Myosin light chain 1 atrial isoform (MLC1A) is expressed in pre-B cell
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《核酸研究医学期刊》
Department of Physiological Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany and 1 Pathologisches Institut, Universit?t Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
*To whom correspondence should be addressed. Tel: +49 731 502 3270; Fax: +49 731 502 2892; Email: thomas.wirth@medizin.uni-ulm.de
ABSTRACT
The BOB.1/OBF.1 protein is a B-cell-specific coactivator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of the Oct proteins. However, animals deficient for BOB.1/OBF.1 showed virtually normal expression of genes that contain octamer motifs in their regulatory regions. To identify new genes that are regulated by BOB.1/OBF.1, we took advantage of a previously described cell system. RNAs differentially expressed in a BOB.1/OBF.1-deficient pre-B cell line and a derivative of this cell line expressing a hormone dependent BOB.1/OBF.1-estrogene receptor (BobER) fusion protein were isolated. Using the cDNA representational difference analysis method we could identify myosin light chain 1 atrial (MLC1A) isoform as a gene regulated by BOB.1/OBF.1. MLC1A was so far unknown to be expressed in tissues other than muscle. Here we demonstrate that MLC1A is indeed expressed in mouse pre-B cells. Analysis of the expressed mRNA revealed an alternative 5' promoter element and an alternative splice product, which had not yet been described for the murine gene. Cotransfection experiments with reporter constructs driven by the MLC1A promoter suggest that the regulation by BOB.1/OBF.1 is indirect. Consistent with this conclusion is the observation that transcriptional induction of the endogenous MLC1A gene by BOB.1/OBF.1 requires de novo protein synthesis.
INTRODUCTION
The octamer motif is a critical regulatory element for B-cell-specific transcription (1,2). It is conserved in virtually all immunoglobulin heavy and light chain gene promoters as well as in several immunoglobulin enhancer elements. It is essential for the B-cell-specific promoter function and contributes to B-cell-specific enhancer activity. However, a variety of ubiquitously expressed genes, such as the histone H2B gene and most U sn RNA genes, require the octamer motif for efficient expression (3,4).
B cells express two octamer binding transcription factors, Oct1 and Oct2 (5). Transcriptional activity conferred by the octamer motif requires additional coactivators (6–8). The BOB.1/OBF.1 (also named Bob.1, OBF-1 or OCA-B) coactivator interacts with either Oct1 or Oct2 and thereby activates transcription from promoter-proximal positions (9–12). Activity from distal enhancer positions specifically requires the presence of Oct2 and a further still unknown B-cell-restricted coactivator (8,13). BOB.1/OBF.1 itself does not bind to the octamer motif with high affinity, but rather is recruited into the active transcription complexes via protein–protein interactions with the Oct proteins. It does, however, increase the selectivity of the ternary complexes for a subset of octamer motifs by making contacts with the major groove of DNA within the octamer motif and with the POU-specific domain and the POU homeodomain (14–17). Furthermore, by contacting both POU subdomains simultaneously, BOB.1/OBF.1 can act as a molecular clamp that holds together these subdomains on DNA (18). A BOB.1/OBF.1 protein isoform bearing an N-terminal extension is myristylated in vivo and localized in the membrane fraction. The exact function of this BOB.1/OBF.1 isoform is not known (19).
BOB.1/OBF.1 is expressed at all stages of B-cell development. Expression levels are highest in germinal center B cells and can be induced in resting B cells by appropriate stimulation (20,21). In B cells, expression of BOB.1/OBF.1 is not only regulated at the level of transcription but also at the level of protein stability (22,23). Furthermore, expression and function of BOB.1/OBF.1 can be induced by co-stimulation of T lymphocytes (24).
Inactivation of the gene coding for the BOB.1/OBF.1 coactivator affects several stages of B-cell development. In bone marrow a reduction of transitional B cells is observed (25). Additionally, numbers of B cells in the periphery, as well as of recirculating B cells in the bone marrow, are reduced. The most striking characteristic of BOB.1/OBF.1-deficient mice is the complete failure to form germinal centers in spleens and lymph nodes (26–28). Notably, expression of the immunoglobulin heavy and light chain genes is not impaired in B cells from BOB.1/OBF.1-deficient mice. As a consequence, normal levels of IgM are produced, whereas the amounts of switched immunoglobulin isotypes are strongly reduced. In addition, development of marginal zone B cells is disturbed (29) and the number of apoptotic B cells is increased (25). A bcl2 transgene can rescue all aspects of early B-cell development in BOB.1/OBF.1-deficient mice. However, terminal differentiation and function is still defective (30). Surprisingly, in mice simultaneously deficient for BOB.1/OBF.1 and Oct2, B cells develop up to the IgM-positive B-cell stage and immunoglobulin gene transcription was not affected (31).
So far, only a few genes have been identified which are regulated by BOB.1/OBF.1. In B cells the expression of the BLR1 gene (32) was shown to be regulated cooperatively by the transcription factors NF-B and Oct2, as well as the cofactor BOB.1/OBF.1. The CCR-5 gene expression (33) was shown to be controlled by BOB.1/OBF.1 and Oct2 in T cells. Expression of both of these genes was only partially affected by the lack of BOB.1/OBF.1. We recently searched for BOB.1/OBF.1 target genes by Chip-based expression profiling and identified a set of genes both induced and repressed by BOB.1/OBF.1 (34). The products of these genes are involved in various aspects of B-cell physiology, such as cellular metabolism, cell adhesion and differentiation. In the present study, we used a parallel approach to search for BOB.1/OBF.1 target genes, employing the cDNA representational difference analysis (RDA) method (35). We identified a gene coding for the myosin light chain 1 atrial (MLC1A) isoform, which was previously described as being specifically expressed in the early development of mouse muscle cells in the embryo (MLC1emb), and in the adult mouse in heart atria (MLC1A) (36–39). Here, we show that MLC1A is expressed in murine pre-B cell lines as well as in primary pre-B cells in a BOB.1/OBF.1-dependent manner. In B cells, MLC1A transcripts are expressed using an alternative 5'UTR as compared with the one utilized in mouse muscle cells.
