Brutons tyrosine kinase (Btk) enhances transcriptional co-activation activity of BAM11, a Btk-associated molecule of a subunit of SWI/SNF complexes
Masayuki Hirano1,
Yuji Kikuchi1,3,
Sazuku Nisitani1,
Akiko Yamaguchi1,
Atsushi Satoh1,
Taiji Ito2,
Hideo Iba2 and
Kiyoshi Takatsu1
Divisions of 1 Immunology and 2 HostParasite Infection, Department of Microbiology and Immunology, the Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 3 Laboratory of Immunoregulation, Department of Infection Control and Immunology, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
The first two authors contributed equally to this work
Correspondence to: K. Takatsu; E-mail: takatsuk{at}ims.u-tokyo.ac.jp
Transmitting editor: K. Sugamura
 |
Abstract
|
---|
Brutons tyrosine kinase (Btk) is required for B cell development and signal transduction through cell-surface molecules such as BCR and IL-5 receptor. We have identified a Btk-associated molecule, BAM11 (hereafter referred to as BAM) that binds to the pleckstrin homology (PH) domain of Btk, and inhibits Btk activity both in vivo and in vitro. In this study, we demonstrate BAMs transcriptional co-activation activity and its functional interaction with Btk. By using transient transcription assays, we demonstrate that the enforced expression of BAM enhances transcriptional activity of the synthetic reporter gene. The C-terminus of BAM is essential for the transcriptional co-activation activity. The ectopic expression of Btk together with BAM enhances BAMs transcriptional co-activation activity. BAMs transcriptional co-activation activity is enhanced through interaction with Btk, and requires both its intact PH domain and functional kinase activity. We also show that enforced expression of TFII-I, another Btk-binding protein with transcriptional activity, together with BAM and Btk, further augments BAM- and Btk-dependent transcriptional co-activation. Furthermore, BAM can be co-immunoprecipitated with the INI1/SNF5 protein, a member of the SWI/SNF complex that remodels chromatin and activates transcription. We propose a model in which Btk regulates gene transcription in B cells by activating BAM and the SWI/SNF transcriptional complex via TFII-I activation.
Keywords: Brutons tyrosine kinase, LTG19/ENL/MLLT1, TFII-I
 |
Introduction
|
---|
Brutons tyrosine kinase (Btk) plays a pivotal role in signal transduction pathways regulating survival, activation, proliferation and differentiation of B lineage cells (1,2). Following ligation of BCR, Syk, phosphatidylinositol-3-kinase, Btk, BLNK, phospholipase C (PLC)-
2 and NF-
B can be activated (37). Upon BCR stimulation, Btk is targeted to the plasma membrane by the binding of phosphatidylinositol-3,4,5-triphosphate (PIP3) to its pleckstrin homology (PH) domain (8,9). Binding of Btk to BLNK is crucial for phosphorylation and activation of PLC-
2, implicating Btk as a mediator of BCR-mediated calcium mobilization (3,5,1012). It has recently been shown that Btk is involved in BCR-induced NF-
B activation (1316). The PH domain of Btk is involved in signal transduction by interacting with various molecules, including ß
complexes of the heterotrimeric G protein (17), protein kinase C (PKC) (18) and PIP3 (8,9).
Mutations in the PH domain of human and mouse Btk cause maturational blocks at early stages of B cell ontogeny leading to X-linked agammaglobulinemia (XLA) in humans (1921), and defective B cell development and function leading to X-linked immunodeficiency (Xid) in mice (2224). In order to dissect how the PH domain is involved in Btk-derived signaling pathways, we isolated Btk-associated molecule, BAM11 (hereafter referred to as BAM), which binds to the PH domain of Btk, but not to Itk or Tec (25). Although the cellular function of BAM remains unknown, forced expression of the Btk-binding region of BAM (amino acids 246368) in an IL-5-dependent early B cell line, Y16, suppresses IL-5-induced Btk activation and cell proliferation. Using a green fluorescence protein (GFP)-fused Btk protein we also demonstrated that a significant proportion of Btk is capable of localizing to the nucleus (25). BAM has
90% homology to human LTG19/ENL/MLLT1, which was initially identified as part of a chromosome translocation, t(11;19),(q23;p19.3), involving the MLL/ALL-1/HRX gene in a case of human acute leukemia (26,27).
In addition to the sequential activation of molecules that follows Btk activation, Btk also operates as a nucleocytoplasmic shuttle (28) regulating potential targets inside the nucleus that result in the up-regulation or down-regulation of certain genes. Accumulating evidence suggests that Btk regulates activation of NF-
B (14,16) and the nuclear localization as well as the transcriptional activity of the multifunctional transcription factor BAP-135/TFII-I (2931). TFII-I is ubiquitously expressed, and has broad biological function which may include involvement in the WilliamsBeuren syndrome and XLA (32). Based on its unique interaction at both the Inr element and upstream regulatory sites (3335), TFII-I is postulated to be a transcriptional cofactor that integrates signals from regulatory components to the basal machinery (3537). In B cells, a significant fraction of cytoplasmic TFII-I is constitutively associated with Btk (29,30). Upon BCR cross-linking, Btk transiently tyrosine-phosphorylates TFII-I, thus enabling its release and nuclear translocation (30,31,38); this suggests that TFII-I transcriptional activity could be regulated by alteration of its subcellular localization.
The SWI/SNF family of chromatin-remodeling complexes has been identified in many species (39). Genetic and biochemical data indicate that the SWI/SNF complex increases the accessibility of several transcription factors and histone-modifying complexes to DNA (39,40). Several members of the 912 subunit-containing human SWI/SNF ATP-dependent chromatin-remodeling complexes have been described (4144). Recently, ENL/LTG19, a homolog of the yeast SWI/SNF subunit TFG3/TAF30/ANC1, was found to be a new member of a specific subset of the human SWI/SNF complex. ENL/LTG19 associates and cooperates with the SWI/SNF complex to activate transcription of the HoxA7 promoter, a downstream element essential for the oncogenic activity of MLL-ENL (45). It remains unclear whether BAM, the murine counterpart of ENL/LTG19, is involved in the SWI/SNF complex.
In this study, we investigated the involvement of BAM transcriptional activity and its regulatory role in conjunction with Btk. Here, we report that BAM has transcriptional co-activator activity (i.e. as a transactivator) which is up-regulated by Btks interaction with its C-terminus. Furthermore, TFII-I further augments Btk-dependent enhancement of BAMs transcriptional co-activation activity. Moreover, BAM is co-immunoprecipitated with INI1/SNF5 protein, a member of the SWI/SNF complex. These results suggest that Btk acts as a positive regulator of BAM transcriptional co-activation activity when BAMBtk complexes are co-localized in the nucleus.
 |
Methods
|
---|
Cell lines and antibodies
The COS7 cell line was maintained in RPMI 1640, and supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µM 2-mercaptoethanol. All cells were cultured at 37°C in a humidified 5% CO2 incubator. Anti-INI1/hSNF5 (BD Biosciences PharMingen, San Diego, CA), anti-Btk (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-myc (clone 9E10; ATCC, Rockville, MD), anti-TFII-I (Santa Cruz Biotechnology) and anti-FLAG (clone M2; Sigma, St Louis, MO) antibodies were purchased.
