Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M512052200 on April 28, 2006

J. Biol. Chem., Vol. 281, Issue 29, 20233-20241, July 21, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/29/20233    most recent
M512052200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de la Serna, I. L.
Right arrow Articles by Imbalzano, A. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de la Serna, I. L.
Right arrow Articles by Imbalzano, A. N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Microphthalmia-associated Transcription Factor Requires SWI/SNF Enzymes to Activate Melanocyte-specific Genes*

Ivana L. de la Serna{ddagger}§1, Yasuyuki Ohkawa{ddagger}, Chiduru Higashi, Chaitali Dutta{ddagger}, Jules Osias{ddagger}, Naveen Kommajosyula{ddagger}, Taro Tachibana, and Anthony N. Imbalzano{ddagger}

From the {ddagger}Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, the §Department of Biochemistry and Cancer Biology, Medical University of Ohio, Toledo, Ohio 43614, and the Department of Bioengineering, Osaka City University, Osaka 558-8585, Japan

Received for publication, November 8, 2005 , and in revised form, April 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The microphthalmia transcription factor (Mitf) activates melanocyte-specific gene expression, is critical for survival and proliferation of melanocytes during development, and has been described as an oncogene in malignant melanoma. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that play a role in many developmental processes. To determine the requirement for SWI/SNF enzymes in melanocyte differentiation, we introduced Mitf into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, Brahma or Brahma-related gene 1 (BRG1). These dominant negative SWI/SNF components have been shown to inhibit gene activation events that normally require SWI/SNF enzymes. We found that Mitf-mediated activation of a subset of endogenous melanocyte-specific genes required SWI/SNF enzymes but that cell-cycle regulation occurred independently of SWI/SNF function. Activation of tyrosinase-related protein 1, a melanocyte-specific gene, correlated with SWI/SNF-dependent changes in chromatin accessibility at the endogenous locus. Both BRG1 and Mitf could be localized to the tyrosinase-related protein 1 and tyrosinase promoters by chromatin immunoprecipitation, whereas immunofluorescence and immunoprecipitation experiments indicate that Mitf and BRG1 co-localized in the nucleus and physically interacted. Together these results suggest that Mitf can recruit SWI/SNF enzymes to melanocyte-specific promoters for the activation of gene expression via induced changes in chromatin structure at endogenous loci.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Melanocytes are pigment-producing cells that are developmentally derived from the neural crest and that comprise 1-2% of the epidermis (1). They are also present on the epithelial surfaces of mucous membranes, hair follicles, the cochlea of the inner ear, and both the uvea and conjunctiva of the eye (2, 3). On the skin, they play a photoprotective role by synthesizing and distributing melanin (4). Excessive exposure to UV radiation has been linked to the transformation of cutaneous melanocytes to melanoma, a cancer that has steadily increased in frequency and is difficult to treat (5, 6).

Microphthalmia-associated transcription factor (Mitf)2 is the "master regulator" of melanocyte differentiation and was elegantly shown to convert fibroblasts into dendritic cells that express melanocyte-specific genes (7). It is important for the commitment, proliferation, and survival of melanocytes during neural crest cell migration, and null mutations of the mouse Mitf gene result in complete absence of melanocytes and lack of pigmentation in the skin, eyes, and inner ear (8, 9). Two human diseases resulting from mutations in the MITF gene are Waardenburg type 2 syndrome and Tietz syndrome, both of which are characterized by pigmentary disturbances and sensineural deafness (8).

Mitf is a basic helix-loop-helix leucine zipper transcription factor that binds DNA either as a homodimer or as a heterodimer with TFE3, TFEB, or TFEC to conserved E boxes (CAC(G/A)TG) in the promoters of its target genes, which include genes encoding enzymes involved in melanin synthesis such as tyrosinase, tyrosinase-related protein 1 (Trp1) and tyrosinase-related protein 2 (Trp2 or Dct) (1, 10, 11). Several isoforms of Mitf with cell-specific distribution and activities have been identified in human and mouse cells, including the melanocyte-specific isoform, Mitf-M (12, 13). Moreover, Mitf can be alternatively spliced and post-translationally modified by phosphorylation and sumoylation; each variation affects activity and interactions with other proteins (14).

In addition to melanocyte-specific genes, Mitf has been shown to regulate a number of other genes, including those involved in cell proliferation, and likely plays a role in melanoma etiology and progression. Mitf can promote melanocyte proliferation by direct activation of the cdk2 promoter (15). Conversely, Mitf promotes cell-cycle arrest by direct activation of the p21Cip1 and p16Ink4A promoters and by counteracting B-RAF-stimulated melanocyte and melanoma proliferation (16-18). Mitf has also been designated a "lineage survival oncogene," because it can activate expression of the anti-apoptotic factor, Bcl2, and, with activated B-RAF, can transform primary human melanocytes (19, 20). Cleavage of Mitf by caspases produces a C-terminal product that can promote apoptosis (21). Recently, the alternatively spliced forms of Mitf have been shown to differ in their ability to inhibit cellular proliferation (22). Thus, the regulation of Mitf activity in melanocyte survival and proliferation is complex and most likely depends on the one or more particular isoforms expressed, post-translational modifications, interactions with other proteins, as well as the specific cellular context.

The coordinate activity of gene-specific activators and SWI/SNF chromatin-remodeling enzymes has been shown to be important for the activation of genes during cellular differentiation and for regulation of proliferation (23-30). Mammalian SWI/SNF enzymes are evolutionarily conserved, multiprotein complexes that contain one of two closely related ATPases, BRM or BRG1, and utilize the energy of ATP to disrupt chromatin structure (31, 32). The Brg1 subunit and some other SWI/SNF subunits, including Ini1, and Srg3/Baf155, have been shown to be essential for mouse development (33-37). Furthermore, SWI/SNF enzymes are critical for myogenesis, adipogenesis, neurogenesis, osteogenesis, and myeloid differentiation (23-29) and are involved in the regulation of the cell cycle (38-43).

Although Mitf has been shown to regulate a number of melanocyte-specific genes, little is known about how these genes are activated within their endogenous chromatin context. Mitf can interact with the histone acetyl transferase CBP/p300 to activate target genes; however, it is unclear whether activation of gene expression results from acetylation of histone proteins or by other mechanisms (44-46). In the current study, we demonstrate that SWI/SNF chromatin-remodeling enzymes are necessary for Mitf-mediated activation of melanocyte-specific genes. We show that BRG1 co-localizes and interacts with Mitf and is recruited to and remodels chromatin structure on the promoter of a melanocyte-specific gene. The data establish a requirement for specific chromatin remodeling enzymes during melanocyte-specific gene expression and melanocyte differentiation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Plasmids—The mouse plus isoform of the melanocyte-specific Mitf (Mitf-M) was generously provided by Thomas Hornyak (NCI, National Institutes of Health) and subcloned into the pBabe vector (9, 47). Control (tetVP16), dominant negative BRM (H17), and dominant negative BRG1 (B22) cell lines inducibly express ATPase-deficient, dominant negative alleles of BRM or BRG1 in a tetracycline-dependent manner (48). Cells were cultured in the presence (dominant negative expression OFF) or absence (dominant negative expression ON) of tetracycline for 3 days and were infected at 50% confluence with pBabe-Mitf retrovirus or with retrovirus generated from the empty pBabe vector as previously described (28) for 30 h. A low serum medium containing 2% horse serum and 2 µg/ml puromycin was then added, and cells were harvested 40 h later. B16 (F0) mouse melanoma cells were purchased from ATCC and maintained in media with 10% fetal calf serum.