MATERIALS AND METHODS
Cell culture and transfection
The BOB.1/OBF.1-deficient Abelson virus-transformed pre-B cells were kept in Iscoves modified Dulbecco’s medium (IMDM; Life Technologies, Inc.), supplemented with 10% fetal bovine serum, penicillin/streptomycin, L-glutamate and 50 μM ?-mercaptoethanol and grown at 37°C and 10% CO2. All other cell lines were kept in Dulbecco’s modified Eagle’s medium/glutamax (DMEM; Life Technologies, Inc.) supplemented with 10% fetal bovine serum, penicillin/streptomycin and 50 μM ?-mercaptoethanol and grown at 37°C and 5% CO2. For hydroxytamoxifen (OHT) induction experiments, cells were treated with 200 nM OHT (BioTrend Chemikalien GmbH) and, as indicated, pre-incubated for 30 min with 1 μg/ml cycloheximide (CHX). NIH/3T3 cells were transiently transfected by electroporation (450 V, 250 μF in PBS), pRL-CMV (Promega) was cotransfected in all experiments and used for normalization of different transfection efficiencies in the individual experiments.
Plasmids
Different MLC1A promoter constructs (–630, –3000) were recloned from the originally MLC1A-CAT constructs or amplified by PCR (–680) and cloned into the pTKL/2 vector containing the HSV-thymidine kinase promoter (–105 to +52) from the pBLCAT2 in front of the firefly luciferase coding region. The HSV-thymidine kinase promoter was excised by a restriction endonuclease digest and replaced by the MLC1A promoter sequences. The wild-type BOB.1/OBF.1 cDNA was cloned into the expression vector pCDNA3. For luciferase reporter assays the octamer-dependent reporter plasmid μET1 was used as an internal control (40).
cDNA RDA
Poly(A)+ RNA was prepared from the indicated cell lines as described (40). cDNA was prepared using the cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). The cDNA RDA protocol was performed exactly as described by Hubank and Schatz (35). MB10-mock cDNA was used as driver, MB10-BobER cDNA as tester. For the three subsequent rounds of hybridizations the tester/driver ratios were 1:100, 1:800, 1: 500 000. The difference product III (DPIII) was DpnII digested, cloned into BamHI cut pBluescript and 160 randomly picked cDNAs were sequenced using the T7 primer.
RACE protocol
5' Rapid amplification of cDNA ends (RACE) was performed using the RACE-kit (Life Technologies, Inc.) according to the instructions of the manufacturer. The following primers were used: myGSP1 (cca ttg gca tcc tcc), myGSP2 (tcc acc tct gcc tcg ct) and myGSP3 (ccg cag ccc ctc cac gaa g).
RT–PCR analysis
CD38-negative and -positive cells from human tonsils were isolated as described before (21,42). Total RNA of the respective cells or cell lines was used to prepare cDNA using SuperscriptII or M-MLV reverse transcriptase (Life Technologies, Inc.; Gibco). The cDNAs were used to analyze the expression of different MLC1A isoforms. PCR was performed as follows: 3 min at 94°C, 30 cycles of 30 s at 94°C, 60 s at 60°C, 90 s at 72°C. PCR products were analyzed on a 1.2% agarose gel. The following primers were used: MLC1A-3.1 (tgc tcc acc tct gcc tcg ctc atc), MLC1A-5.1 (gcc aga acc cca cca acg cag ag), MLC1A-5.2 (ccc acc tcc act gga gag cc), MLC1A-5.3 (cct ctg tgg ggg ctc cta ccc), MLC1A-5.4 (cga gcc acc tct cct cct ttg g), MLC1A-5.5 (ccc acc cct ctc tgg gtt tcc). For the simultaneous detection of murine and human MLC1A expression, the following primers were used: m/hMLC1A forward, gga gag atg aag atc acc tac gg; m/hMLC1A reverse, ggt gcc ctg ctc ctt gtt gc.
Northern blot analysis
Preparation and analysis of total cytoplasmic RNA, poly(A)+ RNA and northern blots were performed as described (40). For analysis of MLC1A expression an MLC1A cDNA fragment was amplified using the MLC1A-5.1 and MLC1A-3.1 primers.
RESULTS AND DISCUSSION
In order to identify BOB.1/OBF.1-dependent target genes in BOB.1/OBF.1-deficient B cells, we used a previously described Abelson virus-transformed pre-B cell line established from the bone marrow of BOB.1/OBF.1-deficient mice. In addition, they were transduced with a retrovirus, which resulted in the expression of an inducible BobER fusion protein (40). In this cell line (MB10-BobER), octamer-dependent synthetic promoter and immunoglobulin promoter/enhancer driven reporter constructs were inactive, unless the activity of the BobER protein was induced by the hormone OHT (40). The use of this system allows us to examine B cells at the same stage of differentiation. Using this approach, differential gene expression should be caused by the transcriptional activity of the BOB.1/OBF.1 protein.
The MB10-BobER cells and, as control, the MB10-mock cell line, which was infected with the empty expression vector, were induced with OHT to avoid potential side effects of OHT. RNA was prepared from the induced cell lines and a cDNA RDA screen was performed (35). The MB10-BobER cDNA was used as tester, the MB10-mock cDNA as driver. After three rounds of hybridization/amplification the resulting cDNAs (DPIII) were cloned into the pBluescript vector and subsequently 160 clones were sequenced.
Among these clones, we frequently found estrogen receptor sequences (50%), confirming that the cDNA RDA protocol enriched differentially expressed clones. Most of the remaining clones were single cDNA isolates and appeared not to be regulated by BOB.1/OBF.1 (data not shown). The cDNA identified most frequently (12 times) beside the estrogen receptor sequences was derived from the MLC1A gene.
The MLC1A gene was originally identified as an essential myosin light chain, specifically expressed in the murine heart atria (MLC1A, ‘atrial’ isoform). Additionally, it was described as the first myosin light chain during early embryonic development in embryonic skeletal muscle, therefore, also designated as MLC1emb (‘embryonic’ isoform) (36–38). The MLC1A gene was differentially expressed as confirmed by northern blot (Fig. 1A). In addition to an unspecific band already present in the mock-infected cell line (see below), a second, more slowly migrating band, was observed in cells expressing the active BobER fusion protein.
Figure 1. The expression of MLC1A is dependent on BOB.1/OBF.1. (A–C) Northern blot analysis using an MLC1A-specific probe and 2 μg of poly(A)+ RNA or 10 μg of total RNA of the indicated cell line. MB10, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells; MB10-mock, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells mock infected; MB10-BobER, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells BobER infected; PD31, wild-type Abelson virus-transformed pre-B cells; preB Oct2–/–, preB BOB.1/OBF.1–/–, preB wt, IL-7 and stroma cell-dependent primary pre-B cells from Oct2–/–, BOB.1/OBF.1–/– or wild-type mice derived from fetal liver. (D–F) Detection of MLC1A transcripts by RT–PCR. Genomic localization of the used primer pair (5.1 and 3.1) used in (D) is described in Figure 3A. Total RNA was prepared from atria, from total spleen of wild-type or BOB.1/OBF.1–/– mice, from CD38– or CD38+ tonsilar B cells, or from cell lines indicated, transcribed into cDNA and subsequently analyzed by RT–PCR. MB10-mock, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells mock infected; MB10-BobER, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells BobER infected; NIH/3T3, mouse fibroblast cell line.
