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Oct 6, 1988 - was provided by Brian Pollok. The neomycin (neo) .... Mak,T.W., Kearney,J.F., Perry,R.P. and Bosma,M.J. (1986) Cell, 46,. 963-972. Sen,R. and ...
The EMBO Journal vol.7 no.13 pp.4213-4220, 1988

Complementation between two cell lines lacking enhancer activity: implications for the developmental control of immunoglobulin transcription x

Michael L.Atchison and Robert P.Perry Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA

Communicated by R.P.Perry

Plasmacytoma S107 and the pre-B cell line 3-1 both lack immunoglobulin x (Igx) enhancer activity due to the absence of the active form of a trans-acting nuclear factor, NF-xB, which binds to and activates the x enhancer. PreB cells possess the factor in a masked form and can activate it by a post-translational mechanism after treatment with specific inducing agents. In the experiments presented here somatic cell hybrids were used to determine whether S107 cells also possess NF-xB in a masked form, or alternatively, whether they possess the activation system but lack the factor. We observed that hybrids between S107 and pre-B cells produce the active form of NF-xB and exhibit transcriptional activation of previously silent x loci. These results demonstrate that S107 cells totally lack factor NF-xB but not the ability to activate it. Treatment of the hybrid cells with bacterial lipopolysaccharide (LPS), increases the NF-xB titer 4to 5-fold and causes a concomitant 4- to 5-fold increase in x enhancer activity within these cells. However, the expression of the activated x loci remains unchanged after LPS-treatment, indicating that they are no longer under the control of the x enhancer. Therefore, a two-step transcriptional process occurs in these cells. First, the silent x loci are activated by the production of factor NFxB. Subsequently, a second transcriptional mechanism overrides the dependence on the x enhancer and maintains x transcription at a constant level regardless of the level of x enhancer activity within the cell. Demethylation of the transcriptionally activated x loci was observed in these hybrids, consistent with a possible link between alterations in chromatin structure and

enhancer-independent transcription. Key words: demethylation/immunoglobulin/x enhancer/ NF-xB/transcription

Introduction Enhancers are cis-acting DNA elements that increase transcription from nearby promoters in an orientation and distance independent manner (reviewed in Khoury and Gruss, 1983; Serfling et al., 1985; Atchison, 1988). Immunoglobulin x (Igx) genes contain a transcriptional enhancer element located within the intron that separates the joining (Jx) from constant region (Cx) exons (Queen and Baltimore, 1983; Picard and Schaffner, 1984; Queen and Stafford, 1984). This enhancer controls the transcriptional activity of the Igx locus at certain stages of lymphoid development (Atchison and Perry, 1987; Lenardo et al., 1987). Thus, pre-B cells lack x enhancer activity (Atchison ©IRL Press Limited, Oxford, England

and Perry, 1987) and the x locus is transcriptionally silent and heavily methylated (Nelson et al., 1984, 1985). However, pre-B cells can be induced by treatment with bacterial lipopolysaccharide (LPS) to initiate transcription from a cryptic promoter element (x°P) that lies upstream of Cx (Van Ness et al., 1981; Nelson et al., 1984; 1985). Transcription from the xo promoter yields an 8 kb primary transcript which is processed into a 1 kb cytoplasmic derivative (Kelley et al., 1988). This induction is accompanied by the appearance of a DNAse I-hypersensitive site that maps to the enhancer region (Parslow and Granner, 1982, 1983). LPS treatment of pre-B cells has also been shown to cause the appearance of a nuclear factor, NF-xB (Sen and Baltimore, 1986a,b), which binds to and controls the activity of the x enhancer (Sen and Baltimore, 1986a,b; Atchison and Perry, 1987; Lenardo et al., 1987). NF-xB, and concomitantly x locus transcription, can be induced in pre-B cells by LPS even in the presence of inhibitors of protein synthesis (Nelson et al., 1985; Sen and Baltimore, 1986b). Thus, NF-xB is present in an inactive form in preB cells and is activated by a post-translational mechanism, perhaps by protein kinase C (Sen and Baltimore, 1986b). This induction apparently releases NF-xB from interaction with a cytoplasmic repressor allowing the factor to enter the nucleus and bind to the enhancer (Baeuerle and Baltimore, 1988). Recent evidence indicates that the mechanism of transcriptional regulation at the Ig loci (i.e. heavy chain and x chain genes) changes during development. Although the Ig enhancers are absolutely required to initiate transcriptional competence, it now appears that at certain stages of lymphoid development the enhancers can be either deleted or rendered non-functional without the loss of Ig transcriptional activity (Wabl and Burrows, 1984; Klein et al., 1984; 1985; Eckhardt and Birshtein, 1985; Zaller and Eckhardt, 1985; Atchison and Perry, 1987). Thus, a developmentally controlled transcriptional mechanism is capable of maintaining transcription in the absence of enhancer activity. The cis-acting DNA sequences and regulatory factors responsible for this mechanism are still unknown. Possible modes of action include the formation of a stable, heritable transcription complex, or the restructuring of the chromatin surrounding the x locus into a transcriptionally active state. Indeed, the coupling of transcriptional activity and demethylation of the x locus has been found to correlate with enhancer-independent transcription of Igx genes (Kelley et al., 1988). Previously, we identified a plasmacytoma variant, S 107, which lacks detectable factor NF-xB in nuclear extracts and therefore lacks x enhancer activity (Atchison and Perry, 1987). Like pre-B cells, this plasmacytoma is incapable of expressing x genes introduced by transfection (Atchison and Perry, 1987; Kelley et al., 1988). However, unlike pre-B cells the endogenous x genes in this plasmacytoma are transcriptionally active indicating that they are no longer 4213