Vectors and plasmid constructs
The pME18S-myc mammalian expression vector, which allows for in-frame fusion with the human myc epitope tag, was generously provided by T. Yamamoto (University of Tokyo, Japan). The p146 vector expressing the GSTTFII-I fusion protein (31) was kindly provided by A. L. Roy (Tufts University, Boston, MA). To generate pMEmyc-BAM (pME18S-myc containing the BAM cDNA), a DNA fragment encoding BAM was amplified by PCR using primers incorporating EcoRI sites at the 5' ends. The amplified product was cloned into the EcoRI sites of pMEl8S-myc. To produce the GST fusion construct, DNA fragments encoding BAM (full length; amino acids 1547), BAM-N (amino acids 1186), BAM-BB (amino acids 131256), BAM-B (amino acids 240368) and BAM-C (amino acids 363547) were amplified by PCR, and cloned into the EcoRI sites of pGEX-4T (Amersham Biosciences, Piscataway, NJ); these constructs were designated pGEX-BAM, pGEX-BAM-N, pGEX-BAM-BB, pGEX-BAM-B and pGEX-BAM-C respectively. The construct pGEX-BAM-NC expressing the deletion mutant of BAM (BAM-NC; amino acids 1186 ligated with amino acids 363547) was constructed from plasmids containing BAM-N and BAM-C. Plasmids encoding GAL4 fusion proteins were derived from pBIND vector (Promega, Madison, WI), which expresses the GAL4 DNA-binding domain (amino acids 1-147) under control of the human cytomegalovirus immediate early promoter. To produce GAL4BAM fusion constructs, DNA fragments encoding BAM, BAM-N, BAM-BB, BAM-B, BAM-C and BAM-NC were amplified by PCR. Amplified products were cloned into the BamHI and XbaI sites of the pBIND vector downstream and in-frame with GAL4, resulting in pBIND-BAM, pBIND-BAM-N, pBIND-BAM-BB, pBIND-BAM-B, pBIND-BAM-C and pBIND-BAM-NC respectively. The pG5luc vector (Promega), containing five tandem copies of the GAL4 consensus binding site, cloned upstream of a firefly luciferase gene driven by the major late promoter of adenovirus, was used as a reporter construct. The pG5luc-
Inr vector was generated by three-step PCR, and was cloned into the EcoRI and BclI sites of the pG5luc vector. In the first PCR reaction, the sense primer 5'-CCGCGAATTCCGGAGTACTGTCC-3' and antisense primer 5'-CGCAGATCTCGGCTGAGGACGA-3' were applied to the pG5luc as template, and in the second PCR reaction, the sense primer 5'-TCGTCCTCAGCCGAGATCTGCG-3' and antisense primer 5'-CATGATCAGTGCAATTGTCTTGT-3' were applied to the pG5luc template. The two PCR products were complementary to each other and used as templates in the third PCR reaction together with the primary sense and the secondary antisense primers. The vectors pME-Btk and pME-Btk(
PH) that express wild-type Btk and the Btk mutant lacking the PH domain were previously described (16).
Two different lines of Btk mutant constructs were used. R28C and K430R mutant constructs, referred to as Btk(Xid) and Btk(K430R) respectively, were generated by PCR-based site-directed mutagenesis. The resulting mutated cDNA were subcloned into pME18S, resulting in pME-Btk(Xid) and pME-Btk(K430R) respectively. To produce GFP fusion constructs, the DNA fragments encoding Btk and Btk(R28C) were cloned between the XhoI and BamHI sites of the pEGFP-Nl vector (Clontech, Palo Alto, CA); these constructs were designated pEGFP-Btk and pEGFP-Btk(Xid) respectively. All constructs involving PCR manipulation were sequenced to ensure the absence of mutation.
Luciferase reporter assay
Transfections were carried out with the Superfect Transfection Reagent (Qiagen, Hilden, Germany) in accordance with the manufacturers protocol. A series of pBIND-BAM truncated constructs were transfected either alone or with 100 ng of pME-Btk, pME-Btk(Xid), pME-Btk(K430R), pME-Btk(
PH) or p146. All transfections were performed with 2 µg of reporter plasmid (pG5luc or pG5luc-
Inr). Briefly, COS7 cells (1 x 105) were grown in RPMI 1640 containing 10% FCS to
50% confluency in a six-well plate (Corning, Corning, NY). The indicated DNA constructs were added to 150 µl of DMEM, and 30 µl of the Superfect Transfection Reagent was added to the DNA solution and incubated for 10 min at room temperature. During this incubation period, COS7 cells were washed once with 4 ml of Dulbeccos PBS. After the incubation, 1 ml of DMEM containing 10% FCS was added to the mixture, which was then added to the washed COS7 cells. After 2 h of incubation, the medium was aspirated and the cells were washed once with PBS. Then, 2 ml of DMEM containing 10% FCS was added to the plates and the cultures were incubated for an additional 48 h. For reporter assays, luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega). All raw firefly luciferase values were normalized to the activity of Renilla luciferase values. The normalized value was then divided by the luciferase activity obtained by co-transfection of the reporter with pBIND alone. The ratio was expressed as the relative luciferase activity.
Immunoprecipitation and western blot analysis
Immunoprecipitation and western blot analyses were carried out as previously described (25). Briefly, COS7 cells (1 x 106) were resuspended in 800 µl of electroporation buffer [120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 (pH 7.6), 25 mM HEPES (pH 7.6), 2 mM EGTA (pH 7.6), 5 mM MgCl2, 2 mM ATP and 5 mM glutathione] containing 5 µg of the appropriate expression plasmid and electroporated at 300 V, 960 µF. After 48 h of incubation, cells were collected by scraping in 50 ml of PBS, pelleted and lysed on ice in lysis buffer [1% NP-40, 10% glycerol, 150 mM NaCl, 20 mM TrisHCl (pH 7.4), 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 100 U/ml aprotinin, 10 mM iodoacetoamide, 25 µg/ml p-nitrophenyl-p'-guanidinobenzoate]. In some experiments, cytosol and nuclear fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Pierce, Rockford, IL). Samples were precleared with Protein GSepharose 4B and incubated at 4°C overnight with 10 µg of the proper antibody. Immune complexes were then precipitated with 20 µl of Protein GSepharose during 60 min incubation at 4°C, washed 5 times with lysis buffer and boiled for 5 min with 2 x Laemmlis sample buffer.