In addition, several other cDNA clones were isolated repeatedly but less abundant, like alpha enolase, translation initiation factor 4E, complement protein C7 and several dehydrogenase motives. Notably, the transcripts for aldehyde dehydrogenase-2 like, the aldoketoreductase AKR1C13 and several other genes had been identified as BOB.1/OBF.1 regulated genes using a recently applied gene expression profiling approach and have been already described (34). Here we will focus on the analysis of the MLC1A gene.
We analyzed the influence of the hormone OHT on MLC1A expression in BobER-expressing cells. RNA was prepared from either uninfected MB10 cells, mock-infected MB10 cells or MB10 cells expressing the BobER allele (MB10-BobER), treated with OHT or left uninduced, and analyzed with the MLC1A-specific probe in northern blot experiments (Fig. 1B). As a control, RNA from PD31, an Abelson virus-transformed wild-type pre-B cell line, was included in this study. In all analyzed samples, the faster migrating non-specific band was visible, albeit at somewhat variable levels. Additionally, the MLC1A-specific (slower) band could be detected in the case of BOB.1/OBF.1-expressing PD31 cells and in OHT-treated MB10-BobER cells. In the case of PD31 cells, this expression was OHT independent as expected, whereas in BobER-expressing cells MB10-BobER, the expression of this additional band was regulated by treatment with OHT. Identical results were obtained analysing a second BobER-expressing cell clone (data not shown). To exclude a non-physiological expression of MLC1A in the Abelson virus-transformed pre-B cell lines we next analyzed primary mouse pre-B cells cultured in the presence of the cytokine IL-7 on a stromal cell layer (Fig. 1C). Northern blot analysis revealed a strong MLC1A expression in wild-type pre-B cells. Notably, MLC1A was also detected in Oct2-deficient pre-B cells, whereas in BOB.1/OBF.1-deficient B cells no MLC1A was detectable confirming the BOB.1/OBF.1 dependence of its expression.
Since we always observed two bands in the BOB.1/OBF.1-expressing pre-B cells we asked if both of these bands are MLC1A specific. MLC1A expression in adult mice was reported to be restricted to the atrium (36–39) and should not be expressed in other tissues, for example, in fibroblasts. When we analyzed MLC1A expression in NIH/3T3 cells, in 10T fibroblast cells and in ST2 stroma cells by northern blots, only the slower migrating band could be detected (data not shown). These data indicate that the lower band is rather an unspecific hybridization than an MLC1A-specific signal. To corroborate this conclusion we used MLC1A gene-specific primers to analyze MLC1A transcripts by RT–PCR. At high numbers of PCR cycles we observed low MLC1A expression in MB10-mock cells and a massive increase of MLC1A in MB10-BobER cells. However, no PCR product could be detected in the fibroblast cell line NIH/3T3 (Fig. 1D). The specificity of the RT–PCR was verified by sequencing the PCR products and by performing RT–PCR with mouse atria RNA that gave a product of the same size (data not shown). Therefore, we conclude that the upper band observed in northern blot experiments corresponds specifically to MLC1A, whereas the lower band results from an unspecific hybridization.
As mentioned above, MLC1A, so far, was described as being expressed specifically in atria but not in other adult tissues. We were interested to determine whether MLC1A was expressed throughout the B-cell lineage and/or even in other hematopoietic cells. Therefore, we performed northern blot analysis with several additional B cell lines representing different stages of B-cell development. However, whereas the MLC1A band was present in pre-B cells, no expression was seen in cell lines representing immature B cells (WEHI 231), plasma cell stage (S194), or in T cells irrespective of whether they express BOB.1/OBF.1 (EL4) or not (BW 5147) (data not shown). In addition, we analyzed several other mature B and plasma cell lines but could not detect MLC1A expression. Furthermore, analysing lymphocytes derived from total spleen of wild-type or BOB.1/OBF.1-deficient mice by RT–PCR no MLC1A expression could be detected even in wild-type splenocytes. In parallel, mouse atria were analyzed where MLC1A expression was observed (Fig. 1E). Since the expression level of BOB.1/OBF.1 is the highest in germinal centers, we asked whether MLC1A is also upregulated in mature IgMlowIgDhigh CD38+ primary human germinal center B cells. Using primers specific for the mouse, as well as for the human, MLC1A gene, RT–PCRs were performed with cDNAs from mouse atria as a positive control or from human CD38+ germinal center and CD38– non-germinal center mature B cells (Fig. 1F). No MLC1A expression could be detected either in mature germinal center cells or in non-germinal center B cells and other mature B cells. Therefore, MLC1A expression seems to be restricted to the pre-B-cell stage. This observation is not surprising, since BOB.1/OBF.1 was shown to be required at several stages of B-cell development including the pre-B-cell stage. Additionally, the target gene screen was performed using pre-B cells. Thus, identified BOB.1/OBF.1-dependent genes might be expressed and regulated only at this stage of B-cell development. This observation is reminiscent of the Oct2 target gene CRISP-3, which is expressed in pre-B cells and has not been found at later developmental B-cell stages (43).
MLC1A was described as being expressed in mouse atria and in the mouse skeletal muscle cell line C2C12. We wanted to compare the expression levels of MLC1A in pre-B cells with that in muscle cells. Northern blot experiments indeed confirmed a very strong MLC1A expression in mouse muscle cells, especially in the atria (Fig. 2A). This muscle-specific expression (in atrium and C2C12 cells) was significantly stronger than the MLC1A expression level found in PD31 cells. Interestingly, when we compared the sizes of MLC1A-specific bands it became clear that the pre-B-cell-specific band was slightly larger than that in the muscle cells. We reasoned that MLC1A might be alternatively spliced in mouse pre-B cells. In human skeletal muscle cells, different 5'UTR and 3'UTRs of the MLC1A gene are expressed (44,45), whereas in the mouse, so far, only one 5'UTR has been described. We performed 5'-RACE with RNA from the MB10-BobER cell line induced with OHT. A dominant 5'UTR from pre-B cells was identified, which we named distal 5'UTR since it results from an alternative upstream promoter and consequently from a splice process within the MLC1A 5'UTR. The previously described 5'UTR from mouse muscle cells was named proximal 5'UTR (sequences of both 5'UTRs are compared in Fig. 2B). Therefore, the situation in mouse pre-B cells resembles that in human muscle cells, where distinct MLC1A and MLC1emb mRNAs are expressed differing in their 5'UTRs (44,45). The function of the alternative transcript in humans is still unclear.