RNA Analysis—RNA was isolated and reverse transcribed as previously described except that 1 µl of cDNA was amplified using Qiagen Master Mix (49). Mitf was amplified for 23 cycles; tyrosinase, Trp1, and Pmel17 were amplified for 29 cycles, Mc-1r for 31 cycles, Dct for 32 cycles, and Hprt for 27 cycles in the presence of [32P]dATP. The conditions for PCR were: 94 °C for 15 min, followed by the indicated number of cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min. Radiolabeled products were resolved on polyacrylamide gels and detected and quantified via PhosphorImager (Amersham Biosciences). The following primers used for reverse transcription-PCR have been previously described: Mitf (50); tyrosinase, Trp1, and Dct (7); Mc-1r (51); Pmel17 (52); and Hprt (49).

Fluorescence-activated Cell Sorting—Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry as described (49). The data were analyzed with FlowJo software (Tree Star Inc.).

Antibodies, Protein Extracts, and Western Analysis—Antibodies used were Mitf (C5, Abcam), phosphatidylinositol 3-kinase (06-496, Upstate), tetra-acetylated histone H4 (06-866, Upstate), and rabbit antisera against the FLAG epitope. Rabbit antisera to BRG1, INI1, (48), and BAF57 (53), MyoD (Santa Cruz) were used for immunoprecipitations, Westerns, and ChIP experiments. Supernatant from a rat hybridoma generated against a GST-BRG1 fusion protein (48) was used for immunocytochemistry without dilution. Isolation of protein and Western analysis were performed as previously described (48).

Immunoprecipitations—Cells were washed two times with phosphate-buffered saline (PBS) and lysed in TLB buffer (20 mM Tris-HCl, pH 7.8, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 0.5 mM dithiothreitol, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml bovine serum albumin) by passing cells through a syringe with a 27-gauge needle. Lysate was then centrifuged at 13,000 x g for 15 min. The supernatant was rocked with 8 µl of rabbit antisera against BRG1, BAF57, Ini1, or with the irrelevant antibody, MyoD, for 12 h, followed by the addition of protein A-Sepharose (Amersham Biosciences) and an additional incubation while rocking for 2 h. Beads were washed three times in lysis buffer and eluted with 2% SDS gel loading buffer.

Immunocytochemistry—Cells were plated on coverslips, washed twice with 0.1% Tween 20 in PBS, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, and washed twice with ice-cold PBS/0.1% Tween 20.A1h incubation with blocking buffer (10% goat serum/0.1% Tween 20 in PBS) was followed by an overnight incubation with rat anti-BRG1 monoclonal antibody and mouse anti-Mitf antibody (C-5, Abcam) at 4 °C. Coverslips were then washed three times with ice-cold PBS/0.1% Tween 20 and incubated with Alexa 488-labeled goat anti-mouse antibody and Alexa568-labeled goat anti rat antibody (Molecular Probes) for 30 min at room temperature. Coverslips were again washed three times in 0.1% Tween 20 in PBS and mounted in Prolong (Molecular Probes). Images were visualized using a confocal laser microscope (Leica). Co-localization efficiency was analyzed as described (54), except that the cross-correlation function (CCF) was calculated by rotating the red image over an angle {Delta}x°inthe x orientation with respect to the green image such that -180 ≤ {Delta}x ≤ 180. A negative value of {Delta}x indicates that the red image was rotated in the counterclockwise direction, and a positive value indicates a rotation in the clockwise direction. Pearson's correlation coefficient ({gamma}p) was calculated for each value of {Delta}x and plotted against {Delta}x to get the CCF (54).


Figure 1
View larger version (71K):
[in this window]
[in a new window]
  		FIGURE 1.
Dominant negative BRM and BRG1 inhibit induction of a subset of melanocyte-specific genes by Mitf. Control cell lines (tetVP16), or cell lines that express dominant negative BRM (H17) or BRG1 (B22) were either infected with a pBabe control vector or pBabe-Mitf in the presence or absence of tetracycline and then cultured in low serum media to promote melanocyte differentiation. A, Western blot showing expression of Mitf in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRM and BRG1 in the absence of tetracycline. Phosphati-dylinositol 3-kinase was used as a loading control. B, reverse transcription-PCR of Mitf and Mitf target genes from the same pBabe- or pBabe-Mitf-infected cells. Hprt was used as a loading control. The data shown is representative of at least three independent experiments.

 
ChIP Experiments—ChIP experiments were performed as previously described (55) with the antibodies listed above. Primers to amplify promoter regions were 5'-CAGCGTCTCTAATACATCTTCC-3' and 5'-GCCCGAAGAGATTTTCTGCCAGAC-3' for Trp1, 5'-AGTCATGTGCTTTGCAGAAGAT-3' and 5'-CAGCCAAGAACATTTTCTCCTT-3' for tyrosinase, and 5'-CACTACACATCCCTGGCACA-3' and 5'-CCCAAGTGGCTCAGGTAGAG-3' for Mc-1r. Primers to the immunoglobulin H (IgH) enhancer were previously described (56).

ChIP experiments were also quantified by real-time PCR using a Hot Start Sybr Green Master Mix (Qiagen) and amplified on a 7500 real-time Thermocycler (ABI). Threshold cycle values were determined for each ChIP and standardized to their respective inputs using the 7500 SDS software.

Restriction Enzyme Accessibility—Restriction enzyme accessibility experiments using a ligation-mediated (LM)-PCR protocol were performed as previously described (55). Briefly, nuclei were isolated and digested with either DraI or PvuII, and the genomic DNA was purified. One microgram of digested DNA was ligated to the LM-PCR1 and LM-PCR2 adaptors using the Takara Ligation Kit version 2 (57). PCRs were performed at 94 °C for 15 min, followed by 24 cycles of 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min in the presence of [32P]dATP; the products were run on a 5% polyacrylamide gel then detected and quantified with a PhosphorImager (Amersham Biosciences). The primers used for detection of Trp1 were LM-PCR1 (57) and a primer in the upstream region of Trp1: 5'-GAGGCTGGCATCCATATGTCAGTCAAAG-3'. Inputs were monitored by amplifying the same samples used for LM-PCR with the primers used in the ChIP assay. The primers to myogenin were previously described (55).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transcription of a Subset of Melanocyte-specific Genes Is Inhibited by Dominant Negative BRM and BRG1—Mitf has been shown to regulate a number of melanocyte-specific genes, including those involved in melanin biosynthesis (tyrosinase, Trp1, and Dct). Mitf also regulates the melanocortin-1 receptor (Mc-1r) and Pmel17, a membrane glycoprotein important for melanosome structure (58-61). Ectopic expression of Mitf into NIH 3T3 fibroblasts causes differentiation into melanocyte-like cells that are dendritic and that express melanocyte-specific genes (7).

We previously described fibroblast cell lines that inducibly express dominant negative versions of BRM or BRG1 under the control of the tetVP16 activator (48). To determine whether SWI/SNF enzymes play a role in melanocyte-specific gene expression, we introduced Mitf by retroviral infection into a control tetVP16 cell line, a dominant negative BRM cell line (H17), and a dominant negative BRG1 cell line (B22) that had been grown in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. Fig. 1A shows FLAG-tagged dominant negative BRM and BRG1 expression in H17 and B22 cells, respectively, when the cells were cultured in the absence of tetracycline and demonstrates that ectopic expression of the Mitf protein occurred. Mitf is phosphorylated at multiple sites, and the two bands that we detected most likely represent the hypo- and hyperphosphorylated forms (62-65). Dominant negative BRM and BRG1 expression did not inhibit the ectopic expression of Mitf, consistent with previous work showing that these dominant negative proteins did not affect expression of numerous regulatory factors introduced by pBABE-derived retroviruses (28, 29, 49, 55, 66).