For western blotting, samples were electrophoresed on SDSpolyacrylamide gels (8%). Wet transfer to Immobilon-P membrane (Nihon Millipore, Tokyo, Japan) was accomplished by electrophoresis in buffer containing 25 mM Tris/192 mM glycine and 10% methanol for 1 h at 60 V. After blocking with Tris-buffered saline [TBS, 20 mM Tris (pH 7.6) and 150 mM NaCl] containing 5% BSA, the membranes were incubated with the appropriate primary antibody and washed in TBS containing 0.05% Tween 20 (TBS-T). After incubation with horseradish peroxidase-coupled goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies, the membranes were washed with TBS-T and subjected to an ECL detection system (Amersham Biosciences). For removal of immune complexes from membranes, each blot was incubated with 62.5 mM Tris (pH 6.5)/2% SDS/100 mM 2-mercaptoethanol for 30 min at 55°C. The membrane was washed twice in TBS and re-blocked as required for the next round of western blot analysis.
Binding assay for GST fusion proteins with Btk
GST fusion protein was expressed in the protease-deficient Escherichia coli strain BL21 and purified on glutathioneSepharose beads (Amersham Biosciences) (25). An equal amount of GST fusion protein was incubated with the bacterial cell lysate expressing FLAG-Btk for 4 h at 4°C and washed with PBS containing 0.1% of Triton X-100. After adding the SDSPAGE sample buffer, precipitates bound to the GST fusion protein were eluted by boiling and examined by immunoblotting with the anti-FLAG mAb.
Histochemistry
The pEGFP-Btk, pEGFP-Btk(Xid) and pMEmyc-BAM constructs were introduced into COS7 cells by electroporation. After 48 h, cells were collected and fixed onto 0.1% poly-L-lysine-treated glass slides. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.3% Triton X-100 and blocked with 5% normal rabbit serum (Santa Cruz Biotechnology). For the staining of myc-BAM, glass slides were incubated with mouse anti-myc antibody (9E10) followed by Cy5-conjugated rabbit anti-mouse IgG (Chemicon, Temecula, CA). Some samples were incubated with 0.1 mg/ml of propidium iodide for 15 min at room temperature. We monitored the localization of GFP fusion proteins and BAM using confocal laser scanning microscopy (Bio-Rad, Hercules, CA). Images were obtained with Noran Intervision 2D Image Analysis modules. A composite image of fluorescein and propidium iodide stains was generated.
 |
Results
|
---|
Nuclear co-localization of BAM and Btk
We and other investigators have reported that Btk is able to shuttle from the cytoplasm to the nucleus (25,28). To examine whether Btk(Xid) localizes to the nucleus like wild-type Btk, we transfected COS7 cells with BtkGFP or Btk(Xid)GFP and analyzed the cells by confocal immunostaining microscopy. Most Btk as well as Btk(Xid) was localized in the cytoplasm, but a significant proportion was also found in the nucleus (Fig. 1A and B) in accordance with the published reports (25). Because it has been reported that BAM is a Btk-binding molecule whose gene has five nuclear localization signals (NLS) (25), BAM may co-localize with Btk in the nucleus. To address this hypothesis, we ectopically expressed myc-BAM alone or myc-BAM and BtkGFP in COS7 cells and analyzed their localization by confocal microscopy. Staining of myc-BAM-transfected COS7 cells with anti-myc and Cy5- conjugated anti-mouse IgG antibodies revealed intense red nuclear staining, indicating the preferential localization of BAM to the nucleus as described (25,28) (data not shown). When we analyzed COS7 cells transfected with BtkGFP and myc-BAM, large proportions of Btk and BAM expression were identified in the cytoplasm and nucleus respectively. When we merged the expression of Btk and BAM, a significant proportion of scattered discrete yellow spots was seen in the nucleus (Fig. 1C), indicating the co-localization of BAM and Btk to the nucleus.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1. Subcellular localization of Btk and BAM in COS7 cells. COS7 cells were transfected with pEGFP-Btk (A), pEGFP-Btk(Xid) (B), pEGFP-Btk and pMEmyc-BAM (C) plasmids (5 µg) by electroporation. After 48 h, protein localization was analyzed by confocal laser scanning microscopy. The nuclei were stained with propidium iodide (A and B) and myc-BAM was stained with a mouse anti-myc antibody followed by a Cy5-conjugated anti-mouse IgG antibody (C).
|
|
BAM acts as a transcriptional co-activator
It has been reported that the human LTG19/ENL/MLLT1 protein shows transcriptional transactivator potential in lymphoid and myeloid cells (46). Because BAM and Btk are capable of interacting with each other and co-localizing to the nucleus (Fig. 1), we assumed that BAM and Btk may regulate the transcription of certain genes. To gain insight into the functional significance of BtkBAM interactions and to avoid complication from the presence of endogenous Btk and BAM, we chose to express Btk and BAM ectopically in COS7 cells that do not express endogenous Btk.
At first, we co-transfected COS7 cells with the expression vector pBIND-BAM, which consists of a GAL4 DNA-binding domain fused to BAM (GAL4BAM), and pG5luc, a firefly luciferase reporter cassette containing GAL4-binding sites. The transcriptional co-activating activity of BAM was assessed by a luciferase reporter assay and expressed as relative luciferase activity. Transfectants expressing GAL4BAM, GAL4BAM-C and GAL4BAM-NC together with pG5luc showed enhanced luciferase activity, while transfectants expressing GAL4 and pG5luc showed a low level of luciferase activity (Fig. 2B). GAL4BAM expression demonstrated a slightly higher level of luciferase activity than those of GAL4BAM-C and GAL4BAM-NC. In contrast, transfectants expressing GAL4BAM-N, GAL-4BAM-BB or GAL4BAM-B did not show an enhancement of transactivator activity. BAM, BAM-BB and BAM-B were capable of binding to Btk, while BAM-N, BAM-C and BAM-NC were incapable of binding to Btk (Fig. 2A). These results indicate that BAM has transcriptional co-activation activity. Interestingly, the C-terminus of BAM is indispensable for its transactivator activity, while the Btk-binding region of BAM is not necessary for its transactivator activity.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2. (A) Association between BAM and Btk. Schematic representation of GSTBAM fusion protein deletion mutants used for the binding assay (upper panel). Black boxes and hatched boxes represent the regions rich in serines and prolines respectively. Equal amounts of GSTBAM fusion protein deletion mutants were bound to glutathione-coupled Sepharose beads and incubated with bacterial cell lysates expressing FLAG-Btk. The precipitated complexes were then separated using SDSPAGE, transferred to membranes and blotted with an anti-FLAG antibody (lower panel). (B) Transcriptional co-activation activity of BAM. COS7 cells were co-transfected with the pG5luc reporter plasmid and a series of pBIND-BAM mutant constructs using the Superfect Transfection Reagent as described in Methods. For reporter assays, luciferase activity was assessed using the Dual-Luciferase Reporter Assay System. All raw values obtained from firefly luciferase were normalized to the activity of Renilla luciferase values. The normalized value was then divided by the luciferase activity obtained by co-transfection of the reporter with pBIND alone. Data are given as means of three independent experiments and SD.