Figure 2. Comparison of MLC1A expression in mouse muscle and pre-B cells. (A) Northern blot analysis using an MLC1A-specific probe and 10 μg of total RNA of the indicated cell lines and tissue: atria, mouse atrium; PD31, wild-type Abelson virus-transformed pre-B cells; C2C12, mouse skeletal muscle cell line; NIH/3T3, mouse fibroblast cell lines. (B) Sequence of the 5' region of the mouse MLC1A gene. Alignment of the proximal 5'UTR identified by 5'-RACE from MB10-K10 cell cDNA and the known distal 5'UTR.
In order to analyze if both 5'UTRs are expressed in mouse B cells, we prepared primer pairs in order to distinguish the different 5'UTRs by RT–PCR (Fig. 3A). Using the primer 5.1 (located in MLC1A exon 3) or 5.2 (located nearby the translation initiation signal), an MLC1A-specific PCR product should be observed in all cases, independent of the specific 5'UTRs used. The 5.3 primer was a negative control since it is located in the region between distal and proximal promoters that is spliced out. Consequently, no PCR product should be observed. Using the primer 5.5 or 5.4 we can distinguish which 5'UTR was used. As expected, the primers 5.1 (Fig. 1D) and 5.2 (Fig. 3B) revealed a signal in MB10 cells, whereas the primer 5.3 did not show a signal. The signal was much stronger in cells expressing a functional BOB.1/OBF.1 protein. Although we could detect some transcript originating from the proximal promoter, the majority of MLC1A transcripts in pre-B cells derived from the distal promoter. The use of another 5'UTR in B cells other than in muscle cells suggests that different promoter regions are responsible for the regulation of MLC1A expression in B cells versus muscle cells.
Figure 3. Analyses of the expression of different 5'UTRs in pre-B cells. (A) Schematic representation of the genomic locus of the MLC1A gene, corresponding proximal and distal promoters and 5'UTR in mouse pre-B cells as well as location of the used RT–PCR primers. (B) RT–PCR using the indicated primer pairs and cDNAs from the cell lines indicated. As a control, ?-actin PCR was performed.
We then addressed the question whether regulation of MLC1A expression by BOB.1/OBF.1 is direct or involves an intermediate. We generated luciferase reporter constructs, differing in the length of the MLC1A promoter –630, –680 or –3000 bp. The activity of all these reporters was increased by BOB.1/OBF.1 cotransfection in NIH/3T3 cells (Fig. 4A). The highest induced activity was found in the case of the shortest 630 bp promoter, which is consistent with the earlier observation in muscle cells that 630 bp of the mouse MLC1A promoter are sufficient to achieve a regulated expression of the gene (41). However, the basal activity of the –630 bp promoter was also higher compared with the longer promoter constructs. Determining the absolute induction of analyzed promoters by BOB.1/OBF.1, all constructs were induced equally 2–3-fold. Surprisingly, when the activity of these constructs was analyzed in MB10-BobER cells, no OHT induction of luciferase activity could be observed analyzing cells 24 h after transfection, while some induction of luciferase activity was observed upon longer incubation (data not shown). This result suggested that the activation of MLC1A gene transcription by BOB.1/OBF.1 might be indirect. Since BobER cells originally used for the presented RDA were induced with OHT for 20 h, we cannot exclude an indirect regulation of identified genes by BOB.1/OBF.1. Recently, two E-box motives were shown to be critically involved in the regulation of the MLC1A promoter, binding MyoD/E12 and myogenin/E12 heterodimer (41,46). Importantly, the known MLC1A promoter sequences do not contain recognizable octamer motifs which could mediate the regulation via BOB.1/OBF.1.
Figure 4. Regulation of MLC1A promoter activity as well as gene expression by BOB.1/OBF.1 is indirect. (A) Transient transfection of NIH/3T3 cells using 5 μg of different MLC1A promoter constructs as indicated, either alone or together with 10 μg of a BOB.1/OBF.1 expression vector (each experiment was repeated three times). Additionally, as an internal control, the BOB.1/OBF.1-dependent μET1 promoter was analyzed. The value of the μET1 promoter vector was set to one. The fold induction was determined. (B) Northern blot analysis using an MLC1A-specific probe and 10 μg of total RNA of MB10-BobER pre-B cell line. Cells were induced with OHT (200 nM) for the indicated time or pre-treated with CHX (1 μg/ml), as indicated, for 30 min, RNA was prepared and subsequently subjected to northern blot analyses.
To address this issue, MB10-BobER cells were pre-incubated with the protein synthesis inhibitor CHX and induced for 12 h with OHT. Northern blot analyses revealed that CHX prevents the OHT-mediated induction of MLC1A by BOB.1/OBF.1 (Fig. 4B), indicating that MLC1A indeed is indirectly regulated by BOB.1/OBF.1.
The actin–myosin complex is the main contractile component of the muscle sarcomers. Furthermore, actin and myosin undergo reorganization in cultured mammalian non-muscle cells in response to cellular signals during various motile processes in cells, such as chemotaxis, phagocytosis, capping of surface receptors, vesicle transport and cytokinesis (47–50).
Many functions of myosins in non-muscle cells have been described, such as cytoskeletal dynamics, which in turn are essential for cell migration and motility. Immune cells are characterized by cellular movement between compartments. Immature (transitional) B cells emerge from the bone marrow to the periphery and migrate into specific microenvironments of peripheral secondary lymphoid tissues, such as spleen and lymph nodes. Although the physical sites of B-cell precursor development and differentiation in the bone marrow remain poorly defined, it is likely that the actin–myosin complex is also essential for B-cell motility of earlier developmental stages, e.g. to build contacts with the stromal layer, important for progenitor B cells. This hypothesis is supported by recent findings demonstrating the important role of cytoskeleton reorganization of hematopoietic stem/progenitor cells. These processes affect adhesion and mobilization from the medullary cavity into the blood circulation and migration into lymph nodes or sites of inflammation (51,52). Additionally, it was shown that the cytoskeletal organization that contributes to the shape of hematopoietic cells influences the transduction of growth signals through a number of externally activating signaling pathways (53). B cells deficient for BOB.1/OBF.1 have several defects in early development in the bone marrow and cannot properly develop into transitional B cells (25). Taking together, the BOB.1/OBF.1-dependent expression of an essential myosin light chain in pre-B cells could be a novel mechanism to achieve a regulation of potential functions of actin–myosin motility in pre-B cells, essential for development and function.
