An Upstream Oct-1- and Oct-2-Binding Silencer Governs B29 (Igβ) Gene Expression Cindy Sue Malone, Lisa Patrone, Kent L. Buchanan, Carol F. Webb and Randolph Wall This information is current as of September 24, 2021. J Immunol 2000; 164:2550-2556; ; doi: 10.4049/jimmunol.164.5.2550 http://www.jimmunol.org/content/164/5/2550 Downloaded from References This article cites 55 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/164/5/2550.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 24, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. An Upstream Oct-1- and Oct-2-Binding Silencer Governs B29 (Ig␤) Gene Expression1 Cindy Sue Malone,* Lisa Patrone,* Kent L. Buchanan,2† Carol F. Webb,† and Randolph Wall3* The B cell-specific B29 (Ig␤) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5؅ of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A؉T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A؉T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This Downloaded from B29 A؉T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A؉T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function. The Journal of Immunology, 2000, 164: 2550–2556. ␤ he product of the B29 (Ig ) gene is an essential compo- Oct-1 and Oct-2 transcription factors (6). This octamer motif is a http://www.jimmunol.org/ nent of the B cell receptor (BCR)4 that plays an indis- major determinant of B29 promoter activity in that mutations that T pensable role in B cell development. The B29 gene prod- eliminated Oct-1 and Oct-2 binding also abolished B29 promoter uct is disulfide-linked to the mb-1 (Ig␣) gene product, and this function (5). heterodimer is associated with Ig to form functional BCR com- Expression of the B29 minimal promoter is also regulated by plexes on B cells. B29-mb-1 heterodimers control VDJH recombi- upstream transcription control elements with inhibitory activities. nation, allelic exclusion, surface translocation of Ig, and signal We previously identified two silencer elements, FROG and transduction events that occur through the BCR (reviewed in Refs. TOAD, upstream of the B29 minimal promoter that coordinately 1–3). govern its level of expression (7). To date, the FROG- and TOAD- Due to the critical role the B29 gene plays in B cell maturation interacting proteins have not been identified. These B29 silencer by guest on September 24, 2021 and function, we have characterized the features regulating its tran- elements are equally active in both orientations, are position-inde- scription and B cell specificity. Previously, we demonstrated that pendent, can affect heterologous promoters, and are not tissue re- the B cell specificity of B29 is determined by a minimal promoter stricted (7). These features of the B29 silencers resemble other that contains multiple transcription factor motifs that collectively functionally defined cis-acting negative regulatory DNA se- determine its tissue specificity and transcriptional activity. These quences that down-regulate gene transcription and belong to the motifs include early B cell factor, Ikaros, ETS, SP1, and octamer class of silencer elements that restrict levels of gene expression (4, 5). The B29 octamer motif was shown to interact with both rather than impart tissue specificity. Other examples of silencers reported to govern gene expression like the B29 silencer are *Department of Microbiology and Immunology, and Molecular Biology Institute, present in the bcl-2 (8), ETS-1 (9), and ␭5 (10) genes. It has been University of California, School of Medicine, Los Angeles, CA 90095; and †Immu- nobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma postulated that this class of inhibitory transcription control ele- City, OK 73104 ments functions in maintaining the level of specific gene expres- Received for publication October 18,1999. Accepted for publication December sion, thereby preventing deleterious consequences of overexpres- 16, 1999. sion (7). For example, moderate overexpression of bcl-2 is linked The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance to the prolonged survival of neoplastic B cells in follicular lym- with 18 U.S.C. Section 1734 solely to indicate this fact. phoma and chronic lymphocytic leukemia (11, 12). bcl-2 is gov- 1 This work was supported by National Institutes of Health Grants CA12800 and erned by silencer elements similar in nature to the silencers of the GM40185 and University of California Amgen STAR Biotechnology Project S96-02 B29 gene in that they affect bcl-2 expression levels but do not (to R.W.); and by National Institutes of Health Grants GM46462 and AI44215 (to ⑀ C.F.W.). C.S.M. was supported by Public Health Service National Service Award impart tissue specificity (8). Elevated CD3 expression in T cells AI07126, the Dr. Ursula Mandel Scholarship, and the University of California Dis- blocked early T cell development (13) and in prothymocytes func- sertation Year Fellowship. L.P. was supported by Public Health Service National ⑀ Research Service Award 2T32 CA 09120. tioned as an oncogene (14). These consequences of CD3 gene overexpression are particularly relevant in that CD3⑀ fulfills the 2 Current address: Department of Microbiology and Immunology, Tulane University Medical Center, New Orleans, LA 70112 same role in the TCR as B29 does in the BCR. 3 Address correspondence and reprint requests to Dr. Randolph Wall, University of Further functional analysis of the region upstream of the B29 California, 611 Charles E. Young Drive East, 529 Molecular Biology Institute, Los minimal promoter has identified a new region of potent silencer Angeles, CA 90095. E-mail address: [email protected] activity 5Ј of the previously identified FROG and TOAD silencer 4 Abbreviations used in this paper: BCR, B cell receptor; MAR, matrix attachment region; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; Bright, B elements. Like these previously characterized B29 silencer ele- cell regulator of IgH transcription. ments, this new 5Ј B29 silencer is not B29 promoter-restricted. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 2551 DNase I protection assays over this region delineated a well-de- fined protected area with an extended central AϩT-rich sequence. This central AϩT-rich sequence is homologous to the degenerate AϩT-rich motifs recognized by octamer transcription factor fam- ily members (15). Both Oct-1 and Oct-2 factors bound the B29 AϩT-rich sequence in EMSA. Site-directed mutations incorpo- rated into this AϩT-rich predicted octamer motif eliminated Oct-1 and Oct-2 binding, which in turn abolished the silencer activity of this region. The B29 AϩT-rich sequence also conforms to all the consensus sequence criteria for nuclear matrix attachment regions (MARs). MARs mediate chromatin association with the nuclear matrix (16, 17), are usually located within or near transcriptional regulatory elements (18–22), and are postulated to function in regulating gene expression by acting as boundary elements for transcription (20), by creating altered nucleosome environments (23, 24), and by af- FIGURE 1. B29 AϩT-rich motif contributes to the negative regulation fecting enhancer and silencer functions by impacting chromatin of the B29 minimal (Ϫ164) promoter. Transient transfection to detect tran- structure (25–27). The B29 AϩT-rich sequence selectively inter- scriptional expression of B29 promoter deletion constructs were conducted acted with nuclear matrix proteins, suggesting that this segment in the M12 B cell line. The activity of each construct is expressed as a Downloaded from may also function as a MAR in B29 gene control. percentage of the CAT activity obtained with the B29 (Ϫ164) minimal promoter construct (100%). Deletion construct Ϫ411 was created to sep- Materials and Methods arate the activities of the AϩT-rich motif from the FROG motif silencer ϩ Plasmid construction and mutagenesis element to determine transcriptional activity of the A T-rich motif region.