A number of Mitf target genes were induced in the control tetVP16 cell lines differentiated in the presence or absence of tetracycline and in the dominant negative cell lines differentiated in the presence of tetracycline. However, when H17 and B22 cells were differentiated in the absence of tetracycline, expression of most of these genes was inhibited by the presence of dominant negative BRM and BRG1 (Fig. 1B). Mc-1r was the only gene examined that was expressed but not inhibited by the presence of dominant negative BRM or BRG1 (Fig. 1B). We conclude that a subset of Mitf target genes require SWI/SNF enzymes for activation.

Cell-cycle Arrest Occurs Independently of SWI/SNF Enzymes—For many cell types, terminal differentiation is characterized by withdrawal from the cell cycle in G1. Mitf induces p21CIP1 and p16Ink4A (16, 17), both inhibitors of cell-cycle progression, however, it is not known if this is required for expression of melanocyte-specific genes. Mitf has been shown to interact with the retinoblastoma protein (pRb), and it was previously reported that low serum conditions promote expression of melanocyte-specific genes when Mitf is introduced into fibroblasts, suggesting that cell-cycle arrest is important (7, 67). SWI/SNF enzymes also have been shown to activate both the p21CIP1 and p16Ink4A promoters and to promote pRb-mediated cell-cycle arrest in some cell types (40, 41, 68, 69). Therefore, to determine whether SWI/SNF enzymes are required for cell-cycle withdrawal during Mitf-mediated differentiation, we differentiated cells with Mitf in low serum media in the presence or absence of dominant negative BRM or BRG1 and stained cells with propidium iodide to determine the number of cells in the different phases of the cell cycle. Fig. 2 shows that the expression of dominant negative BRM or BRG1 did not affect the ability of differentiated cells to arrest in the G1 phase of the cell cycle and suggests that SWI/SNF enzymes can promote activation of melanocyte-specific genes by Mitf but do not mediate changes in gene expression that promote cell-cycle withdrawal.


Figure 2
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2.
SWI/SNF enzymes are not required for cell-cycle withdrawal during Mitf-mediated differentiation. TetVP16, H17, and B22 cells were infected with either an empty pBabe vector or with pBabe-Mitf in the presence or absence of tetracycline and differentiated. As a control, proliferating cells were cultured in growth media. Propidium iodine-stained samples were sorted (with fluorescence-activated cell sorting) and analyzed using FlowJo (Tree Star Inc.). Data are presented as bar graphs showing the averages and standard deviations of three independent experiments.

 


Figure 3
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3.
Mitf induces an increase in restriction enzyme accessibility at the endogenous Trp1 gene in a SWI/SNF-dependent manner. Nuclei were isolated from uninfected or Mitf-infected B22 cells that were differentiated in low serum media in the presence or absence of tetracycline for 40 h. Nuclei were digested with either DraI or PvuII, and cleaved genomic DNA was then visualized using a modified LM-PCR protocol as previously described (55). A, a restriction map of the upstream and downstream regions flanking the start site of the Trp1 gene. The positions of the DraI and PvuII sites and the Mitf binding site are shown relative to the start site of transcription. The arrows indicate the positions of the upstream and downstream primers used for PCR amplification. B, PCR products resulting from cleaved and ligated samples. To monitor input DNA, 5% of the purified, cleaved DNA used for LM-PCR was subjected to PCR with primers flanking the site of ligation between -134 and +100. These data are representative of three independent experiments. C, as a control, the PvuII-digested samples were amplified with primers to the transcriptionally silent myogenin locus (55).

 
SWI/SNF Enzymes Promote Mitf-dependent Chromatin Remodeling on a Melanocyte-specific Gene—To determine whether the requirement for SWI/SNF enzymes in the activation of melanocyte-specific genes was due to direct remodeling of chromatin structure on the promoters of these genes, we analyzed the chromatin structure at the endogenous Trp1 promoter by performing in vivo restriction enzyme accessibility experiments. We found that the expression of Mitf resulted in an increase in restriction enzyme accessibility upstream of the start site of transcription at a DraI cleavage site located at -271 and at a PvuII cleavage site downstream in the coding region at +107 and that expression of dominant negative BRG1 inhibited the increase in accessibility at both sites (Fig. 3, A and B). In contrast, no increase in accessibility was observed at a PvuII site located in the promoter region of the endogenous myogenin locus in these cells (Fig. 3C). This observation is consistent with the lack of myogenin expression in Mitf-differentiated cells (data not shown). Therefore, SWI/SNF-dependent activation of a melanocyte-specific gene by Mitf is correlated with remodeling of chromatin structure at its promoter.

Mitf Promotes Recruitment of SWI/SNF Enzymes to and Histone Hyperacetylation of a Melanocyte-specific Promoter—To further support the idea that SWI/SNF enzymes activate melanocyte-specific gene expression by directly remodeling chromatin structure, we performed chromatin immunoprecipitation (ChIP) experiments to determine whether the Brg1 ATPase was interacting with the Trp1 promoter. Semiquantitative ChIP analysis in Fig. 4A shows that Brg1 was associated with the Trp1 promoter in the presence or absence of tetracycline and that the association was dependent on the expression of Mitf. Epitope (FLAG)-tagged dominant negative BRG1 expressed in cells grown in the absence of tetracycline was also bound to the Trp1 promoter in Mitf-differentiated cells. Furthermore, the interaction of Mitf with the Trp1 promoter occurred independently of functional SWI/SNF enzymes, suggesting that SWI/SNF enzymes are recruited to the Trp1 promoter by Mitf. The specificity of these ChIP experiments is demonstrated by the lack of association with the IgH enhancer, even though this region contains a potential Mitf binding site (Fig. 4B).

We also analyzed binding of Brg1 to the Trp1, tyrosinase, and Mc-1r promoters using real-time PCR and found that Brg1 was associated with both the Trp1 and tyrosinase promoters but not with the Mc-1r promoter (Fig. 4C). This is consistent with data showing that Trp1 and tyrosinase gene expression required functional SWI/SNF enzymes, whereas Mc-1r expression occurred independently of functional SWI/SNF enzymes (Fig. 1B). The Brg1 antibody, which recognizes both wild-type Brg1 and dominant negative BRG1, detected Brg1 on the Trp1 and tyrosinase promoters irrespective of the presence or absence of tetracycline, suggesting that dominant negative BRG1 is associated with its target promoters.


Figure 4
View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 4.
Brg1 and hyperacetylated histone H4 are associated with the Trp1 promoter as a result of Mitf-mediated differentiation. Chromatin immunoprecipitations were performed with an irrelevant rabbit polyclonal antibody (MyoD, which is not expressed in these cells) or with antisera or antibodies to Mitf, Brg1, FLAG-dnBRG1, and tetra-acetylated histone H4 (AcH4). A and B, ChIP experiments were performed on undifferentiated or Mitf-differentiated B22 cells that had been cultured in the presence or absence of tetracycline. Following differentiation in low serum media for 40 h, samples were harvested. 1% of the input DNA is shown. A 2-fold titration of input DNA was performed to demonstrate that the PCR was in the linear range. A, detection of protein interactions on the Trp1 promoter. These data are representative of two independent experiments each analyzed in duplicate. B, as a control, the silent IgH enhancer was amplified. C, real-time analysis of ChIP experiments from two independent experiments done in duplicate. The average and standard deviations are shown for the protein interactions on the Trp1, tyrosinase, Mc-1r, and IgH regulatory regions.