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*To whom correspondence should be addressed. Tel: +49 731 502 3270; Fax: +49 731 502 2892; Email: thomas.wirth@medizin.uni-ulm.de
ABSTRACT
The BOB.1/OBF.1 protein is a B-cell-specific coactivator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of the Oct proteins. However, animals deficient for BOB.1/OBF.1 showed virtually normal expression of genes that contain octamer motifs in their regulatory regions. To identify new genes that are regulated by BOB.1/OBF.1, we took advantage of a previously described cell system. RNAs differentially expressed in a BOB.1/OBF.1-deficient pre-B cell line and a derivative of this cell line expressing a hormone dependent BOB.1/OBF.1-estrogene receptor (BobER) fusion protein were isolated. Using the cDNA representational difference analysis method we could identify myosin light chain 1 atrial (MLC1A) isoform as a gene regulated by BOB.1/OBF.1. MLC1A was so far unknown to be expressed in tissues other than muscle. Here we demonstrate that MLC1A is indeed expressed in mouse pre-B cells. Analysis of the expressed mRNA revealed an alternative 5' promoter element and an alternative splice product, which had not yet been described for the murine gene. Cotransfection experiments with reporter constructs driven by the MLC1A promoter suggest that the regulation by BOB.1/OBF.1 is indirect. Consistent with this conclusion is the observation that transcriptional induction of the endogenous MLC1A gene by BOB.1/OBF.1 requires de novo protein synthesis.
INTRODUCTION
The octamer motif is a critical regulatory element for B-cell-specific transcription (1,2). It is conserved in virtually all immunoglobulin heavy and light chain gene promoters as well as in several immunoglobulin enhancer elements. It is essential for the B-cell-specific promoter function and contributes to B-cell-specific enhancer activity. However, a variety of ubiquitously expressed genes, such as the histone H2B gene and most U sn RNA genes, require the octamer motif for efficient expression (3,4).
B cells express two octamer binding transcription factors, Oct1 and Oct2 (5). Transcriptional activity conferred by the octamer motif requires additional coactivators (6–8). The BOB.1/OBF.1 (also named Bob.1, OBF-1 or OCA-B) coactivator interacts with either Oct1 or Oct2 and thereby activates transcription from promoter-proximal positions (9–12). Activity from distal enhancer positions specifically requires the presence of Oct2 and a further still unknown B-cell-restricted coactivator (8,13). BOB.1/OBF.1 itself does not bind to the octamer motif with high affinity, but rather is recruited into the active transcription complexes via protein–protein interactions with the Oct proteins. It does, however, increase the selectivity of the ternary complexes for a subset of octamer motifs by making contacts with the major groove of DNA within the octamer motif and with the POU-specific domain and the POU homeodomain (14–17). Furthermore, by contacting both POU subdomains simultaneously, BOB.1/OBF.1 can act as a molecular clamp that holds together these subdomains on DNA (18). A BOB.1/OBF.1 protein isoform bearing an N-terminal extension is myristylated in vivo and localized in the membrane fraction. The exact function of this BOB.1/OBF.1 isoform is not known (19).
BOB.1/OBF.1 is expressed at all stages of B-cell development. Expression levels are highest in germinal center B cells and can be induced in resting B cells by appropriate stimulation (20,21). In B cells, expression of BOB.1/OBF.1 is not only regulated at the level of transcription but also at the level of protein stability (22,23). Furthermore, expression and function of BOB.1/OBF.1 can be induced by co-stimulation of T lymphocytes (24).
Inactivation of the gene coding for the BOB.1/OBF.1 coactivator affects several stages of B-cell development. In bone marrow a reduction of transitional B cells is observed (25). Additionally, numbers of B cells in the periphery, as well as of recirculating B cells in the bone marrow, are reduced. The most striking characteristic of BOB.1/OBF.1-deficient mice is the complete failure to form germinal centers in spleens and lymph nodes (26–28). Notably, expression of the immunoglobulin heavy and light chain genes is not impaired in B cells from BOB.1/OBF.1-deficient mice. As a consequence, normal levels of IgM are produced, whereas the amounts of switched immunoglobulin isotypes are strongly reduced. In addition, development of marginal zone B cells is disturbed (29) and the number of apoptotic B cells is increased (25). A bcl2 transgene can rescue all aspects of early B-cell development in BOB.1/OBF.1-deficient mice. However, terminal differentiation and function is still defective (30). Surprisingly, in mice simultaneously deficient for BOB.1/OBF.1 and Oct2, B cells develop up to the IgM-positive B-cell stage and immunoglobulin gene transcription was not affected (31).
So far, only a few genes have been identified which are regulated by BOB.1/OBF.1. In B cells the expression of the BLR1 gene (32) was shown to be regulated cooperatively by the transcription factors NF-B and Oct2, as well as the cofactor BOB.1/OBF.1. The CCR-5 gene expression (33) was shown to be controlled by BOB.1/OBF.1 and Oct2 in T cells. Expression of both of these genes was only partially affected by the lack of BOB.1/OBF.1. We recently searched for BOB.1/OBF.1 target genes by Chip-based expression profiling and identified a set of genes both induced and repressed by BOB.1/OBF.1 (34). The products of these genes are involved in various aspects of B-cell physiology, such as cellular metabolism, cell adhesion and differentiation. In the present study, we used a parallel approach to search for BOB.1/OBF.1 target genes, employing the cDNA representational difference analysis (RDA) method (35). We identified a gene coding for the myosin light chain 1 atrial (MLC1A) isoform, which was previously described as being specifically expressed in the early development of mouse muscle cells in the embryo (MLC1emb), and in the adult mouse in heart atria (MLC1A) (36–39). Here, we show that MLC1A is expressed in murine pre-B cell lines as well as in primary pre-B cells in a BOB.1/OBF.1-dependent manner. In B cells, MLC1A transcripts are expressed using an alternative 5'UTR as compared with the one utilized in mouse muscle cells.
MATERIALS AND METHODS
Cell culture and transfection
The BOB.1/OBF.1-deficient Abelson virus-transformed pre-B cells were kept in Iscoves modified Dulbecco’s medium (IMDM; Life Technologies, Inc.), supplemented with 10% fetal bovine serum, penicillin/streptomycin, L-glutamate and 50 μM ?-mercaptoethanol and grown at 37°C and 10% CO2. All other cell lines were kept in Dulbecco’s modified Eagle’s medium/glutamax (DMEM; Life Technologies, Inc.) supplemented with 10% fetal bovine serum, penicillin/streptomycin and 50 μM ?-mercaptoethanol and grown at 37°C and 5% CO2. For hydroxytamoxifen (OHT) induction experiments, cells were treated with 200 nM OHT (BioTrend Chemikalien GmbH) and, as indicated, pre-incubated for 30 min with 1 μg/ml cycloheximide (CHX). NIH/3T3 cells were transiently transfected by electroporation (450 V, 250 μF in PBS), pRL-CMV (Promega) was cotransfected in all experiments and used for normalization of different transfection efficiencies in the individual experiments.
