Restoration of Mitf isoform expression in Mitf^−/− BMMCs. Mitf^−/− BMMCs were transduced with modified pMMP retroviruses that express the mast cell isoform (Mitf-mc), the e-isoform (Mitf-e), and the a-isoform (Mitf-a) (e-isoform). Transduced cells were stably selected in puromycin. The Western blot analysis shows protein expression of each of the individual Mitf isoforms and the vector control. Protein expression in wild-type (Mitf^+/+) and Mitf^−/− BMMCs are also shown. The Western blot for anti-tubulin (α tubulin) expression demonstrated comparable loading of samples.

Mitf isoforms restore SCF-directed migration in Mitf^−/− cells. BMMCs were placed in Transwell chambers coated with fibronectin or BSA as a control (described in Material and Methods). Chambers were placed in medium with or without SCF. Migration and binding to fibronectin was quantified by counting adherent cells on an inverted microscope at X40 and indicated on y-axis by cells migrated per HPF (high-powered field). In wild-type cells (Mitf^+/+), binding to fibronectin was dependent on SCF treatment (□); in Mitf^−/− cells, binding was severely impaired. Expression of the mast cell isoform (Mitf-mc), the e-isoform (Mitf-e), and the a-isoform (Mitf-a) restored SCF-dependent binding. Data are expressed as the mean result of experiments performed in triplicate. Error bars indicate SD.

Mitf isoforms regulate protease gene expression. Quantitative PCR analysis of protease genes in Mitf isoform cells compared with vector alone, wild-type (Mitf ^+/+), and Mitf^−/− cells. Expression is normalized to β-actin. All Mitf isoforms regulate the expression of mMCP-1, -2, -4, -5, -6, and granzyme B. mMCP-8 and cathepsin G are preferentially regulated by the mast cell and e-isoforms. Values (Relative Expression) are expressed as relative ratios to expression of Mitf^−/− cells on the y-axis (described in Materials and Methods). Data are expressed as the mean result of experiments performed in triplicate. Error bars indicate SD.

Mitf isoforms regulate genes involved in cell adhesion and migration. A, Quantitative PCR analysis of cell adhesion and migration genes in Mitf isoform cells compared with vector alone, wild-type (Mitf ^+/+), and Mitf^−/− cells. Expression is normalized to β-actin. All Mitf isoforms regulate the expression of SgIGSF4 and α_E integrin. However, only the mast cell and e-isoforms restores α₄ integrin expression. Values (Relative Expression) are expressed as relative ratios to expression of Mitf^−/− cells on the y-axis (described in Materials and Methods). Data are expressed as the mean result of experiments performed in triplicate. Error bars indicate SD. B, Flow cytometry analysis for α₄ integrin (Vla4) shows a moderate protein expression in Mitf ^+/+ cells and no detectable expression in Mitf^−/− cells. The mast cell and e-isoforms up-regulate α₄ expression, whereas the a-isoform does not, consistent with quantitative PCR data. Dark shaded histogram denotes isotype Ab control.

Mitf isoform expression in mast cells. A, RT-PCR analysis of in vitro-differentiated mast cells from murine embryonic stem cells shows expression of the mast cell isoform (mast), e-isoform (E-form), and a-isoform (A-form). Wright-Geimsa stain of cytospin preparations from differentiated cells at weekly time points above (original magnification, X60). Mast cell isoform and e-isoform expression is detectable with the appearance of morphologically identifiable mast cells at wk 3 and wk 4. B, RT-PCR analysis of specific Mitf isoforms from various tissue sources. Primary bone marrow-derived wild-type (Mitf^+/+) mast cells (1°BMMC), intestinal mast cells, and the mast cell line, C57, show expression of mast cell, e- and a-isoforms. The heart isoform is also detected from intestinal cells. The melanoma cell line (B16), macrophage cell line (RAW), and heart tissue are used for controls for the heart, melanocyte, and a-isoforms. Specific isoforms are not detected from peritoneal mast cells, and no Mitf expression is detectable from Mitf^−/− BMMCs. C, Schematic representation of protein domains of the Mitf isoforms expressed in mast cells show the unique amino-terminal domains of the mast cell isoform (▤, 43 aa) and the a-isoform (▨, 35 aa). The contiguous B-domain ( , 83 aa) is shared between mast cell and a-isoform. The e-isoform harbors a truncated B domain amino terminus (66 aa). All isoforms share the transactivation domain (TAD), as well as the DNA binding basic domain and helix-loop-helix leucine zipper dimerization motif (b-hlh-zip).

Correction of hypogranular phenotype by Mitf isoform expression in Mitf^−/− mast cells. A, Toluidine blue stains of cytospin preparations of wild-type (Mitf^+/+) and Mitf^−/− BMMCs demonstrate the hypogranular phenotype of Mitf^−/− cells. BMMCs expressing the mast cell isoform (Mitf-mc), the e-isoform (Mitf-e), and the a-isoform (Mitf-a) demonstrate restoration of the granular phenotype by all isoforms, but most notably by the mast cell and a-isoform. Magnification at X60. B, Flow cytometry analysis demonstrates heterogeneous granule expression by the wide range of SSC signal in Mitf ^+/+ cells. All Mitf isoforms restore heterogenous granule expression as shown by SSC signal. FSC denotes forward scatter, which is a measure of cell size.

Microarray analysis of BMMCs expressing Mitf isoforms. Gene sets that were up-regulated by individual Mitf isoforms compared with the vector control were identified by microarray analysis using the Affymetrix MOE430A chips, which contain probe sets for 22,223 genes. The Venn diagram shows the number of shared and unique gene sets regulated by the Mitf isoforms. Probe sets were annotated with the GeneSpring software. The shared genes (white) are listed in Table I. Unique gene sets are available in the supplemental material.

Mitf isoforms regulate genes involved in signaling. A, Quantitative PCR analysis of genes encoding signaling proteins in Mitf isoform-cells compared with vector alone, wild-type (Mitf ^+/+), and Mitf^−/− cells. Expression is normalized to β-actin. All Mitf isoforms regulate the expression of RSK and SLAP. The receptor tyrosine kinase, c-Kit mRNA is increased about 4-fold in wild-type cells (^+/+) compared with Mitf^−/− cells; however, only the a-isoform restores c-Kit expression to wild-type levels. Values (Relative Expression) are expressed as relative ratios to expression of Mitf^−/− cells on the y-axis (described in Materials and Methods). Data are expressed as the mean result of experiments performed in triplicate. Error bars indicate SD. B, Flow cytometry analysis for c-Kit protein expression shows a moderate impairment of c-Kit in Mitf^−/− cells (dark line) compared with Mitf ^+/+ cells (shaded histogram). Only the a-isoform restores c-Kit expression to wild-type levels, consistent with quantitative PCR data. Dark shaded histogram denotes isotype Ab control.