|
|
Btk up-regulates the transactivation activity of BAM
To examine the effect of Btk on BAM transcriptional co-activation activity, BAM, GAL4BAM or GAL4BAM-NC and Btk expression plasmids were co-transfected into COS7 cells with pG5luc. As controls, transfectants expressing GAL4, GAL4BAM or GAL4BAM-NC together with pG5luc were also analyzed. Interestingly, BAM transcriptional co-activation activity was enhanced up to
2.5-fold when Btk was co-expressed (Fig. 3, upper panel). Importantly, BAM-NC showed significant transcriptional co-activation activity; however, the activity was not affected by co-expression of Btk. As BAM-NC is unable to associate with Btk (Fig. 2A), direct interaction between BAM and Btk is required for enhancing BAM activity. The co-expression of GAL4-VP16 and Btk in COS7 cells did not alter the transcriptional activity of VP16 (Fig. 3, lower panel). These results further indicate that Btk up-regulates the transcriptional co-activation activity of a Btk-binding protein.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3. Effects of Btk on the transcriptional co-activation activity of BAM. COS7 cells were co-transfected with the pG5luc reporter plasmid and pBIND-BAM, pBIND-BAM-NC (upper panel) or pBIND-VP16 (lower panel) with or without pME-Btk. After 48 h of incubation, luciferase activities in the cell lysates were measured and indicated as Fig. 1. Data are given as means of three independent experiments and SD.
|
|
The intact PH domain and kinase activity of Btk are indispensable for enhancing BAM transcriptional co-activation activity
BAM is capable of binding similarly to Btk(Xid) and the kinase-inactive Btk [Btk(K430R)] mutant as it is to wild-type Btk (25). We co-transfected expression plasmids for GAL4BAM and mutant Btk constructs such as the PH-deleted Btk [Btk(
PH)], Btk(Xid) or Btk(K430R), together with pG5luc to assess up-regulation of BAM transcriptional co-activation activity. We also analyzed COS7 transfectants expressing DNA-binding domain of GAL4, GAL4 and Btk as well as GAL4BAM and Itk. The expression levels of wild-type Btk and mutant Btk proteins in each of the transfectants were comparable, as estimated by a western blot of the extracts probed with an anti-Btk antibody (data not shown). Results revealed that the luciferase activity of GAL4 transfectants and that of Btk and GAL4 transfectants were comparable at baseline levels, indicating that by itself Btk does not exert transcriptional co-activation activity. Co-expression of Btk together with GAL4BAM enhanced the transcriptional co-activation activity of BAM, while co-expression of Btk(
PH), Btk(Xid), Btk(K430R) or Itk with GAL4BAM did not affect the transcriptional co-activation activity of BAM (Fig. 4). These results indicate that both the intact PH domain and the kinase activity of Btk are indispensable for enhancing the transactivator activity of BAM. As the Btk(Xid) mutant capable of associating with BAM failed to enhance the BAM activity, results shown in Fig. 4 suggest that another PH domain-binding protein, which is sensitive to the Xid mutation, is involved in the Btk-mediated enhancement of the transactivator activity of BAM.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4. Effects of Xid mutation, PH domain deletion and Btk kinase activity on the regulation of BAM transcriptional co-activation activity. COS7 cells were co-transfected with the pG5luc reporter plasmid and pBIND-BAM with or without pME-Btk, pME-Btk (Xid), pME-Btk (K430R) or pME-Btk ( PH). After 48 h of incubation, luciferase activities from cell lysates were measured and indicated as in Fig. 1. Data are given as means of three independent experiments and SD.
|
|
TFII-I augments the Btk-mediated enhancement of BAM activity regardless of the existence of the Inr element
Novina et al. have reported that wild-type Btk and Btk(K430E), but not Btk(Xid), constitutively associate with TFII-I, and that wild-type Btk, but not Btk(K430E), enhances its transcriptional activity (30). It can be argued that Btk-induced enhancement of BAM transcriptional co-activation activity may be influenced by TFII-I. When we examined COS7 transfectants expressing GAL4 and TFII-I together with pG5luc, significant transcriptional activity was observed compared with GAL4 and pG5luc transfectants. In contrast, the luciferase activity of GAL4 transfectants and that of Btk and GAL4 transfectants were comparable at baseline levels (Fig. 5), indicating that by itself Btk does not exert transcriptional activity. Consistent with the results reported by Novina et al. (30), the transcriptional activity of TFII-I was further enhanced
2-fold by Btk co-expression (Fig. 5). Co-expression of Btk(Xid) and Btk(K430R) was ineffective in enhancing the transcriptional activity of TFII-I.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5. Effects of Btk on TFII-I transcriptional activity. COS7 cells were co-transfected with the pG5luc reporter plasmid, pBIND and p146, with or without pME-Btk (Xid) or pME-Btk (K430R). After 48 h of incubation, luciferase activities from cell lysates were measured and indicated as in Fig. 1. Data are given as means of three independent experiments and SD.
|
|
To gain insight into the molecular basis of TFII-I activity for Btk-mediated enhancement of BAM transcriptional co-activation, we prepared mutant plasmids that deleted the TFII-I-binding element (Inr element) from the pG5luc plasmid (designated as pG5luc-
Inr). We co-transfected COS7 cells with GAL4BAM, Btk and TFII-I expression plasmids with pG5luc or pG5luc-
Inr. As controls, COS7 transfectants of GAL4BAM and TFII-I and pG5luc or pG5luc-
Inr were also analyzed. Results of BAM transcriptional co-activation activity revealed that TFII-I slightly enhanced the transcriptional co-activation activity of BAM, but to a lesser extent than Btk (Fig. 6, upper panel). The enhancing effect of TFII-I was diminished when pG5luc-
Inr was expressed in place of pG5luc (Fig. 6, lower panel), indicating the importance of the Inr element for TFII-I activity. Importantly, the enhancing effect of Btk on BAM activity was observed in both transfectants expressing pG5luc and pG5luc-
Inr, regardless of the existence of the Inr element (Fig. 6). Co-expression of TFII-I with GAL4BAM and Btk further enhanced BAM activity in transfectants expressing pG5luc-
Inr to a similar extent to those expressing pG5luc (Fig. 6, lower panel). Btk(Xid) and Btk(K430R) did not show an enhancing effect on the transcriptional co-activation activity of BAM in the presence of TFII-I. These results imply that TFII-I is capable of augmenting Btk-induced enhancement of BAM transcriptional co-activation activity through a non-Inr element.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6. Assay to determine BAM transcriptional co-activation activity using an Inr-deleted reporter plasmid. COS7 cells were co-transfected with reporter plasmid, pG5luc (upper panel) or pG5luc- Inr (lower panel) and pBIND, with or without p146 or pME-Btk. After 48 h of incubation, luciferase activities from cell lysates were measured and indicated as in Fig. 1. Data are given as means of three independent experiments and SD.
|
|
BAM is a mouse homolog of yeast TFG3/TAF30/ANC1 that is co-immunoprecipitated with INI1
Using the BLAST algorithm we searched for a BAM gene homolog. In this query we identified yeast TFG3/TAF30/ANC1, a component of the SWI/SNF complex, which strikingly had 20 identical and 11 similar amino acids in common among BAM amino acids 4998 (Supplementary Fig. 1).