Plasmids
Different MLC1A promoter constructs (–630, –3000) were recloned from the originally MLC1A-CAT constructs or amplified by PCR (–680) and cloned into the pTKL/2 vector containing the HSV-thymidine kinase promoter (–105 to +52) from the pBLCAT2 in front of the firefly luciferase coding region. The HSV-thymidine kinase promoter was excised by a restriction endonuclease digest and replaced by the MLC1A promoter sequences. The wild-type BOB.1/OBF.1 cDNA was cloned into the expression vector pCDNA3. For luciferase reporter assays the octamer-dependent reporter plasmid μET1 was used as an internal control (40).
cDNA RDA
Poly(A)+ RNA was prepared from the indicated cell lines as described (40). cDNA was prepared using the cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). The cDNA RDA protocol was performed exactly as described by Hubank and Schatz (35). MB10-mock cDNA was used as driver, MB10-BobER cDNA as tester. For the three subsequent rounds of hybridizations the tester/driver ratios were 1:100, 1:800, 1: 500 000. The difference product III (DPIII) was DpnII digested, cloned into BamHI cut pBluescript and 160 randomly picked cDNAs were sequenced using the T7 primer.
RACE protocol
5' Rapid amplification of cDNA ends (RACE) was performed using the RACE-kit (Life Technologies, Inc.) according to the instructions of the manufacturer. The following primers were used: myGSP1 (cca ttg gca tcc tcc), myGSP2 (tcc acc tct gcc tcg ct) and myGSP3 (ccg cag ccc ctc cac gaa g).
RT–PCR analysis
CD38-negative and -positive cells from human tonsils were isolated as described before (21,42). Total RNA of the respective cells or cell lines was used to prepare cDNA using SuperscriptII or M-MLV reverse transcriptase (Life Technologies, Inc.; Gibco). The cDNAs were used to analyze the expression of different MLC1A isoforms. PCR was performed as follows: 3 min at 94°C, 30 cycles of 30 s at 94°C, 60 s at 60°C, 90 s at 72°C. PCR products were analyzed on a 1.2% agarose gel. The following primers were used: MLC1A-3.1 (tgc tcc acc tct gcc tcg ctc atc), MLC1A-5.1 (gcc aga acc cca cca acg cag ag), MLC1A-5.2 (ccc acc tcc act gga gag cc), MLC1A-5.3 (cct ctg tgg ggg ctc cta ccc), MLC1A-5.4 (cga gcc acc tct cct cct ttg g), MLC1A-5.5 (ccc acc cct ctc tgg gtt tcc). For the simultaneous detection of murine and human MLC1A expression, the following primers were used: m/hMLC1A forward, gga gag atg aag atc acc tac gg; m/hMLC1A reverse, ggt gcc ctg ctc ctt gtt gc.
Northern blot analysis
Preparation and analysis of total cytoplasmic RNA, poly(A)+ RNA and northern blots were performed as described (40). For analysis of MLC1A expression an MLC1A cDNA fragment was amplified using the MLC1A-5.1 and MLC1A-3.1 primers.
RESULTS AND DISCUSSION
In order to identify BOB.1/OBF.1-dependent target genes in BOB.1/OBF.1-deficient B cells, we used a previously described Abelson virus-transformed pre-B cell line established from the bone marrow of BOB.1/OBF.1-deficient mice. In addition, they were transduced with a retrovirus, which resulted in the expression of an inducible BobER fusion protein (40). In this cell line (MB10-BobER), octamer-dependent synthetic promoter and immunoglobulin promoter/enhancer driven reporter constructs were inactive, unless the activity of the BobER protein was induced by the hormone OHT (40). The use of this system allows us to examine B cells at the same stage of differentiation. Using this approach, differential gene expression should be caused by the transcriptional activity of the BOB.1/OBF.1 protein.
The MB10-BobER cells and, as control, the MB10-mock cell line, which was infected with the empty expression vector, were induced with OHT to avoid potential side effects of OHT. RNA was prepared from the induced cell lines and a cDNA RDA screen was performed (35). The MB10-BobER cDNA was used as tester, the MB10-mock cDNA as driver. After three rounds of hybridization/amplification the resulting cDNAs (DPIII) were cloned into the pBluescript vector and subsequently 160 clones were sequenced.
Among these clones, we frequently found estrogen receptor sequences (50%), confirming that the cDNA RDA protocol enriched differentially expressed clones. Most of the remaining clones were single cDNA isolates and appeared not to be regulated by BOB.1/OBF.1 (data not shown). The cDNA identified most frequently (12 times) beside the estrogen receptor sequences was derived from the MLC1A gene.
The MLC1A gene was originally identified as an essential myosin light chain, specifically expressed in the murine heart atria (MLC1A, ‘atrial’ isoform). Additionally, it was described as the first myosin light chain during early embryonic development in embryonic skeletal muscle, therefore, also designated as MLC1emb (‘embryonic’ isoform) (36–38). The MLC1A gene was differentially expressed as confirmed by northern blot (Fig. 1A). In addition to an unspecific band already present in the mock-infected cell line (see below), a second, more slowly migrating band, was observed in cells expressing the active BobER fusion protein.
Figure 1. The expression of MLC1A is dependent on BOB.1/OBF.1. (A–C) Northern blot analysis using an MLC1A-specific probe and 2 μg of poly(A)+ RNA or 10 μg of total RNA of the indicated cell line. MB10, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells; MB10-mock, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells mock infected; MB10-BobER, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells BobER infected; PD31, wild-type Abelson virus-transformed pre-B cells; preB Oct2–/–, preB BOB.1/OBF.1–/–, preB wt, IL-7 and stroma cell-dependent primary pre-B cells from Oct2–/–, BOB.1/OBF.1–/– or wild-type mice derived from fetal liver. (D–F) Detection of MLC1A transcripts by RT–PCR. Genomic localization of the used primer pair (5.1 and 3.1) used in (D) is described in Figure 3A. Total RNA was prepared from atria, from total spleen of wild-type or BOB.1/OBF.1–/– mice, from CD38– or CD38+ tonsilar B cells, or from cell lines indicated, transcribed into cDNA and subsequently analyzed by RT–PCR. MB10-mock, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells mock infected; MB10-BobER, BOB.1/OBF.1–/– Abelson virus-transformed pre-B cells BobER infected; NIH/3T3, mouse fibroblast cell line.