 
To confirm that dominant negative BRG1 was associated with the Trp1 and tyrosinase promoters, we performed ChIP experiments using a FLAG antibody that detects the FLAG-tagged dominant negative protein. We found that FLAG-dnBRG1 was localized on the tyrosinase and Trp1 promoters but not on the Mc-1r promoter (Fig. 4C). This observation is consistent with previous data showing that dominant negative BRG1 can form a complex with other SWI/SNF components and with gene-specific activators that recruit a non-functional SWI/SNF complex to target genes (48, 55). Because SWI/SNF complexes with a mutation in the ATPase subunit are non-functional, they cannot remodel chromatin structure and their presence inhibits gene activation events that require its chromatin-remodeling activity.

Hyperacetylation of histones H3 and H4 is often correlated with gene activation, and the bromodomains of ATP-dependent chromatin-remodeling enzymes have been shown to preferentially interact with acetylated histone proteins (70). We found that activation of gene expression by Mitf correlated with the association of Mitf and hyperacetylation of histone H4 on the Trp1 and tyrosinase promoters in both the presence and absence of tetracycline. Thus, H4 hyperacetylation occurred independently of SWI/SNF enzyme function, consistent with previous studies showing that, in mammalian cells, histone acetylation occurred at specific regulatory sequences prior to the association of SWI/SNF enzymes (29, 55, 71-74).

Consistent with previous work showing that Mitf activates and binds to a promoter region containing a potential Mitf binding site (58, 59), Mitf was also associated with the endogenous Mc-1r promoter, and activation of the Mc-1r gene correlated with hyperacetylation of histone H4. Thus, Mitf may recruit histone acetyltransferases but not SWI/SNF enzymes to the Mc-1r promoter. The specificity of these promoter interactions was demonstrated by the lack of any interaction at the IgH enhancer, which contains an E box but is not known to be regulated by Mitf (Fig. 4C) (56).

To corroborate our results in the Mitf-differentiated fibroblasts, we investigated whether SWI/SNF enzymes could interact with the Trp1 promoter in a melanocyte-derived cell line. The B16 cell line is a mouse melanoma cell line that is pigmented and that expresses Trp1 (7). Initial semiquantitative ChIP analysis using a Brg1 antibody showed that Brg1 was associated with the Trp1 promoter in B16 cells (Fig. 5A), suggesting that SWI/SNF enzymes are involved in the expression of Trp1 in cells that normally express this gene.

Additional ChIP experiments analyzed with real-time PCR showed that two other SWI/SNF components, BAF 57 and Ini1, as well as Brg1 were associated with the Trp1 promoter (Fig. 5B). The association of these SWI/SNF subunits correlated with localization of Mitf on the Trp1 promoter, suggesting that Mitf recruits the SWI/SNF complex to target genes.

Mitf Co-localizes and Physically Interacts with SWI/SNF Enzymes—SWI/SNF enzymes do not possess DNA binding sequence specificity and are thought to be recruited to promoters by gene-specific activators. Both chromatin remodeling and localization of Brg1 on the Trp1 promoter were Mitf-dependent, suggesting that Mitf recruits SWI/SNF to its target promoters. To address this possibility, we used immunocytochemistry and immunoprecipitation to determine whether Mitf and Brg1 co-localize in the nucleus and physically interact. Immunocytochemistry revealed that both Mitf and Brg1 are associated with euchromatic regions of the nucleus and that some Mitf co-localized with Brg1 both in the presence and absence of tetracycline (Fig. 6A). Limited co-localization would be expected, because Brg1 has been reported to regulate many cellular genes. To determine whether the observed overlap between Mitf and BRG1 was randomly distributed or positively correlated, we quantified the degree of co-localization by cross-correlation analysis (54). In cross-correlation analysis, a cross-correlation function (CCF) with a negative peak indicates exclusion, a flat CCF indicates random distribution, and a CCF with a positive peak indicates non-random overlap (54). The CCFs obtained show positive peaks, for cells differentiated in both the presence and absence of tetracycline, which indicates that there was significant, non-random overlap between the red (Brg1) and the green (Mitf). Furthermore, the results show that dominant negative BRG1 did not affect co-localization (Fig. 6A).


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 5.
In B16 cells, the interaction of SWI/SNF proteins with the Trp1 promoter correlates with Mitf binding. Chromatin immunoprecipitations were performed with an irrelevant rabbit polyclonal antibody (MyoD, which is not expressed in these cells) or with antisera or antibodies to Brg1, Baf 57, Ini1, or Mitf. A, ChIP indicating the presence of Brg1 on the Trp1 promoter but not on the control IgH enhancer in B16 melanoma cells. B, real-time PCR analysis of Brg1, Baf57, Ini1, and Mitf ChIP experiments on the Trp1 promoter. The values presented are relative to that on the IgH enhancer.

 
To determine whether Mitf and Brg1 can physically interact, we immunoprecipitated Brg1 and looked for the presence of Mitf. We found that endogenous Mitf could be co-immunoprecipitated with endogenous Brg1, Baf57, and Ini1, thereby demonstrating an interaction between Mitf and multiple members of the SWI/SNF complex (Fig. 6B). The co-localization of Mitf and Brg1 as well as the co-immunoprecipitation of Mitf, Brg1, Baf57, and Ini1 support the hypothesis that Mitf recruits SWI/SNF enzymes to melanocyte-specific genes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Ectopic expression of the melanocyte-specific isoform of human MITF was previously shown to convert fibroblasts into cells with melanocytic characteristics (7). Since that initial demonstration, there have been many studies describing activation of reporter genes by ectopic expression of Mitf into non-melanocytic cells, but there have only been a few additional reports describing induction of endogenous genes. To determine the requirement for SWI/SNF chromatin-remodeling enzymes in melanocyte differentiation, we expressed mouse Mitf-M in fibroblasts that inducibly express dominant negative versions of BRM or BRG1 and looked at the expression of endogenous melanocyte-specific genes. Similar to the original report using human MITF, we found that mouse Mitf could activate expression of tyrosinase and Trp1, two members of the tyrosinase gene family that encode enzymes involved in melanin biosynthesis. We also found that dopachrome tautomerase (Dct/Trp2), a third member of this gene family, was activated, whereas the original report did not detect any activation of Dct (7). Dct is activated earlier in development than tyrosinase or Trp1, and its regulation may differ from that of the other two members of the tyrosinase gene family (75). Transfection of human MITF into some human cell lines was unable to activate the Dct promoter unless Lef-1 was co-transfected, whereas mouse Mitf could significantly activate expression of Dct (76-78). Thus, the mouse and human forms of Mitf may differ in their ability to transactivate the Dct promoter. Recently, Mitf, Sox10, and Lef-1 have been shown to synergistically transactivate the Dct promoter to much higher levels than Mitf alone (77, 79). Regardless, our results clearly show that the activation of Dct and the other members of the tyrosinase gene family by Mitf was highly dependent on SWI/SNF enzymes.

We found that ectopic expression of Mitf could also induce expression of Pmel17. Pmel17 has been shown to be a direct target of Mitf and to be induced by ectopic expression of Mitf in fibroblasts (52, 61). Pmel17 is the product of the silver locus and plays a role in melanosome structure (80). The human protein has been used as a target in melanoma immunotherapy, therefore regulation of Pmel17 expression is of particular interest (81). Our work further describes the regulation of Pmel17 by showing that Pmel17 gene expression requires the activity of SWI/SNF enzymes.

The {alpha}-melanocyte-stimulating hormone is required for expression of many melanocyte-specific genes. It binds to the melanocortin-1 receptor (Mc-1r), a member of a subfamily of G protein-coupled receptors that is expressed in melanocytes and other cells (82). Mitf has been shown to activate the Mc-1r promoter in both melanocytes and mast cells (58, 59); therefore, we tested whether Mitf could activate Mc-1r expression in fibroblasts and whether its expression required SWI/SNF enzymes. We found that Mc-1r was expressed in fibroblasts at low levels and was activated by Mitf in an SWI/SNF-independent manner. ChIP analysis confirmed the association of Mitf with Mc-1r but did not show significant enrichment of Brg1 on the Mc-1r promoter, suggesting that its association with the promoter is either less stable than with the Trp1 and tyrosinase promoters or that Mitf does not recruit SWI/SNF enzymes to the Mc-1r promoter.