Yeast TFG3/TAF30/ANC1 biochemical data suggests a strong association between TFG3/TAF30/ANC1 and INI1/hSNF5 (47). We therefore postulated that mouse BAM may associate with INI1/SNF5. COS7 cells were transfected with myc-BAM and their nuclear extracts were immunoprecipitated with an anti-myc antibody. The immunoprecipitates were analyzed by SDSPAGE followed by immunoblotting with anti-INI1, anti-TFII-I or anti-Btk antibodies. Results revealed that INI1/SNF5 is co-immunoprecipitated with myc-BAM (Fig. 7A), suggesting that BAM is a component of the mouse SWI/SNF complex and associates with INI1/SNF5. In contrast, a sizable amount of TFII-I could not be co-immunoprecipitated with BAM from the nuclear extracts of COS7 cells transfected with myc-BAM (Fig. 7B). BtkBAMINI1/SNF trimers were not detected in the nuclear fraction, possibly due to a low sensitivity to detect a tiny amount of trimers in the nucleus (data not shown). These results suggest that TFII-I does not directly associate with BAM in the nucleus. COS7 cells transfected with TFII-I and Btk confirmed their cytoplasmic association (Fig. 7C) (30).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7. Association of BAM with INI1/SNF5. COS7 cells were transfected with pMEmyc-BAM (A), pMEmyc-BAM and p146 (B) by electroporation. After 48 h, nuclear extracts were prepared and immunoprecipitated with an anti-myc antibody. Precipitates were subjected to western blotting using anti-INI1/hSNF5 (A) or anti-TFII-I (B) antibodies. The blot was stripped and reprobed with an anti-myc antibody (lower panel). (C) COS7 cells were transfected with pME-Btk and p146. After 48 h, cells were lysed and immunoprecipitated with an anti-Btk antibody. Immune complexes were subjected to western blotting using an anti-TFII-I antibody. The same blot was stripped and reprobed with an anti-Btk antibody (lower panel).
|
|
 |
Discussion
|
---|
Btk is required for B cell development, maturation and signal transduction through the BCR and cytokine receptors including the IL-5 receptor. The PH domain of Btk has been implicated as a protein interaction domain, and plays an important role in Btk-mediated signal transduction.
We identified and characterized a molecule, BAM, that binds to the PH domain of Btk (25). The human homolog of mouse BAM is LTG19/ENL/MLLT1 that encodes a transcriptional regulator with unknown function (46,48,49). In leukemia cells, it has been suggested that the t(11;19) chromosomal translocation fuses the AT-hook, a DNA binding domain from the MLL/ALL-1/HRX protein, with the LTG19/ENL/MLLT1 protein, resulting in the formation of a new transcription factor that may play an important role in leukemogenesis. Doty et al. reported that the targeted disruption of the murine Mllt1 gene, which maps to mouse chromosome 17, leads to embryonic lethality, a finding which indicates its importance in early development (50).
In this study, we have examined BAM transcriptional co-activation activity and demonstrated five major findings. (i) By using a GFPBtk fusion protein, we demonstrate nuclear co-localization of Btk and BAM. (ii) Forced expression of GAL4BAM in COS7 cells together with pG5luc, a reporter gene that contains a GAL4-binding domain, demonstrates BAM transcriptional co-activation activity which is dependent on its C-terminal region. (iii) This is further shown by Btk enhancement of BAM transcriptional co-activation activity in experiments employing the Btk(Xid) and Btk(K430R) mutants which are unable to enhance BAM transcriptional co-activation activity, even though they are still able to associate with BAM. (iv) TFII-I augments Btk-mediated enhancement of BAM transactivation activity. (v) BAM is co-immunoprecipitated with INI1, a homologous component of the SWI/SNF complex.
BAM contains a NLS and is capable of associating with Btk. Expression of a myc-tagged form of BAM was restricted to the nucleus as was an epitope-tagged form of LTG19/ENL/MLLT1 (46) which co-localized to the nucleus with Btk when co-expressed with BtkGFP (Fig. 1). It is of note that BAM has two major types of NLS: (i) a single stretch of 56 basic amino acids, that is similarly present in the SV40 large T antigen NLS (four out of five NLS), and (ii) a bipartite NLS composed of 2 basic amino acids and a spacer region of 1012 amino acids, that is typical nucleoplasmin (one out of five NLS). More interestingly, BAM also contains a consensus sequence for a nuclear export signal (NES) defined as a set of critically spaced hydrophobic residues, usually leucines (LXXLXXLXL, where X indicates any residue) (40).
BAM does not appear to contain a DNA-binding consensus sequence such as a helixloophelix motif or zinc finger motif. Search of an EST library for BAM homologs revealed that BAM does not belong to a known family of transcriptional factors. We were also unable to find BAM DNA-binding activity (data not shown). Rubnitz et al. (46) reported that LTG19/ENL/MLLT1 could activate transcription from synthetic reporter genes in both lymphoid and myeloid cells. Their results support the notion that BAM is a transcriptional regulator that binds transcriptional factor(s) and regulates expression of certain gene(s) indirectly.
In this study, we showed BAM transcriptional co-activation activity in a transient expression system using the pG5luc reporter gene as a substrate (Figs 26). Experiments using truncated forms of BAM showed that the C-terminus is necessary and sufficient for the transcriptional co-activation activity (Fig. 2B). This is in sharp contrast to the fact that amino acids 186363 of BAM are indispensable for Btk binding (Fig. 2A). BAM seems to have at least two functional domainsa domain for interacting with the PH domain of Btk and C-terminus for transcriptional co-activation. The C-terminus of BAM is a serine-rich region. This is reminiscent of the Sox family of proteins which require both serine-rich and C-terminus regions for transactivation (51). We do not yet know which molecules interact with the C-terminus or serine-rich regions of BAM. As reported by Garcia-Cuellar et al. (52), it is interesting that the C-terminal hydrophobic domain of human LTG19/ENL/MLLT1 protein interacts with Polycomb 3. Together with the trithorax group of proteins, the polycomb group of proteins maintains a stable transcriptional state despite changing chromatin architecture (53). We need further molecular analyses to clarify the role of the C-terminus region and/or serine-rich regions of BAM in BAM activity.
