Downloaded from genesdev.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press A thymus-specific member of the HMG protein family regulates the human T cell receptor enhancer Marian L. Waterman, Wolfgang H. Fischer, 1 and Katherine A. Jones 2 Molecular Biology and Virology Laboratory and Regulatory Biology Laboratory, 1peptide Biology Laboratory, The Salk Institute, La Jolla, California 92037 USA The human T cell-specific transcription factor TCF-la plays a key role in the tissue-specific activation of the T cell receptor (TCR) Ca enhancer and binds to pyrimidine-rich elements (5'-PyCTTTG-3') present in a variety of other T cell-specific control regions. Using amino acid sequence information derived from the DNA affinity-purified protein, we have now isolated cDNA clones encoding TCF-la. The TCF-Ia cDNA contains a single 68-amino-acid domain that is homologous to a region conserved among high-mobility group (HMG) and nonhistone chromosomal proteins. Expression of full-length and mutant cDNA clones in bacteria reveal that the single HMG motif, which is predicted to contain two extended a-helical segments, is sufficient to direct the sequence-specific binding of TCF-lc~ to DNA. Northern blot experiments demonstrate further that TCF-I,~ mRNA is highly tissue specific, found primarily in the thymus or T cell lines. The immature CEM T cell line expresses relatively low levels of TCF-la mRNA, which are increased upon activation of these cells by phorbol esters. Interestingly, the cloned TCF-I¢~ protein is a potent transcriptional activator of the human TCRa enhancer in nonlymphoid cell lines, whereas the activity of the endogenous protein in T cell lines is strongly dependent on an additional T cell-specific protein that interacts with the core enhancer. TCF-let is currently unique among the newly emerging family of DNA-binding regulatory proteins that share the HMG motif in that it is a highly tissue-specific RNA polymerase II transcription factor. [Key Words: TCRa enhancer~ HMG protein~ T cell-specific transcription factor] Received January 2, 1991~ revised version accepted January 25, 1991. The development of pluripotent stem cells into highly with transcriptional regulation mediated by potent T specialized T cell lymphocytes can be defined by the cell-specific enhancers that have been mapped down- appearance and assembly at the cell surface of multicom- stream of many of these genes {Krimperdort et al. 1988~ ponent complexes containing the T cell receptor {TCR) McDougall et al. 1988~ Ho et al. 1989~ Winoto and Bal- and CD3 antigens. Functionally distinct T cell lineages timore 1989a~ Redondo et al. 1990}. bear either the et/B or ~//~ heterodimeric forms of the T The best-characterized of the TCR enhancers lies 4.5 cell receptor [Davis and Bjorkman 1988). TCR~t/B + cells kb downstream of the cx-chain gene (Chien et al. 1987} predominate in mature peripheral blood T cells {includ- and is recognized by a complex array of DNA-binding ing helper and cytotoxic cell subtypes) and function to proteins, although a minimal core segment spanning 100 mediate major histocompatability complex {MHC)- bp is sufficient for high-level activity in mature ot/B + T restricted antigen recognition in combination with ei- cell lines {Ho et al. 1989~ Winoto and Baltimore 1989a). ther the CD4 or CD8 antigens {for review, see Marrack This core segment interacts with three DNA-binding and Kappler 1987; Strominger 1989}. The TCR genes, proteins: a ubiquitously distributed member of the like other members of the immunoglobulin gene super- cAMP-responsive {CREB/ATF} transcription factor fam- family, are rearranged and expressed developmentally in ily (Ho et al. 1989), the T cell-specific TCF-lcx factor a stage-specific manner that begins with the ~//~-chain {Waterman and Jones 19901, and a distinct T cell-specific genes and follows with the B-chain and lastly the (x-chain protein (TCF-2~t} that may be a member of the c-ets pro- genes {Furley et al. 1986; Pardoll et al. 1987; Havran and tein family (Ho et al. 19901. The core TCRct enhancer is Allison 1988). The coordinate synthesis of the members a potent T cell-specific control region that can activate of the TCR-CD3 complex is accomplished by a combi- heterologous promoters in mature T cell lines, and each nation of both transcriptional and post-transcriptional of the three protein-binding sites is necessary for en- steps {Wilkinson and MacLeod 1988; Paillard et al. 1990}, hancer activity in vivo (Winoto and Baltimore 1989a~ Ho et al. 1990; Waterman and ]ones 19901. Interestingly, a 2Corresponding author. subfragment of the enhancer lacking the CRE motif re- 656 GENES & DEVELOPMENT 5:656-669 © 1991 by Cold Spring Harbor Laboratory ISSN 0890-9369/91 $3.00 Downloaded from genesdev.