M.L.Atchison and R.P.Perry

under control of the x enhancer. The lack of measureable NF-xB in S107 cells might be similar to the case in pre-B cells which actually possess the factor but are unable to activate it, or alternatively, the S107 cells might possess the activation system but not the factor. We show here that somatic cell fusions between S107 and pre-B cells, both of which lack x enhancer activity, result in complementation of activities to produce NF-xB and x enhancer activity. Thus, S107 cells lack NF-xB but not the ability to activate it. We also show that after transcriptional activation of a previously silent x locus in these hybrids, transcription is no longer controlled by the x enhancer and the x locus DNA becomes demethylated. Therefore, a two-step transcriptional process occurs in these cells. Transcription is first activated by production of functional NF-xB and x enhancer activity. Subsequently, a second transcriptional mechanism overrides the control of the x enhancer and maintains transcription at a constant level regardless of the level of x enhancer activity within the cells.

Results

Inducers of NF-xB in pre-B cells do not induce NF-xB in S107 cells Previously we demonstrated that NF-xB is not present in nuclear extracts prepared from S107 cells (Atchison and

Perry, 1987). The absence of active NF-xB in these cells could simply be due to the factor being bound by a repressor, identical to the situation observed in pre-B cells. To test this possibility, S107 cells were treated with agents [LPS or phorbol 12-myristate 13-acetate (PMA)] known to release NF-xB from its repressor in pre-B cells (Sen and Baltimore, 1986b; Baeuerle and Baltimore, 1988). S107 cells were treated with either LPS or PMA and nuclear extracts sub-

A

sequently prepared were assayed for the presence of NF-xB by gel mobility shift assays (Fried and Crothers, 1981). A nuclear extract prepared from S194 plasmacytoma cells served as a positive control. Factor NF-xB was readily detectable in the extract derived from S194 cells (Figure IA, lane 1) but was absent from extracts prepared from S107 or LPS-treated S107 cells (Figure IA, lanes 2 and 3). The absence of NF-xB in the S107 extracts was not due to proteolytic degradation because x enhancer factor NF-xE3 (Sen and Baltimore, 1986a) and promoter factor NF-Al (Singh et al., 1986) were easily detectable (Figure IA, lanes 5, 6, 8 and 9). Similar results were observed with extracts prepared from PMA-treated S107 cells. Although treatment of pre-B cell line 3-1 with PMA resulted in the appearance of NF-xB in nuclear extracts, no induction was observed in S107 cells (Figure lB, lanes 1-4). Again, the presence of NF-xE3 in the S107 extracts verified extract integrity (Figure iB, lanes 5-8). The inability of the above agents to induce NF-xB in S107 cells suggested that it was unlikely that the cells possessed factor NF-xB in an inactive form as in pre-B cells. Rather, S107 cells may be capable of activating NF-xB (i.e. releasing it from its repressor) but are totally lacking the factor. If this is indeed the case, one might expect that somatic hybrids between pre-B and S107 cells would yield active NF-xB due to the modifying system present in S107 cells activating the masked NF-xB present in pre-B cells. Production of NF-xB should also result in concomitant transcriptional activation of the silent xo loci derived from the pre-B cells. Alternatively, if S107 cells are incapable of activating the masked NF-xB in pre-B cells, no active NF-xB would be formed in the hybrids and the x° loci would remain transcriptionally silent. A diagram of this model is shown in Figure 2.

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