Several studies have reported that other molecules which bind to Btk control its activation. Full Btk activation appears to depend on transphosphorylation of Tyr551 by protein tyrosine kinases (54). Yao et al. (18) provided evidence that multiple isoforms of PKC interact with Btk and that PKC-mediated phosphorylation down-regulates the enzymatic activity of Btk. In our previous study, forced expression of the Btk-binding region of BAM in an IL-5-dependent early B cell line, Y16, suppressed IL-5-induced Btk activation and cell proliferation while full-length BAM showed little suppressive effect, if any (25). We carefully re-examined the effect of full-length BAM on IL-5-induced proliferation and Btk activation using other IL-5-dependent mouse early B cell lines, and found that enforced expression of full-length of BAM in T88-M significantly suppressed IL-5-induced Btk activation and cell proliferation (unpublished observation). We think that full-length BAM is capable of suppressing Btk activity. As we clearly demonstrated in Fig. 3, Btk enhances BAM transcriptional co-activation activity in COS7 cells. Activation of kinase activity of Btk is induced by transfection of Btk expression plasmid into COS7 cells (30). This enhancing effect was not observed in Btk(
PH), Btk(Xid) and Btk(K430R), indicating an essential role for the functional interaction between Btk and BAM as well as Btks kinase activity for the Btk-mediated enhancement of BAM activity. Thus, mutations impairing the physical and/or functional association between BAM and Btk may result in diminished BAM-dependent transcription. BAM contains three possible tyrosine phosphorylation sites; however, in vitro tyrosine phosphorylation of BAM was not induced by Btk (data not shown). These results may suggest that BAM is not a major substrate of Btk and the Btk-mediated phosphorylation of BAM is not required for the enhancement of BAM activity. We speculate that Btk regulates the BAM activity indirectly, because Btk(Xid) can associate with BAM. To examine the functional interaction between BAM and Btk in mature B cells, we transfected siRNA matching a 22-nucleotide sequence of BAM (BAMsiRNA) into a mouse mature B cell line, A20/2J, and examined anti-IgM-induced calcium mobilization. Transfection of BAMsiRNA into A20/2J induced a marked reduction of endogenous expression of BAM mRNA and a 1520% reduction of BCR-mediated calcium mobilization (data not shown), suggesting that BAM is involved to a certain extent in BCR-mediated signaling in mature B cells.
TFII-I is a multifunctional transcription initiation factor that is expressed ubiquitously and is tyrosine phosphorylated shortly after BCR stimulation (30). TFII-I can function both as a basal factor through interaction with the Inr element and as an activator in the absence of a functional Inr element. TFII-I functionally interacts with Btk and Btk(K430R), but not with Btk(Xid). Reported data indicate that in a heterologous expression system Btk, but not Btk(Xid) or Btk(K430R), augments the transcriptional activity of ectopically expressed TFII-I, suggesting the necessity of both Btk kinase activity and its ability to directly associate with TFII-I to function as a positive regulator of TFII-I transcriptional activity. It is evident that a Btk enhancing effect was observed in COS7 transfectants expressing GAL4BAM, Btk and pG5luc-
Inr as well as pG5luc (Fig. 6). Co-expression of Btk(Xid) and Btk(K430R) with GAL4BAM and pG5luc-
Inr or pG5luc was not effective. Importantly, TFII-I is able to augment the transcriptional co-activation activity of BAM together with Btk in the absence of the Inr element (Fig. 6, lower panel). These results indicate that TFII-I is indispensable for Btk-mediated enhancement of BAM activity and further suggest that TFII-I functions as an activator of transcription, but not as a basal factor, in our system. BAM may regulate gene expression in B cells during their development or triggering through the SWI/SNF complex that is enhanced by Btk and TFII-I. Another possibility is that BAM may regulate TFII-I-mediated transcription together with Btk.
SWI/SNF chromatin remodeling complexes are large multisubunit enzymes with a molecular mass of 12 MDa. In mammals, SWI/SNF complexes have been shown to participate in the transcriptional regulation of many genes, some of which are critical for the normal growth of organisms (39). In mice, heterozygous mutations of some SWI/SNF components result in an increased risk of cancer, whereas homozygous mutations cause embryonic lethality (41,55). Genetic and biochemical data indicate that the SWI/SNF complex can increase transcription factor as well as histone-modifying complex access to DNA (56). Mutations in SWI/SNF component genes could be suppressed by mutations that alter histone gene expression, histone structure or non-histone chromatin proteins. This leads to the suggestion that these gene products facilitate transcriptional activation by altering chromatin structure (5759). Genome-wide expression studies have shown that SWI/SNF is involved in both transcriptional activation and repression (57,58,60,61). Several members of the human ATP-dependent chromatin-remodeling SWI/SNF complex family, which consist of 912 subunits, have previously been described (4144). Recently, LTG19/ENL/MLLT1 was found to be a new member of SWI/SNF complex (45). SNF5 is reported to bind to TFG3/TAF30/ANC1, a component of the yeast SWI/SNF complex (47). It is intriguing that BAM can be co-immunoprecipitated with INI1 when overexpressed in COS7 cells. INI1/SNF5 has a NES that mediates hCRM1/exportin1-dependent nuclear export (62). This result and our data suggest that BAM and INI1/SNF5 cooperate to export Btk to the cytosol. The BAM-INI1/SNF5 association and the high sequence identify between BAM and TFG3/TAF30/ANC1 support the idea that BAM is the mouse homolog of yeast TFG3/TAF30/ANC1 and a component of the mouse SWI/SNF complex.
In conclusion, by using transient transcription assays, we demonstrate that BAM contains transcriptional co-activation activity that is enhanced by Btk. The functional interaction between BAM and Btk as well as the kinase activity of Btk are essential for Btk-mediated enhancement of BAM activity. BAMs transcriptional co-activation activity can be regulated by TFII-I and the SWI/SNF complex of transcription. Btk may participate in the transcriptional regulation of many genes in B cells either by TFII-I activation together with BAM or SWI/SNF complex activation together with TFII-I. The experimental system described in this study should provide a useful tool for delineating the Btk-mediated BCR and IL-5 receptor signaling pathways; however, it remains elusive which gene(s) and their respective patterns of expression are regulated by BAM.
 |
Supplementary data
|
---|
Supplementary data are available at International Immunology Online.
 |
Acknowledgements
|
---|
We are grateful to Satoshi Takaki and R. S. Davis for valuable suggestions and for reviewing the manuscript. We thank Tadashi Yamamoto and A. L. Roy for providing the pME18S-myc vector and the p146 vector respectively. This work was supported in part by a Research Grant from the Human Frontier Science Program (K. T.), by Special Coordination Funds for Promoting Science and Technology (K. T.), and by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan.
