Cell. Mol. Life Sci. 66 (2009) 1631 – 1646 1420-682X/09/101631-16 Cellular and Molecular Life Sciences DOI 10.1007/s00018-009-8668-7 Birkhuser Verlag, Basel, 2009 Review The Notch signaling pathway: Transcriptional regulation at Notch target genes T. Borggrefe1, * and F. Oswald2 1Department of Cellular and Molecular Immunology, Max-Planck-Institute of Immunobiology, Stbeweg 51, 79108 Freiburg (Germany) Fax: ++49-761-5108-799, e-mail: [email protected] 2Department of Internal Medicine I, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm (Germany) Received 21 October 2008; received after revision 09 December 2008; accepted 15 December 2008 Online First 21 January 2009 Abstract. The Notch gene encodes a transmembrane cruiting a corepressor complex. Here, we review receptor that gave the name to the evolutionary highly Notch signaling with a focus on gene regulatory conserved Notch signaling cascade. It plays a pivotal events at Notch target genes. This is of utmost role in the regulation of many fundamental cellular importance to understand Notch signaling since processes such as proliferation, stem cell maintenance certain RBP-J associated cofactors and particular and differentiation during embryonic and adult devel- epigenetic marks determine the specificity of Notch opment. After specific ligand binding, the intracellu- target gene expression in different cell types. We lar part of the Notch receptor is cleaved off and subsequently summarize the current knowledge about translocates to the nucleus, where it binds to the Notch target genes and the physiological significance transcription factor RBP-J. In the absence of activated of Notch signaling in development and cancer. Notch, RBP-J represses Notch target genes by re- Keywords. Notch signaling, transcription, recombination signal sequence-binding protein Jkappa (RBP-Jk/ CSL), chromatin and epigenetic regulation, leukemia. Introduction the Notch receptor contains 36 epidermal growth factor (EGF)-like repeats essential for ligand binding. The Notch gene was discovered almost 90 years ago by The intracellular domain is involved in cellular signal- Morgan and colleagues who observed that partial loss ing and contains multiple conserved protein domains. of function results in notches at the wing margin in Subsequently, Notch-like molecules have been iden- flies (Drosophila melanogaster) [1]. Decades later in tified from C. elegans (LIN-12) to humans, playing loss of function experiments, Notch was found to important, and apparently conserved, functional roles cause a “neurogenic” phenotype, where cells destined in development. In mammals, four Notch receptors to become epidermis switch fate and give rise to neural (Notch 1–4) and five transmembrane ligands (Jag- tissue, reviewed in [2, 3]. In the early eighties, the ged1, Jagged2, Delta-like1, Delta-like3, and Delta- Notch gene was found to encode a 300kDa single-pass like4) are described so far. Although Notch was transmembrane receptor. The extracellular domain of originally classified as a neurogenic gene even the first characterization of Drosophila embryos made it clear that Notch signals are highly pleiotropic, thereby * Corresponding author. affecting many tissues. 1632 T. Borggrefe and F. Oswald Notch signaling and gene expression The Notch signaling cascade appears remarkably fashion. In the absence of ligand, hence without simple with apparently no second messengers in- nuclear NICD, RBP-J represses Notch target genes volved. However, the role of Notch signaling and the through the recruitment of corepressor complexes. activation of downstream genes in a given tissue NICD binding to RBP-J is crucial for the switch from remain often complex and unpredictable. Here, we repressed to activated state. NICD first displaces review in depth what is known so far about transcrip- corepressors from RBP-J, resulting in derepression of tional regulation mediated by Notch. Subsequently, promoters containing RBP-J binding sites and sub- we summarize the current knowledge about Notch sequently recruits a coactivator complex to activate target genes and, in a broader context, the relevance of transcription of Notch target genes. Notch signaling in cell differentiation and cancer. Notch target genes Although signals mediated through Notch receptors Molecular mechanism of transcriptional regulation at have diverse outcomes, only a fairly limited set of Notch target genes Notch target genes have been identified in various cellular and developmental contexts. The hairy/en- The Notch signaling cascade hancer of split (Hes) genes are highly conserved Notch signaling is activated upon cell-to-cell contact proteins that are regulated by Notch in multiple cell as a result of interactions between Notch receptors types, reviewed