The data also indicate that only a subset of Mitf-regulated genes requires SWI/SNF. These results are in agreement with previous studies indicating that SWI/SNF enzymes are required for the induction of some, but not all genes activated by MyoD during muscle differentiation (55) and are required for the expression of a subset of genes activated during myeloid differentiation (24). The data suggest that SWI/SNF enzymes are required for activation of specific genes during differentiation but not all of the genes that are activated during the specification of a new cell lineage. It will be interesting to determine whether those genes that require SWI/SNF enzymes can be categorized by as yet undetermined common characteristics.


Figure 6
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 6.
Brg1 co-localizes and interacts with Mitf. A, B22 cells grown in the presence or absence of tetracycline were differentiated with Mitf for 40 h and then stained with antibodies to Mitf and Brg1. Confocal images and cross-correlation analysis are shown. B, B16 cells were harvested and either immunoprecipitated with an irrelevant antibody or with rabbit antisera to Brg1, Baf57, or Ini1. The immunoprecipitated material was run on an SDS-polyacrylamide gel and blotted with an antibody to Mitf. The blot was then stripped and re-probed with Brg1 antisera. The total cell extracts are from a darker exposure of the same gel.

 
In addition to activation of melanocyte-specific genes, Mitf is important for the proliferation and survival of melanocytes during migration from the neural crest and has been shown to both promote and to inhibit cell proliferation. A recent report showed that Mitf cooperates with the retinoblastoma protein (pRb) to activate the p21Cip1 gene and induce cell-cycle arrest (16). SWI/SNF enzymes have also been shown to interact with pRb, promote expression of p16INK4A and p21CIP1, and induce cell-cycle arrest (40-42, 68, 69, 83), therefore we were interested to see whether Mitf requires SWI/SNF enzymes for cell-cycle withdrawal associated with melanocyte differentiation. Under the culture conditions used, we did not detect any change in the number of cells arrested in G1 as a result of Mitf expression or dominant negative BRM or BRG1 expression. Although it remains possible that SWI/SNF enzymes might contribute to the ability of Mitf to promote cell-cycle arrest, under the conditions tested, we show that SWI/SNF enzymes are not required for changes in gene expression associated with cell-cycle withdrawal. Similarly SWI/SNF enzymes were not required for the activation of cell-cycle regulators that promote cell-cycle arrest during MyoD-mediated muscle differentiation (49, 66).

During cellular differentiation, the chromatin structure of previously silent genes becomes accessible upon activation of gene expression. SWI/SNF enzymes have been shown to play a critical role in the activation of gene expression during muscle, adipocyte, neural, enterocyte, and erythroid differentiation (23, 27-29, 84, 85). To our knowledge, this is the first report that SWI/SNF enzymes are required for specific aspects of melanocyte differentiation. Moreover, we determined that SWI/SNF enzymes are directly involved in the activation of Mitf target promoters such as Trp1, because the Brg1 ATPase of SWI/SNF enzymes interacts with target promoter sequences, and promotes changes in chromatin structure at an endogenous locus. Consistent with observations made in other systems, histone acetylation at target promoter sequences occurred in a manner that was independent of SWI/SNF function (55). Where examined in mammalian systems, histone acetylation precedes the interaction and function of SWI/SNF enzymes (29, 55, 71-74).

Although we have demonstrated a role for Brg1 in the activation of melanocyte-specific genes, we cannot exclude the possibility that Brm may also be involved in the regulation of these genes, because dominant negative BRG1 and dominant negative BRM can inhibit the activity of SWI/SNF complexes composed of either subunit (48). In addressing the mechanisms by which SWI/SNF enzymes regulate melanocyte-specific gene expression, we focused on Brg1 function, because previous work with Brm knock-out mice did not report a pigmentation phenotype (30), suggesting that Brm is either not required or that Brg1 can compensate for lack of Brm in the regulation of melanocyte-specific genes. We clearly demonstrate that Brg1 is associated with the regulatory regions of melanocyte-specific genes. Future experiments utilizing RNA interference to Brg1 or Brm will more directly address the individual contribution of each of these ATPase subunits, however, work in other cell types showed that RNAi-mediated inhibition of one SWI/SNF subunit caused concomitant down-regulation of other SWI/SNF subunits as well (86, 87).

SWI/SNF enzymes interact with a number of different gene-specific activators (88). The interaction between Brg1 and Mitf, a member of the Myc family of basic helix-loop-helix (bHLH)-Zip transcription factors that share a basic domain for DNA binding and HLH and leucine zipper regions for dimerization, parallels the contribution by SWI/SNF enzymes to the activity of bHLH proteins. MyoD is a member of the bHLH family of transcription factors that heterodimerizes with ubiquitously expressed E proteins and binds cognate E boxes in the promoters of muscle-specific genes and interacts with Brg1 to activate muscle-specific genes (55, 89). Similarly, Brg1 interacts with the bHLH factors NeuroD and Ngnr1 during neurogenesis (27). However, SWI/SNF contributions to activator-mediated gene stimulation are not solely limited to bHLH proteins, because during myeloid differentiation and adipogenesis, the enzymes also promote gene activation by C/EBP family members, which have distinct protein domains (23, 24). In addition, SWI/SNF enzymes promote activation of gene expression by transcriptional activators with zinc fingers, including peroxisome proliferator-activated receptor {gamma} and other nuclear hormone receptors (29, 90-92) as well as with erythroid Kruppel-like factor and GATA-1 (41, 88). Thus, SWI/SNF enzymes can cooperate with gene-specific transcriptional activators having different structural motifs to activate gene expression during differentiation.

The interaction between SWI/SNF enzymes and gene-specific activators appears to be critical for SW/SNF recruitment. During melanocyte differentiation, we found that SWI/SNF enzymes are associated with the Trp1 and tyrosinase promoters, are co-localized with Mitf in the nucleus, and physically interact. Together these results suggest that Mitf recruits SWI/SNF enzymes to the promoters of melanocyte-specific genes where SWI/SNF enzymes remodel chromatin to activate gene expression.

Chromatin remodeling by SWI/SNF enzymes can promote gene expression by promoting different phases of transcription in a gene-specific manner. Our previous work on muscle differentiation showed that SWI/SNF enzymes are required for binding of gene-specific activators to the myogenin promoter (55). During adipocyte differentiation, SWI/SNF enzymes are required for polymerase II pre-initiation complex stability (29). For other genes, SWI/SNF enzymes have been shown to facilitate binding of TATA-binding protein and also to promote later stages of transcription initiation and elongation of transcription (84, 93-95). Future studies will identify the specific requirements for SWI/SNF enzymes during transcription of melanocyte-specific genes.


   FOOTNOTES
 
* This work was funded by an American Heart Association Scientist Development Grant and by a grant from the Medical Foundation through support from the June Rockwell Levy Foundation and the Charles A. King Trust (to I. L. D.), and by National Institutes of Health Grant GM56244 and a Scholar Award from the Leukemia and Lymphoma Society (to A. N. I.). Fluorescence-activated cell sorting core resources were supported in part by the University of Massachusetts Medical School Diabetes Endocrinology Research Center (Grant DK32520). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Cancer Biology, Medical University of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4111; Fax: 419-383-6228; E-mail: idelaserna{at}meduohio.edu.