 |
Abbreviations
|
---|
BAMBtk-associated protein
BtkBrutons tyrosine kinase
GFPgreen fluorescent protein
NESnuclear export signal
NLSnuclear localization signal
PHpleckstrin homology
PIP3phosphatidylinositol-3,4,5-triphosphate
PKCprotein kinase C
TBSTris-buffered saline
TBS-TTBS containing 0.05% Tween 20
XLAX-linked agammaglobulinemia
XidX-linked immunodeficiency
 |
References
|
---|
- Kurosaki, T. 1999. Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17:555.[CrossRef][ISI][Medline]
- Yang, W. C., Ghiotto, M., Castellano, R., Collette, Y., Auphan, N., Nunes, J. A. and Olive, D. 2000. Role of Tec kinase in nuclear factor of activated T cells signaling. Int. Immunol. 12:1547.[Abstract/Free Full Text]
- Kurosaki, T. 2000. Functional dissection of BCR signaling pathways. Curr. Opin. Immunol. 12:276.[CrossRef][ISI][Medline]
- Campbell, K. S. 1999. Signal transduction from the B cell antigen-receptor. Curr. Opin. Immunol. 11:256.[CrossRef][ISI][Medline]
- Kurosaki, T. and Tsukada, S. 2000. BLNK: connecting Syk and Btk to calcium signals. Immunity 12:1.[ISI][Medline]
- Marshall, A. J., Niiro, H., Yun, T. J. and Clark, E. A. 2000. Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase C
pathway. Immunol. Rev. 176:30.[CrossRef][ISI][Medline]
- Katso, R., Okkenhaug, K., Ahmadi, K., White, S., Timms, J. and Waterfield, M. D. 2001. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu. Rev. Cell. Dev. Biol. 17:615.[CrossRef][ISI][Medline]
- Li, Z., Wahl, M. I., Eguinoa, A., Stephens, L. R., Hawkins, P. T. and Witte, O. N. 1997. Phosphatidylinositol 3-kinase-
activates Brutons tyrosine kinase in concert with Src family kinases. Proc. Natl Acad. Sci. USA 94:13820.[Abstract/Free Full Text]
- Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D. and Panayotou, G. 1996. Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Brutons tyrosine kinase. EMBO J. 15:6241.[Abstract]
- Tsukada, S., Baba, Y. and Watanabe, D. 2001. Btk and BLNK in B cell development. Adv. Immunol. 77:123.[ISI][Medline]
- Kurosaki, T., Maeda, A., Ishiai, M., Hashimoto, A., Inabe, K. and Takata, M. 2000. Regulation of the phospholipase C-
2 pathway in B cells. Immunol. Rev. 176:19.[CrossRef][ISI][Medline]
- Rawlings, D. J., Scharenberg, A. M., Park, H., Wahl, M. I., Lin, S., Kato, R. M., Fluckiger, A. C., Witte, O. N. and Kinet, J. P. 1996. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science 271:822.[Abstract]
- Bajpai, U. D., Zhang, K., Teutsch, M., Sen, R. and Wortis, H. H. 2000. Brutons tyrosine kinase links the B cell receptor to nuclear factor
B activation. J. Exp. Med. 191:1735.[Abstract/Free Full Text]
- Petro, J. B., Rahman, S. M., Ballard, D. W. and Khan, W. N. 2000. Brutons tyrosine kinase is required for activation of I
B kinase and nuclear factor
B in response to B cell receptor engagement. J. Exp. Med. 191:1745.[Abstract/Free Full Text]
- Kaku, H., Horikawa, K., Obata, Y., Kato, I., Okamoto, H., Sakaguchi, N., Gerondakis, S. and Takatsu, K. 2002. NF-
B is required for CD38-mediated induction of C(
)1 germline transcripts in murine B lymphocytes. Int. Immunol. 14:1055.[Abstract/Free Full Text]
- Mizuno, T. and Rothstein, T. L. 2003. Cutting edge: CD40 engagement eliminates the need for Brutons tyrosine kinase in B cell receptor signaling for NF-
B. J. Immunol. 170:2806.[Abstract/Free Full Text]
- Tsukada, S., Simon, M. I., Witte, O. N. and Katz, A. 1994. Binding of ß
subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase. Proc. Natl Acad. Sci. USA 91:11256.[Abstract/Free Full Text]
- Yao, L., Kawakami, Y. and Kawakami, T. 1994. The pleckstrin homology domain of Bruton tyrosine kinase interacts with protein kinase C. Proc. Natl Acad. Sci. USA 91:9175.[Abstract]
- Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., et al. 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X- linked agammaglobulinemia. Cell 72:279.[ISI][Medline]
- Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., et al. 1993. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361:226.[CrossRef][ISI][Medline]
- Satterthwaite, A. B., Li, Z. and Witte, O. N. 1998. Btk function in B cell development and response. Semin. Immunol. 10:309.[CrossRef][ISI][Medline]
- Rawlings, D. J., Saffran, D. C., Tsukada, S., Largaespada, D. A., Grimaldi, J. C., Cohen, L., Mohr, R. N., Bazan, J. F., Howard, M., Copeland, N. G., et al. 1993. Mutation of unique region of Brutons tyrosine kinase in immunodeficient XID mice. Science 261:358.[ISI][Medline]
- Thomas, J. D., Sideras, P., Smith, C. I., Vorechovsky, I., Chapman, V. and Paul, W. E. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261:355.[ISI][Medline]
- Tarakhovsky, A. 1997. Xid and Xid-like immunodeficiencies from a signaling point of view. Curr. Opin. Immunol. 9:319.[CrossRef][ISI][Medline]
- Kikuchi, Y., Hirano, M., Seto, M. and Takatsu, K. 2000. Identification and characterization of a molecule, BAM11, that associates with the pleckstrin homology domain of mouse Btk. Int. Immunol. 12:1397.[Abstract/Free Full Text]
- Tkachuk, D. C., Kohler, S. and Cleary, M. L. 1992. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 71:691.[ISI][Medline]
- Yamamoto, K., Seto, M., Komatsu, H., Iida, S., Akao, Y., Kojima, S., Kodera, Y., Nakazawa, S., Ariyoshi, Y., Takahashi, T., et al. 1993. Two distinct portions of LTG19/ENL at 19p13 are involved in t(11;19) leukemia. Oncogene 8:2617.[ISI][Medline]
- Mohamed, A. J., Vargas, L., Nore, B. F., Backesjo, C. M., Christensson, B. and Smith, C. I. 2000. Nucleocytoplasmic shuttling of Brutons tyrosine kinase. J. Biol. Chem. 275:40614.[Abstract/Free Full Text]
- Yang, W. and Desiderio, S. 1997. BAP-135, a target for Brutons tyrosine kinase in response to B cell receptor engagement. Proc. Natl Acad. Sci. USA 94:604.[Abstract/Free Full Text]
- Novina, C. D., Kumar, S., Bajpai, U., Cheriyath, V., Zhang, K., Pillai, S., Wortis, H. H. and Roy, A. L. 1999. Regulation of nuclear localization and transcriptional activity of TFII-I by Brutons tyrosine kinase. Mol. Cell Biol. 19:5014.[Abstract/Free Full Text]
- Cheriyath, V., Desgranges, Z. P. and Roy, A. L. 2002. c-Src-dependent transcriptional activation of TFII-I. J. Biol. Chem. 277:22798.[Abstract/Free Full Text]