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press T cell-specific HMG transcription factor presses transcription from heterologous promoters in a T TCF-lu transcription factor was found to harbor a single cell-specific manner (Ho and Leiden 1990). The a en- 68-amino-acid domain that is homologous to a con- hancer is less active in immature T cell lines (e.g., served region of the high-mobility group (HMG) and CCRF-CEM cells) but can be stimulated in these cells by other nonhistone chromosomal proteins. This HMG mo- treatment with phorbol esters (Winoto and Baltimore tif is shown to be both necessary and sufficient for spe- 1989a), which also induces surface expression of the cific binding of TCF ol u to its conserved pyrimidine-rich TCRu/B complex (Shackelford et al. 1987). DNA element. The cloned TCF-lc, protein can also Rearrangement and expression of the TCRu gene is strongly activate the expression of a minimal promoter regulated in a lineage-specific manner in developing thy- linked to the TCRc~ enhancer in nonlymphoid cells. Ex- mocytes because the a-chain gene is transcribed in pression of TCF-lu mRNA is highly restricted to the TCRa/[3 + but not in TCR~//8 + T cells (for review, see thymus and to T cell lines and can be further induced Alt et al. 1987). In the mouse, the minimal 116- bp TCRu upon activation of an immature T cell line. These data enhancer core was found to be fully active in ~//8+ cell establish that TCF-lu belongs to a new family of regu- lines, whereas larger fragments of the enhancer (5 kb or latory proteins related by the shared HMG box homol- greater) were inactive (Winoto and Baltimore 1989b). ogy. Other members of this family include hUBF, a ubiq- These larger fragments contain multiple "silencer" ele- uitous RNA polymerase I enhancer-binding protein ments reported to extinguish enhancer activity in a va- (Jantzen et al. 1990), and SRY, a testis-specific protein riety of non-~f~ + cell lines. Consequently, the T cell- encoded on the Y chromosome that is implicated genet- specific factors associated with the minimal enhancer ically in human sex determination (Gubbay et al. 1990; appear to be expressed and active in both T cell subtypes Sinclair et al. 1990). TCF-lu is presently unique among but are subject to lineage-specific repression in ~/8 + T this family in that its specific DNA-binding activity is cells. determined by a single HMG motif, and it is required for We recently reported the purification and character- the T cell-specific regulation of RNA polymerase II ization of TCF-lu, a new T cell-specific DNA-binding genes. protein that recognizes the 100-bp core region of the hu- man TCRu enhancer (Waterman and Jones 1990). TCF-1~ was purified from nuclear extracts of Jurkat cells Results and shown to consist of a family of 57- to 53-kD proteins Amino acid sequence analysis of DNA that bind specifically to pyrimidine-rich elements (e.g., affinity-purified TCF-1 a 5'-PyCTTTG-3'), which are present in a variety of T cell- specific control regions, including the TCR~ enhancer, Our previous analysis of the T cell-specific TCF-lc~ fac- the p561ok and CD3 (~/and 8) promoters, and the HIV-1 tor indicated that it was a unique protein that had not long terminal repeat (LTR). More recently, we have been implicated previously in T cell-specific transcrip- found that the TGF-lu protein also interacts with the tional regulation (Waterman and Jones 1990). To isolate TCR8 enhancer (M.L. Waterman and K. Jones, unpubl.), cDNAs encoding TCF-lu and further characterize this as well as with transcriptional regulatory regions of the regulatory protein, -200 pmoles (16 ~g) of TCF-lu was murine CD4 gene (M.L. Waterman, K. Jones, J. Siu, and purified from Jurkat nuclear extracts, using a combina- S. Hedrick, unpubl.). A double point mutation within the tion of conventional gradient and DNA affinity chromo- TCRu enhancer (5'-PyCTTTG-3' to 5'-PyCATAG-3') tography. TCF-lu activity derives from a family of 57- to that eliminates the binding of affinity-purified TCF-lu 53-kD proteins, each of which is capable of binding DNA was found to decrease TCRa enhancer activity specifically in Southwestern blotting as well as gel exci- -100-fold in vivo (Waterman and Jones 1990). This mu- sion and renaturation experiments (Waterman and Jones tation also destroyed the striking synergism observed be- 1990). Three distinct TCF-lu proteins copurified with a tween tandem copies of the TCRR enhancer, effectively nonspecific DNA-binding protein of 116 kD during the eliminating T cell-specific transcriptional activity in purification procedure. The proteins were resolved on a vivo, and reduced enhancer activity in an in vitro tran- preparative denaturing SDS-polyacrylamide gel, trans- scription system derived from Jurkat nuclei.