in [7, 8]. Hairy/Enhancer of split and their ligands (Delta or Jagged). At the molecular family genes were first described as neurogenic genes level, triggering of Notch receptor by ligand binding in Drosophila (like Notch, Deltex and Mastermind), promotes two proteolytic cleavage events at the Notch since embryos lacking the function of these genes receptor (Fig. 1). The first cleavage is catalyzed by the showed an increased number of neuroblasts at the ADAM-family of metalloproteases, whereas the sec- expense of epidermal precursors, reviewed in [2, 9]. ond cleavage is mediated by g-secretase, an enzyme Several lines of evidence have suggested that these complex that contains presenilin, nicastrin, PEN2 and genes are indeed direct Notch target genes: a) The APH1, reviewed in [4]. The second cleavage releases promoters of Hes1, Hes5 and Hes7 as well as Hey1, the Notch intracellular domain (NICD), which then Hey2 and HeyL (subfamily of Hes, related with translocates to the nucleus and acts as a transcriptional YRPW motif) can be activated by a constitutive coactivator. NICD cannot bind directly to DNA but active form of Notch1 [10–12], reviewed in [7]; heterodimerizes with the DNA binding protein RBP-J constitutive active Notch describes a mutant of (recombination signal sequence-binding protein Jk, Notch that is continuously proteolytically processed also called CSL, CBF1, Su(H) and LAG-1) and and migrating to the nucleus, b) endogenous Hey1 and activates transcription of genes containing RBP-J Hey2 show an upregulation by NICD in several binding sites. different cell lines [13], c) in co-culture experiments Interestingly, RBP-J was originally identified as a with Notch-ligand expressing cells, that achieve a repressor of transcription by Vales and colleagues [5]. more physiological level of Notch signaling, these When studying the promoter region of the adenovirus genes are upregulated as well [13–15]; these experi- pIX gene, a 10bp repressive element was character- ments were also performed in the presence of cyclo- ized and it was shown that RBP-J is the factor that hexamide, an inhibitor of protein synthesis, to exclude binds to this element. Subsequently, the RBP-J bind- secondary effects, d) k-secretase inhibitor DAPT, ing site was placed in reporter assays next to other which prevents cleavage of Notch, was added to T- transcriptional activators Gal4-VP16 or SP1, and it cell leukemia cell lines which show constitutive-active was shown that RBP-J could interfere with transcrip- Notch signaling; subsequent microarray analysis tional activation. Other investigators also demonstrat- identified again members of this transcription factor ed the RBP-J repressive activity by placing multiple family as direct Notch target genes [16]. Therefore, in RBP-J binding sites at a TK-promoter element [6]. mammals, the best-described Notch target genes are The RBP-J mediated repression could either be indeed the transcription factors Hes1, Hes5 and Hey1 relieved by addition of the viral activator EBNA2 [8, 17]. Hes and Hey proteins are helix-loop-helix (see also below section on Notch in cancer, Epstein- transcription factors that function as transcriptional Barr virus) or by the fusion of RBP-J to VP16 repressors. Overexpression of Hes1 and Hes5 in bone activation domain. The RBP-J activator/repressor marrow partly inhibits B-cell development [18]. Hes1 paradox was resolved with the realization that repres- deficient mice are not viable and display multiple sion and activation via RBP-J involves the recruit- developmental defects, reviewed in [7]. ment of distinct protein complexes, which influence CD25 (IL2-R and preTa, pre-T-cell receptor alpha- transcription of target genes in a positive or negative chain) were also shown to be Notch target genes in T- Cell. Mol. Life Sci. Vol. 66, 2009 Review Article 1633 cells [19, 20]. In later stages of T-cell development, the Epigenetic regulation of Notch target genes transcription factor GATA3, a master regulator for T- Although precise mechanisms of epigenetic regula- cell development and later for Th1/2 lineage decision, tion at Notch target genes are not completely under- is a direct Notch target gene [21,22]. Flavell and stood, cumulating evidence suggests that histone colleagues could show that Th2-mediated immunity modifications play a pivotal role in this process. The depends on either RBP-J or Notch [22] and that the presence of specific post-translational histone mod- GATA3 promoter contains bona fide RBP-J binding ifications define transcriptionally inactive or active sites. In addition, the Pear lab demonstrated, that the chromatin domains [41–44]. Acetylation and meth- expression of dominant-negative mastermindlike-1 ylation represent the most common modifications