- Perez Jurado, L. A., Wang, Y. K., Peoples, R., Coloma, A., Cruces, J. and Francke, U. 1998. A duplicated gene in the breakpoint regions of the 7q11.23 WilliamsBeuren syndrome deletion encodes the initiator binding protein TFII-I and BAP-135, a phosphorylation target of BTK. Hum. Mol. Genet. 7:325.[Abstract/Free Full Text]
- Roy, A. L., Du, H., Gregor, P. D., Novina, C. D., Martinez, E. and Roeder, R. G. 1997. Cloning of an inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1. EMBO J. 16:7091.[Abstract/Free Full Text]
- Grueneberg, D. A., Henry, R. W., Brauer, A., Novina, C. D., Cheriyath, V., Roy, A. L. and Gilman, M. 1997. A multifunctional DNA-binding protein that promotes the formation of serum response factor/homeodomain complexes: identity to TFII-I. Genes Dev. 11:2482.[Abstract/Free Full Text]
- Roy, A. L., Carruthers, C., Gutjahr, T. and Roeder, R. G. 1993. Direct role for Myc in transcription initiation mediated by interactions with TFII-I. Nature 365:359.[CrossRef][ISI][Medline]
- Roy, A. L., Meisterernst, M., Pognonec, P. and Roeder, R. G. 1991. Cooperative interaction of an initiator-binding transcription initiation factor and the helixloophelix activator USF. Nature 354:245.[CrossRef][ISI][Medline]
- Roy, A. L., Malik, S., Meisterernst, M. and Roeder, R. G. 1993. An alternative pathway for transcription initiation involving TFII-I. Nature 365:355.[CrossRef][ISI][Medline]
- Egloff, A. M. and Desiderio, S. 2001. Identification of phosphorylation sites for Brutons tyrosine kinase within the transcriptional regulator BAP/TFII-I. J. Biol. Chem. 76:27806.[CrossRef]
- Klochendler-Yeivin, A., Muchardt, C. and Yaniv, M. 2002. SWI/SNF chromatin remodeling and cancer. Curr. Opin. Genet. Dev. 12:73.[CrossRef][ISI][Medline]
- Nigg, E. A. 1997. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386:779.[CrossRef][ISI][Medline]
- 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. A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol. Cell. 6:1287.[ISI][Medline]
- Wang, W., Cote, 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. Purification and biochemical heterogeneity of the mammalian SWISNF complex. EMBO J. 15:5370.[Abstract]
- Wong, A. K., Shanahan, F., Chen, Y., Lian, L., Ha, P., Hendricks, K., Ghaffari, S., Iliev, D., Penn, B., Woodland, A. M., Smith, R., Salada, G., Carillo, A., Laity, K., Gupte, J., Swedlund, B., Tavtigian, S. V., Teng, D. H. and Lees, E. 2000. BRG1, a component of the SWISNF complex, is mutated in multiple human tumor cell lines. Cancer Res. 60:6171.[Abstract/Free Full Text]
- Klochendler-Yeivin, A., Fiette, L., Barra, J., Muchardt, C., Babinet, C. and Yaniv, M. 2000. The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression. EMBO Rep. 1:500.[Abstract/Free Full Text]
- Nie, Z., Yan, Z., Chen, E. H., Sechi, S., Ling, C., Zhou, S., Xue, Y., Yang, D., Murray, D., Kanakubo, E., Cleary, M. L. and Wang, W. 2003. Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner. Mol. Cell Biol. 23:2942.[Abstract/Free Full Text]
- Rubnitz, J. E., Morrissey, J., Savage, P. A. and Cleary, M. L. 1994. ENL, the gene fused with HRX in t(11;19) leukemias, encodes a nuclear protein with transcriptional activation potential in lymphoid and myeloid cells. Blood 84:1747.[Abstract/Free Full Text]
- Cairns, B. R., Henry, N. L. and Kornberg, R. D. 1996. TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9. Mol. Cell Biol. 16:3308.[Abstract]
- Slany, R. K., Lavau, C. and Cleary, M. L. 1998. The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX. Mol. Cell Biol. 18:122.[Abstract/Free Full Text]
- Schreiner, S. A., Garcia-Cuellar, M. P., Fey, G. H. and Slany, R. K. 1999. The leukemogenic fusion of MLL with ENL creates a novel transcriptional transactivator. Leukemia 13:1525.[CrossRef][ISI][Medline]
- Doty, R. T., Vanasse, G. J., Disteche, C. M. and Willerford, D. M. 2002. The leukemia-associated gene Mllt1/ENL: characterization of a murine homolog and demonstration of an essential role in embryonic development. Blood Cells Mol. Dis. 28:407.[CrossRef][ISI][Medline]
- Nowling, T. K., Johnson, L. R., Wiebe, M. S. and Rizzino, A. 2000. Identification of the transactivation domain of the transcription factor Sox-2 and an associated co-activator. J. Biol. Chem. 275:3810.[Abstract/Free Full Text]
- Garcia-Cuellar, M. P., Zilles, O., Schreiner, S. A., Birke, M., Winkler, T. H. and Slany, R. K. 2001. The ENL moiety of the childhood leukemia-associated MLL-ENL oncoprotein recruits human Polycomb 3. Oncogene 20:411.[CrossRef][ISI][Medline]
- van Lohuizen, M. 1999. The trithorax-group and polycomb-group chromatin modifiers: implications for disease. Curr. Opin. Genet. Dev. 9:355.[CrossRef][ISI][Medline]
- Wahl, M. I., Fluckiger, A. C., Kato, R. M., Park, H., Witte, O. N. and Rawlings, D. J. 1997. Phosphorylation of two regulatory tyrosine residues in the activation of Brutons tyrosine kinase via alternative receptors. Proc. Natl Acad. Sci. USA 94:11526.[Abstract/Free Full Text]
- Versteege, I., Sevenet, N., Lange, J., Rousseau-Merck, M. F., Ambros, P., Handgretinger, R., Aurias, A. and Delattre, O. 1998. Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394:203.[CrossRef][ISI][Medline]
- Kingston, R. E. and Narlikar, G. J. 1999. ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev. 13:2339.[Free Full Text]
- Cote, J., Quinn, J., Workman, J. L. and Peterson, C. L. 1994. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265:53.[ISI][Medline]
- Imbalzano, A. N., Kwon, H., Green, M. R. and Kingston, R. E. 1994. Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature 370:481.[CrossRef][ISI][Medline]
- Cairns, B. R., Lorch, Y., Li, Y., Zhang, M., Lacomis, L., Erdjument-Bromage, H., Tempst, P., Du, J., Laurent, B. and Kornberg, R. D. 1996. RSC, an essential, abundant chromatin-remodeling complex. Cell 87:1249.[ISI][Medline]
- Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E. and Green, M. R. 1994. Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex. Nature 370:477.[CrossRef][ISI][Medline]
- Owen-Hughes, T. and Workman, J. L. 1996. Remodeling the chromatin structure of a nucleosome array by transcription factor-targeted trans-displacement of histones. EMBO J. 15:4702.[Abstract]
- Craig, E., Zhang, Z. K., Davies, K. P. and Kalpana, G. V. 2002. A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis. EMBO J. 21:31.[Abstract/Free Full Text]