In addition, several other cDNA clones were isolated repeatedly but less abundant, like alpha enolase, translation initiation factor 4E, complement protein C7 and several dehydrogenase motives. Notably, the transcripts for aldehyde dehydrogenase-2 like, the aldoketoreductase AKR1C13 and several other genes had been identified as BOB.1/OBF.1 regulated genes using a recently applied gene expression profiling approach and have been already described (34). Here we will focus on the analysis of the MLC1A gene.
We analyzed the influence of the hormone OHT on MLC1A expression in BobER-expressing cells. RNA was prepared from either uninfected MB10 cells, mock-infected MB10 cells or MB10 cells expressing the BobER allele (MB10-BobER), treated with OHT or left uninduced, and analyzed with the MLC1A-specific probe in northern blot experiments (Fig. 1B). As a control, RNA from PD31, an Abelson virus-transformed wild-type pre-B cell line, was included in this study. In all analyzed samples, the faster migrating non-specific band was visible, albeit at somewhat variable levels. Additionally, the MLC1A-specific (slower) band could be detected in the case of BOB.1/OBF.1-expressing PD31 cells and in OHT-treated MB10-BobER cells. In the case of PD31 cells, this expression was OHT independent as expected, whereas in BobER-expressing cells MB10-BobER, the expression of this additional band was regulated by treatment with OHT. Identical results were obtained analysing a second BobER-expressing cell clone (data not shown). To exclude a non-physiological expression of MLC1A in the Abelson virus-transformed pre-B cell lines we next analyzed primary mouse pre-B cells cultured in the presence of the cytokine IL-7 on a stromal cell layer (Fig. 1C). Northern blot analysis revealed a strong MLC1A expression in wild-type pre-B cells. Notably, MLC1A was also detected in Oct2-deficient pre-B cells, whereas in BOB.1/OBF.1-deficient B cells no MLC1A was detectable confirming the BOB.1/OBF.1 dependence of its expression.
Since we always observed two bands in the BOB.1/OBF.1-expressing pre-B cells we asked if both of these bands are MLC1A specific. MLC1A expression in adult mice was reported to be restricted to the atrium (36–39) and should not be expressed in other tissues, for example, in fibroblasts. When we analyzed MLC1A expression in NIH/3T3 cells, in 10T fibroblast cells and in ST2 stroma cells by northern blots, only the slower migrating band could be detected (data not shown). These data indicate that the lower band is rather an unspecific hybridization than an MLC1A-specific signal. To corroborate this conclusion we used MLC1A gene-specific primers to analyze MLC1A transcripts by RT–PCR. At high numbers of PCR cycles we observed low MLC1A expression in MB10-mock cells and a massive increase of MLC1A in MB10-BobER cells. However, no PCR product could be detected in the fibroblast cell line NIH/3T3 (Fig. 1D). The specificity of the RT–PCR was verified by sequencing the PCR products and by performing RT–PCR with mouse atria RNA that gave a product of the same size (data not shown). Therefore, we conclude that the upper band observed in northern blot experiments corresponds specifically to MLC1A, whereas the lower band results from an unspecific hybridization.
As mentioned above, MLC1A, so far, was described as being expressed specifically in atria but not in other adult tissues. We were interested to determine whether MLC1A was expressed throughout the B-cell lineage and/or even in other hematopoietic cells. Therefore, we performed northern blot analysis with several additional B cell lines representing different stages of B-cell development. However, whereas the MLC1A band was present in pre-B cells, no expression was seen in cell lines representing immature B cells (WEHI 231), plasma cell stage (S194), or in T cells irrespective of whether they express BOB.1/OBF.1 (EL4) or not (BW 5147) (data not shown). In addition, we analyzed several other mature B and plasma cell lines but could not detect MLC1A expression. Furthermore, analysing lymphocytes derived from total spleen of wild-type or BOB.1/OBF.1-deficient mice by RT–PCR no MLC1A expression could be detected even in wild-type splenocytes. In parallel, mouse atria were analyzed where MLC1A expression was observed (Fig. 1E). Since the expression level of BOB.1/OBF.1 is the highest in germinal centers, we asked whether MLC1A is also upregulated in mature IgMlowIgDhigh CD38+ primary human germinal center B cells. Using primers specific for the mouse, as well as for the human, MLC1A gene, RT–PCRs were performed with cDNAs from mouse atria as a positive control or from human CD38+ germinal center and CD38– non-germinal center mature B cells (Fig. 1F). No MLC1A expression could be detected either in mature germinal center cells or in non-germinal center B cells and other mature B cells. Therefore, MLC1A expression seems to be restricted to the pre-B-cell stage. This observation is not surprising, since BOB.1/OBF.1 was shown to be required at several stages of B-cell development including the pre-B-cell stage. Additionally, the target gene screen was performed using pre-B cells. Thus, identified BOB.1/OBF.1-dependent genes might be expressed and regulated only at this stage of B-cell development. This observation is reminiscent of the Oct2 target gene CRISP-3, which is expressed in pre-B cells and has not been found at later developmental B-cell stages (43).
MLC1A was described as being expressed in mouse atria and in the mouse skeletal muscle cell line C2C12. We wanted to compare the expression levels of MLC1A in pre-B cells with that in muscle cells. Northern blot experiments indeed confirmed a very strong MLC1A expression in mouse muscle cells, especially in the atria (Fig. 2A). This muscle-specific expression (in atrium and C2C12 cells) was significantly stronger than the MLC1A expression level found in PD31 cells. Interestingly, when we compared the sizes of MLC1A-specific bands it became clear that the pre-B-cell-specific band was slightly larger than that in the muscle cells. We reasoned that MLC1A might be alternatively spliced in mouse pre-B cells. In human skeletal muscle cells, different 5'UTR and 3'UTRs of the MLC1A gene are expressed (44,45), whereas in the mouse, so far, only one 5'UTR has been described. We performed 5'-RACE with RNA from the MB10-BobER cell line induced with OHT. A dominant 5'UTR from pre-B cells was identified, which we named distal 5'UTR since it results from an alternative upstream promoter and consequently from a splice process within the MLC1A 5'UTR. The previously described 5'UTR from mouse muscle cells was named proximal 5'UTR (sequences of both 5'UTRs are compared in Fig. 2B). Therefore, the situation in mouse pre-B cells resembles that in human muscle cells, where distinct MLC1A and MLC1emb mRNAs are expressed differing in their 5'UTRs (44,45). The function of the alternative transcript in humans is still unclear.