2 The abbreviations used are: Mitf, microphthalmia transcription factor; Trp1, -2, tyrosinase-related proteins 1 and 2; Mitf-M, melanocyte-specific Mitf; PBS, phosphate-buffered saline; CCF, cross-correlation function; ChIP, chromatin immunoprecipitation; Mc-1r, melanocortin-1 receptor; pRb, retinoblastoma protein; bHLH, basic helix-loop-helix; BRM, Brahma; BRG1, Brahma-related gene; LM, ligation-mediated. Back


   ACKNOWLEDGMENTS
 
We thank Thomas Hornyak for providing the mouse Mitf cDNA, Said Sif for providing the Baf57 antisera, and Jeffrey Nickerson for advice about cross-correlation analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Goding, C. R. (2000) Genes Dev. 14, 1712-1728[Free Full Text]
  2. Tachibana, M. (2001) J. Investig. Dermatol. Symp. Proc. 6, 95-98[CrossRef][Medline] [Order article via Infotrieve]
  3. Hu, D. N. (2005) Photochem. Photobiol. 81, 506-509[CrossRef][Medline] [Order article via Infotrieve]
  4. Menon, I. A., and Haberman, H. F. (1977) Br. J. Dermatol. 97, 109-112[Medline] [Order article via Infotrieve]
  5. Jhappan, C., Noonan, F. P., and Merlino, G. (2003) Oncogene 22, 3099-3112[CrossRef][Medline] [Order article via Infotrieve]
  6. Houghton, A. N., and Polsky, D. (2002) Cancer Cell 2, 275-278[CrossRef][Medline] [Order article via Infotrieve]
  7. Tachibana, M., Takeda, K., Nobukuni, Y., Urabe, K., Long, J. E., Meyers, K. A., Aaronson, S. A., and Miki, T. (1996) Nat. Genet. 14, 50-54[CrossRef][Medline] [Order article via Infotrieve]
  8. Widlund, H. R., and Fisher, D. E. (2003) Oncogene 22, 3035-3041[CrossRef][Medline] [Order article via Infotrieve]
  9. Hodgkinson, C. A., Moore, K. J., Nakayama, A., Steingrimsson, E., Copel- and, N. G., Jenkins, N. A., and Arnheiter, H. (1993) Cell 74, 395-404[CrossRef][Medline] [Order article via Infotrieve]
  10. Hemesath, T. J., Steingrimsson, E., McGill, G., Hansen, M. J., Vaught, J., Hodgkinson, C. A., Arnheiter, H., Copeland, N. G., Jenkins, N. A., and Fisher, D. E. (1994) Genes Dev. 8, 2770-2780[Abstract/Free Full Text]
  11. Bentley, N. J., Eisen, T., and Goding, C. R. (1994) Mol. Cell. Biol. 14, 7996-8006[Abstract/Free Full Text]
  12. Udono, T., Yasumoto, K., Takeda, K., Amae, S., Watanabe, K., Saito, H., Fuse, N., Tachibana, M., Takahashi, K., Tamai, M., and Shibahara, S. (2000) Biochim. Biophys. Acta 1491, 205-219[Medline] [Order article via Infotrieve]
  13. Shibahara, S., Takeda, K., Yasumoto, K., Udono, T., Watanabe, K., Saito, H., and Takahashi, K. (2001) J. Investig. Dermatol. Symp. Proc. 6, 99-104[CrossRef][Medline] [Order article via Infotrieve]
  14. Steingrimsson, E., Copeland, N., and Jenkins, N. A. (2004) Annu. Rev. Genet. 38, 365-411[CrossRef][Medline] [Order article via Infotrieve]
  15. Du, J., Wudlund, H. R., Horstmann, M. A., Ramaswamy, S., Ross, K., Huber, W. E., Nishimura, E. K., Golub, T. R., and Fisher, D. E. (2004) Cancer Cell 6, 565-576[CrossRef][Medline] [Order article via Infotrieve]
  16. Carreira, S. G. J., Aksan, I., La Rocca, S. A., Galibert, M. D., Denat, L., Larue, L., and Goding, C. R. (2005) Nature (London) 433, 764-769[CrossRef][Medline] [Order article via Infotrieve]
  17. Loercher, A. E., Tank, E. M., Delston, R. B., and Harbour, J. W. (2005) J. Cell Biol. 168, 35-40[Abstract/Free Full Text]
  18. Wellbrock, C., and Marais, R. (2005) J. Cell Biol. 170, 703-708[Abstract/Free Full Text]
  19. McGill, G. G., Horstmann, M., Widlund, H. R., Du, J., Motyckova, G., Nishimura, E. K., Lin, Y. L., Ramaswamy, S., Avery, W., Ding, H. F., Jordan, S. A., Jackson, I. J., Korsmeyer, S. J., Golub, T. R., and Fisher, D. E. (2002) Cell 109, 707-718[CrossRef][Medline] [Order article via Infotrieve]
  20. Garraway, L., Widlund, H., Rubin, M., Getz, G., Berger, A., Ramaswamy, S., Beroukhim, R., Milner, D., Granter, S., Du, J., Lee, C., Wagner, S., Li, C., Golub, T., Rimm, D., Meyerson, M., Fisher, D., and Sellers, W. (2005) Nature (London) 436, 117-122[CrossRef][Medline] [Order article via Infotrieve]
  21. Larribere, L., Hilmi, C., Khaled, M., Gaggioli, C., Bille, K., Auberger, P., Ortonne, J., Ballotti, R., and Bertolotto, C. (2005) Genes Dev. 19, 1980-1985[Abstract/Free Full Text]
  22. Bismuth, K., Maric, D., and Arnheiter, H. (2005) Pigment Cell Res. 18, 349-359[CrossRef][Medline] [Order article via Infotrieve]
  23. Pedersen, T. A., Kowenz-Leutz, E., Leutz, A., and Nerlov, C. (2001) Genes Dev. 15, 3208-3216[Abstract/Free Full Text]
  24. Kowenz-Leutz, E., and Leutz, A. (1999) Mol. Cell 4, 735-743[CrossRef][Medline] [Order article via Infotrieve]
  25. Young, D. W., Pratap, J., Javed, A., Weiner, B., Ohkawa, Y., van Wijnen, A., Montecino, M., Stein, G. S., Stein, J. L., Imbalzano, A. N., and Lian, J. B. (2005) J. Cell. Biochem. 94, 720-730[CrossRef][Medline] [Order article via Infotrieve]
  26. Holloway, A. F., Rao, S., Chen, X., and Shannon, M. F. (2003) J. Exp. Med. 197, 413-423[Abstract/Free Full Text]
  27. Seo, S., Richardson, G. A., and Kroll, K. L. (2005) Development 132, 105-115[Abstract/Free Full Text]
  28. de la Serna, I. L., Carlson, K. A., and Imbalzano, A. N. (2001) Nat. Genet. 27, 187-190[CrossRef][Medline] [Order article via Infotrieve]
  29. Salma, N., Xiao, H., Mueller, E., and Imbalzano, A. N. (2004) Mol. Cell. Biol. 24, 4651-4663[Abstract/Free Full Text]
  30. Reyes, J. C., Barra, J., Muchardt, C., Camus, A., Babinet, C., and Yaniv, M. (1998) EMBO J. 17, 6979-6991[CrossRef][Medline] [Order article via Infotrieve]
  31. Wang, W., Côte, J., Xue, Y., Zhou, S., Khavari, P. A., Biggar, S. R., Muchardt, C., Kalpana, G. V., Goff, S. P., Yaniv, M., Workman, J. L., and Crabtree, G. R. (1996) EMBO J. 15, 5370-5382[Medline] [Order article via Infotrieve]
  32. Imbalzano, A. N. (1998) Crit. Rev. Eukaryot. Gene Expr. 8, 225-255[Medline] [Order article via Infotrieve]
  33. Bultman, S., Gebuhr, T., Yee, D., La Mantia, C., Nicholson, J., Gilliam, A., Randazzo, F., Metzger, D., Chambon, P., Crabtree, G., and Magnuson, T. (2000) Mol. Cell 6, 1287-1295[CrossRef][Medline] [Order article via Infotrieve]
  34. Roberts, C. W., Galusha, S. A., McMenamin, M. E., Fletcher, C. D., and Orkin, S. H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13796-13800[Abstract/Free Full Text]
  35. Klochendler-Yeivin, A., Fiette, L., Barra, K., Muchardt, C., Babinet, C., and Yaniv, M. (2000) EMBO Rep. 1, 500-506[Medline] [Order article via Infotrieve]
  36. Kim, J. K., Huh, S. O., Choi, H., Lee, K. S., Shin, D., Lee, C., Nam, J. S., Kim, H., Chung, H., Lee, H. W., Park, S. D., and Seong, R. H. (2001) Mol. Cell. Biol. 21, 7787-7795[Abstract/Free Full Text]
  37. Guidi, C. J., Sands, A. T., Zambrowicz, B. P., Turner, T. K., Demers, D. A., Webster, W., Smith, T. W., Imbalzano, A. N., and Jones, S. N. (2001) Mol. Cell. Biol. 21, 3598-35603[Abstract/Free Full Text]
  38. Strobeck, M. W., Knudsen, K. E., Fribourg, A. F., DeCristoforo, M. F., Weissman, B. E., Imbalzano, A. N., and Knudsen, E. S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7748-7753[Abstract/Free Full Text]
  39. Zhang, H. S., Gavin, M., Dahiya, A., Postigo, A. A., Ma, D., Luo, R. X., Harbour, J. W., and Dean, D. C. (2000) Cell 101, 79-89[CrossRef][Medline] [Order article via Infotrieve]
  40. Kang, H., Cui, K., and Zhao, K. (2004) Mol. Cell. Biol. 24, 1188-1199[Abstract/Free Full Text]
  41. Hendricks, K. B., Shanahan, F., and Lees, E. (2004) Mol. Cell. Biol. 24, 362-376[Abstract/Free Full Text]
  42. Dunaief, J. L., Strober, B. E., Guha, S., Khavari, P. A., Ålin, K., Luban, J., Begemann, M., Crabtree, G. R., and Goff, S. P. (1994) Cell 79, 119-130[CrossRef][Medline] [Order article via Infotrieve]
  43. Strober, B. E., Dunaief, J. L., Guha, S., and Goff, S. P. (1996) Mol. Cell. Biol. 16, 1576-1583[Abstract]
  44. Sato, S., Roberts, K., Gambino, G., Cook, A., Kouzarides, T., and Goding, C. R. (1997) Oncogene 14, 3083-3092[CrossRef][Medline] [Order article via Infotrieve]
  45. Weilbaecher, K. N., Motyckova, G., Huber, W. E., Takemoto, C. M., Hemesath, T. J., Xu, Y., Hershey, C. L., Dowland, N. R., Wells, A. G., and Fisher, D. E. (2001) Mol. Cell 8, 749-758[CrossRef][Medline] [Order article via Infotrieve]
  46. Price, E. R., Ding, H. F., Badalian, T., Bhattacharya, S., Takemoto, C., Yao, T. P., Hemesath, T. J., and Fisher, D. E. (1998) J. Biol. Chem. 273, 17983-17986[Abstract/Free Full Text]
  47. Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract/Free Full Text]
  48. de la Serna, I. L., Carlson, K. A., Hill, D. A., Guidi, C. J., Stephenson, R. O., Sif, S., Kingston, R. E., and Imbalzano, A. N. (2000) Mol. Cell. Biol. 20, 2839-2851[Abstract/Free Full Text]
  49. de la Serna, I. L., Roy, K., Carlson, K. A., and Imbalzano, A. N. (2001) J. Biol. Chem. 276, 41486-41491[Abstract/Free Full Text]
  50. Steingrimsson, E., Moore, K. J., Lamoreux, M. L., Ferre-D'Amare, A. R., Burley, S. K., Zimring, D. C., Skow, L. C., Hodgkinson, C. A., Arnheiter, H., and Copeland, N. G., and Jenkins, N. A. (1994) Nat. Genet. 8, 256-263[CrossRef][Medline] [Order article via Infotrieve]
  51. Adachi, S., Nakano, T., Vliagoftis, H., and Metcalfe, D. D. (1999) J. Immunol. 163, 3363-3368[Abstract/Free Full Text]
  52. Joo, A., Aburatani, H., Morii, E., Iba, H., and Yoshimura, A. (2004) Oncogene 23, 726-734[CrossRef][Medline] [Order article via Infotrieve]
  53. Pal, S., Yun, R., Datta, A., Lacomis, L., Erdjument-Bromage, H., Kumar, J., Tempst, P., and Sif, S. (2003) Mol. Cell. Biol. 23, 7475-7487[Abstract/Free Full Text]
  54. van Steensel, B., van Binnendijik, E. P., Hornsby, C. D., van der Voort, H. T. M., Krozowski, Z. S., de Kloet, E. R., and van Driel, R. (1996) J. Cell Sci. 109, 787-792[Abstract]
  55. de la Serna, I. L., Ohkawa, Y., Berkes, C., Bergstrom, D. A., Dacwag, C. S., Tapscott, S. J., and Imbalzano, A. N. (2005) Mol. Cell. Biol. 25, 3997-4009[Abstract/Free Full Text]
  56. Bergstrom, D. A., Penn, B. H., Strand, A., Perry, R. L., Rudnicki, M. A., and Tapscott, S. J. (2002) Mol. Cell 9, 587-600[CrossRef][Medline] [Order article via Infotrieve]
  57. Carey, M., and Smale, S. T. (2000) Transcriptional Regulation in Eukaryotes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  58. Adachi, S., Morii, E., Kim, D., Ogihara, H., Jippo, T., Ito, A., Lee, Y. M., and Kitamura, Y. (2000) J. Immunol. 164, 855-860[Abstract/Free Full Text]
  59. Aoki, H., and Moro, O. (2002) Life Sci. 71, 2171-2179[CrossRef][Medline] [Order article via Infotrieve]
  60. Baxter, L. L., and Pavan, W. J. (2003) Gene Expr. Patterns 3, 703-707[CrossRef][Medline] [Order article via Infotrieve]
  61. Du, J., Miller, A. J., Widlund, H. R., Horstmann, M. A., Ramaswamy, S., and Fisher, D. E. (2003) Am. J. Pathol. 163, 333-343[Abstract/Free Full Text]
  62. Hemesath, T. J., Price, E. R., Takemoto, C., Badalian, T., and Fisher, D. E. (1998) Nature (London) 391, 298-301[CrossRef][Medline] [Order article via Infotrieve]
  63. Wu, M., Hemesath, T. J., Takemoto, C. M., Horstmann, M. A., Wells, A. G., Price, E. R., Fisher, D. Z., and Fisher, D. E. (2000) Genes Dev. 14, 301-312[Abstract/Free Full Text]
  64. Takeda, K., Takemoto, C., Kobayashi, I., Watanabe, A., Nobukuni, Y., Fisher, D. E., and Tachibana, M. (2000) Hum. Mol. Genet. 2000, 125-132
  65. Mansky, K. C., Uma, S., Jiahuai, H., and Ostrowski, M. C. (2002) J. Biol. Chem. 277, 11077-11083[Abstract/Free Full Text]
  66. Roy, K., de la Serna, I. L., and Imbalzano, A. N. (2002) J. Biol. Chem. 277, 33818-33824[Abstract/Free Full Text]
  67. Yavuzer, U., Keenan, E., Lowings, P., Vachtenheim, J., Currie, G., and Goding, C. R. (1995) Oncogene 10, 123-134[Medline] [Order article via Infotrieve]