Figure 2. Comparison of MLC1A expression in mouse muscle and pre-B cells. (A) Northern blot analysis using an MLC1A-specific probe and 10 μg of total RNA of the indicated cell lines and tissue: atria, mouse atrium; PD31, wild-type Abelson virus-transformed pre-B cells; C2C12, mouse skeletal muscle cell line; NIH/3T3, mouse fibroblast cell lines. (B) Sequence of the 5' region of the mouse MLC1A gene. Alignment of the proximal 5'UTR identified by 5'-RACE from MB10-K10 cell cDNA and the known distal 5'UTR.
In order to analyze if both 5'UTRs are expressed in mouse B cells, we prepared primer pairs in order to distinguish the different 5'UTRs by RT–PCR (Fig. 3A). Using the primer 5.1 (located in MLC1A exon 3) or 5.2 (located nearby the translation initiation signal), an MLC1A-specific PCR product should be observed in all cases, independent of the specific 5'UTRs used. The 5.3 primer was a negative control since it is located in the region between distal and proximal promoters that is spliced out. Consequently, no PCR product should be observed. Using the primer 5.5 or 5.4 we can distinguish which 5'UTR was used. As expected, the primers 5.1 (Fig. 1D) and 5.2 (Fig. 3B) revealed a signal in MB10 cells, whereas the primer 5.3 did not show a signal. The signal was much stronger in cells expressing a functional BOB.1/OBF.1 protein. Although we could detect some transcript originating from the proximal promoter, the majority of MLC1A transcripts in pre-B cells derived from the distal promoter. The use of another 5'UTR in B cells other than in muscle cells suggests that different promoter regions are responsible for the regulation of MLC1A expression in B cells versus muscle cells.
Figure 3. Analyses of the expression of different 5'UTRs in pre-B cells. (A) Schematic representation of the genomic locus of the MLC1A gene, corresponding proximal and distal promoters and 5'UTR in mouse pre-B cells as well as location of the used RT–PCR primers. (B) RT–PCR using the indicated primer pairs and cDNAs from the cell lines indicated. As a control, ?-actin PCR was performed.
We then addressed the question whether regulation of MLC1A expression by BOB.1/OBF.1 is direct or involves an intermediate. We generated luciferase reporter constructs, differing in the length of the MLC1A promoter –630, –680 or –3000 bp. The activity of all these reporters was increased by BOB.1/OBF.1 cotransfection in NIH/3T3 cells (Fig. 4A). The highest induced activity was found in the case of the shortest 630 bp promoter, which is consistent with the earlier observation in muscle cells that 630 bp of the mouse MLC1A promoter are sufficient to achieve a regulated expression of the gene (41). However, the basal activity of the –630 bp promoter was also higher compared with the longer promoter constructs. Determining the absolute induction of analyzed promoters by BOB.1/OBF.1, all constructs were induced equally 2–3-fold. Surprisingly, when the activity of these constructs was analyzed in MB10-BobER cells, no OHT induction of luciferase activity could be observed analyzing cells 24 h after transfection, while some induction of luciferase activity was observed upon longer incubation (data not shown). This result suggested that the activation of MLC1A gene transcription by BOB.1/OBF.1 might be indirect. Since BobER cells originally used for the presented RDA were induced with OHT for 20 h, we cannot exclude an indirect regulation of identified genes by BOB.1/OBF.1. Recently, two E-box motives were shown to be critically involved in the regulation of the MLC1A promoter, binding MyoD/E12 and myogenin/E12 heterodimer (41,46). Importantly, the known MLC1A promoter sequences do not contain recognizable octamer motifs which could mediate the regulation via BOB.1/OBF.1.
Figure 4. Regulation of MLC1A promoter activity as well as gene expression by BOB.1/OBF.1 is indirect. (A) Transient transfection of NIH/3T3 cells using 5 μg of different MLC1A promoter constructs as indicated, either alone or together with 10 μg of a BOB.1/OBF.1 expression vector (each experiment was repeated three times). Additionally, as an internal control, the BOB.1/OBF.1-dependent μET1 promoter was analyzed. The value of the μET1 promoter vector was set to one. The fold induction was determined. (B) Northern blot analysis using an MLC1A-specific probe and 10 μg of total RNA of MB10-BobER pre-B cell line. Cells were induced with OHT (200 nM) for the indicated time or pre-treated with CHX (1 μg/ml), as indicated, for 30 min, RNA was prepared and subsequently subjected to northern blot analyses.
To address this issue, MB10-BobER cells were pre-incubated with the protein synthesis inhibitor CHX and induced for 12 h with OHT. Northern blot analyses revealed that CHX prevents the OHT-mediated induction of MLC1A by BOB.1/OBF.1 (Fig. 4B), indicating that MLC1A indeed is indirectly regulated by BOB.1/OBF.1.
The actin–myosin complex is the main contractile component of the muscle sarcomers. Furthermore, actin and myosin undergo reorganization in cultured mammalian non-muscle cells in response to cellular signals during various motile processes in cells, such as chemotaxis, phagocytosis, capping of surface receptors, vesicle transport and cytokinesis (47–50).
Many functions of myosins in non-muscle cells have been described, such as cytoskeletal dynamics, which in turn are essential for cell migration and motility. Immune cells are characterized by cellular movement between compartments. Immature (transitional) B cells emerge from the bone marrow to the periphery and migrate into specific microenvironments of peripheral secondary lymphoid tissues, such as spleen and lymph nodes. Although the physical sites of B-cell precursor development and differentiation in the bone marrow remain poorly defined, it is likely that the actin–myosin complex is also essential for B-cell motility of earlier developmental stages, e.g. to build contacts with the stromal layer, important for progenitor B cells. This hypothesis is supported by recent findings demonstrating the important role of cytoskeleton reorganization of hematopoietic stem/progenitor cells. These processes affect adhesion and mobilization from the medullary cavity into the blood circulation and migration into lymph nodes or sites of inflammation (51,52). Additionally, it was shown that the cytoskeletal organization that contributes to the shape of hematopoietic cells influences the transduction of growth signals through a number of externally activating signaling pathways (53). B cells deficient for BOB.1/OBF.1 have several defects in early development in the bone marrow and cannot properly develop into transitional B cells (25). Taking together, the BOB.1/OBF.1-dependent expression of an essential myosin light chain in pre-B cells could be a novel mechanism to achieve a regulation of potential functions of actin–myosin motility in pre-B cells, essential for development and function.
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