  68. Oruetxebarria, I., Venturini, F., Kekarainen, T., Houweling, A., Zuijderduign, L. M. P., Mohd-Sarip, A., Vries, R. G. J., Hoeben, R. C., and Verrijzer, C. P. (2004) J. Biol. Chem. 279, 3807-3816[Abstract/Free Full Text]
  69. Betz, B. L., Strobeck, M. W., Reisman, D. N., Knudsen, E. S., and Weissman, B. E. (2002) Oncogene 21, 5193-5203[CrossRef][Medline] [Order article via Infotrieve]
  70. Hassan, A. H., Prochasson, P., Neely, K. E., Galasinski, S. C., Chandy, M., Carrozza, M. J., and Workman, J. L. (2002) Cell 111, 369-379[CrossRef][Medline] [Order article via Infotrieve]
  71. Agalioti, T., Lomvardas, S., Parekh, B., Yie, J., Maniatis, T., and Thanos, D. (2000) Cell 103, 667-678[CrossRef][Medline] [Order article via Infotrieve]
  72. Spilianakis, C., Kretsovali, A., Agalioti, T., Makatounakis, T., Thanos, D., and Papamatheakis, J. (2003) EMBO J. 22, 5125-5136[CrossRef][Medline] [Order article via Infotrieve]
  73. Ma, Z., Shah, R. C., Chang, M. J., and Benveniste, E. N. (2004) Mol. Cell. Biol. 24, 5496-5509[Abstract/Free Full Text]
  74. Kemper, J. K., Kim, H., Miao, J., Bhalla, S., and Bae, Y. (2004) Mol. Cell. Biol. 24, 7707-7719[Abstract/Free Full Text]
  75. Steel, K. P., Davidson, D. R., and Jackson, I. J. (1992) Development 115, 1111-1119[Abstract]
  76. Yasumoto, K.-i., Yokoyama, K., Takahashi, K., Tomita, Y., and Shibahara, S. (1997) J. Biol. Chem. 272, 503-509[Abstract/Free Full Text]
  77. Yasumoto, K.-i., Takeda, K., Saito, H., Watanabe, K.-i., Takahashi, K., and Shibahara, S. (2002) EMBO J. 21, 2703-2714[CrossRef][Medline] [Order article via Infotrieve]
  78. Bertolotto, C., Busca, R., Abbe, P., Bille, K., Aberdam, E., Ortonne, J. P., and Ballotti, R. (1998) Mol. Cell. Biol. 18, 694-702[Abstract/Free Full Text]
  79. Jiao, J., Mollaaghababa, R., Pavan, W. J., Antonellis, A., Green, E. D., and Hornyak, T. J. (2004) Pigment Cell Res. 17, 352-362[CrossRef][Medline] [Order article via Infotrieve]
  80. Hearing, V. J. (2005) J. Dermatol. Sci. 37, 3-14[CrossRef][Medline] [Order article via Infotrieve]
  81. Dudley, M. E., Wunderlich, J. R., Robbins, P. F., Yang, J. C., Hwu, P., Schwartzentruber, D. J., Topalian, S. L., Sherry, R., Restifo, N. P., Hubicki, A. M., Robinson, M. R., Raffeld, M., Duray, P., Seipp, C. A., Rogers-Freezer, L., Morton, K. E., Mavroukakis, S. A., White, D. E., and Rosenberg, S. A. (2002) Science 298, 850-854[Abstract/Free Full Text]
  82. Rouzaud, F., and Hearing, V. J. (2005) Peptides 26, 1858-1870[CrossRef][Medline] [Order article via Infotrieve]
  83. Giraud, S., Hurlstone, A., Avril, S., and Coqueret, O. (2004) Oncogene 23, 7391-7398[CrossRef][Medline] [Order article via Infotrieve]
  84. Soutoglou, E., and Talianidis, I. (2002) Science 295, 1901-1904[Abstract/Free Full Text]
  85. Armstrong, J. A., Bieker, J. J., and Emerson, B. M. (1998) Cell 95, 93-104[CrossRef][Medline] [Order article via Infotrieve]
  86. Cui, K., Tailor, P., Liu, H., Chen, X., Ozato, K., and Zhao, K. (2004) Mol. Cell. Biol. 24, 4476-4486[Abstract/Free Full Text]
  87. Chen, J., and Archer, T. K. (2005) Mol. Cell. Biol. 25, 9016-9027[Abstract/Free Full Text]
  88. Kadam, S., and Emerson, B. M. (2003) Mol. Cell 11, 377-389[CrossRef][Medline] [Order article via Infotrieve]
  89. Simone, C., Forcales, S. V., Hill, D. A., Imbalzano, A. N., Latella, L., and Puri, P. L. (2004) Nat. Genet. 36, 738-743[CrossRef][Medline] [Order article via Infotrieve]
  90. Khavari, P. A., Peterson, C. L., Tamkun, J. W., and Crabtree, G. R. (1993) Nature 366, 170-174[CrossRef][Medline] [Order article via Infotrieve]
  91. Chiba, H., Muramatsu, M., Nomoto, A., and Kato, H. (1994) Nucleic Acids Res. 22, 1815-1820[Abstract/Free Full Text]
  92. Fryer, C. J., and Archer, T. K. (1998) Nature 393, 88-91[CrossRef][Medline] [Order article via Infotrieve]
  93. Lomvardas, S., and Thanos, D. (2001) Cell 106, 685-696[CrossRef][Medline] [Order article via Infotrieve]
  94. Sullivan, E. K., Weirich, C. S., Guyon, J. R., Sif, S., and Kingston, R. E. (2001) Mol. Cell. Biol. 21, 5826-5837[Abstract/Free Full Text]
  95. Brown, S. A., Imbalzano, A. N., and Kingston, R. E. (1996) Genes Dev. 10, 1479-1490[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. Chiaverini, L. Beuret, E. Flori, R. Busca, P. Abbe, K. Bille, P. Bahadoran, J.-P. Ortonne, C. Bertolotto, and R. Ballotti
Microphthalmia-associated Transcription Factor Regulates RAB27A Gene Expression and Controls Melanosome Transport
J. Biol. Chem., May 2, 2008; 283(18): 12635 - 12642.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
R. Hu, S. M. Sharma, A. Bronisz, R. Srinivasan, U. Sankar, and M. C. Ostrowski
Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors
Mol. Cell. Biol., June 1, 2007; 27(11): 4018 - 4027.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. M. Sharma, A. Bronisz, R. Hu, K. Patel, K. C. Mansky, S. Sif, and M. C. Ostrowski
MITF and PU.1 Recruit p38 MAPK and NFATc1 to Target Genes during Osteoclast Differentiation
J. Biol. Chem., May 25, 2007; 282(21): 15921 - 15929.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Beuret, E. Flori, C. Denoyelle, K. Bille, R. Busca, M. Picardo, C. Bertolotto, and R. Ballotti
Up-regulation of MET Expression by {alpha}-Melanocyte-stimulating Hormone and MITF Allows Hepatocyte Growth Factor to Protect Melanocytes and Melanoma Cells from Apoptosis
J. Biol. Chem., May 11, 2007; 282(19): 14140 - 14147.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/29/20233    most recent
M512052200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de la Serna, I. L.
Right arrow Articles by Imbalzano, A. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de la Serna, I. L.
Right arrow Articles by Imbalzano, A. N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement