OVERVIEW OF TRANSCRIPTION REGULATION:

Detailed studies of gene transcription regulation in a wide variety of eukaryotic systems has revealed the general principles and mechanisms by which cell- or tissue-specific regulation of differential gene transcription is mediated (reviewed in Naar, 2001. Kadonaga, 2004, Maston, 2006, Barolo, 2002; Roeder, 2005, Rosenfeld, 2006). Of the three major classes of DNA polymerase involved in eukaryotic gene transcription, Polymerase II generally regulates protein-encoding genes. Figure 1 shows a diagram of the various components involved in cell-specific regulation of Pol-II gene transcription.

Core Promoter: Pol II-regulated genes typically have a Core Promoter where Pol II and a variety of general factors bind to specific DNA motifs:
i: the TATA box (TATA DNA sequence), which is bound by the "TATA-binding protein" (TBP).
ii: the Initiator motif (INR), where Pol II and certain other core factors bind, is present in many Pol II-regulated genes.
iii: the Downstream Promoter Element (DPE), which is present in a subset of Pol II genes, and where additional core factors bind.
The core promoter binding factors are generally ubiquitously expressed, although there are exceptions to this.

Proximal Promoter: immediately upstream (5') of the core promoter, Pol II target genes often have a Proximal Promoter region that spans up to 500 base pairs (b.p.), or even to 1000 b.p.. This region contains a number of functional DNA binding sites for a specific set of transcription activator (TA) and transcription repressor (TR) proteins. These TA and TR factors are generally cell- or tissue-specific in expression, rather than ubiquitous, so that the presence of their cognate binding sites in the proximal promoter region programs cell- or tissue-specific expression of the target gene, perhaps in conjunction with TA and TR complexes bound in distal enhancer regions.

Distal Enhancer(s): many or most Pol II regulated genes in higher eukaryotes have one or more distal Enhancer regions which are essential for proper regulation of the gene, often in a cell or tissue-specific pattern. Like the proximal promoter region, each of the distal enhancer regions typically contain a cluster of binding sites for specific TA and/or TR DNA-binding factors, rather than just a single site.

Enhancers generally have three defining characteristics:
i: They can be located very long distances from the promoter of the target gene they regulate, sometimes as far as 100 Kb, or more.
ii: They can be either upstream (5') or downstream (3') of the target gene, including within introns of that gene.
iii: They can function in either orientation in the DNA.

Combinatorial mechanisms of transcription regulation: The specific combination of TA and TR binding sites within the proximal promoter and/or distal enhancer(s) provides a "combinatorial transcription code" that mediates cell- or tissue-specific expression of the associated target gene. Each promoter or enhancer region mediates expression in a specific subset of the overall expression pattern. In at least some cases, each enhancer region functions completely independently of the others, so that the overall expression pattern is a linear combination of the expression patterns of each of the enhancer modules.

Co-Activator and Co-Repressor Complexes: DNA-bound TA and TR proteins typically recruit the assembly of specific Co-Activator (Co-A) and Co-Repressor (Co-R) Complexes, respectively, which are essential for regulating target gene transcription. Both Co-A's and Co-R's are multi-protein complexes that contain several specific protein components.

Co-Activator complexes generally contain at lease one component protein that has Histone Acetyl Transferase (HAT) enzymatic activity. This functions to acetylate Histones and/or other chromatin-associated factors, which typically increases that transcription activation of the target gene. By contrast, Co-Repressor complexes generally contain at lease one component protein that has Histone De-Acetylase (HDAC) enzymatic activity. This functions to de-acetylate Histones and/or other chromatin-associated factors. This typically increases the transcription repression of the target gene.

Adaptor (Mediator) complexes: In addition to the co-activator complexes that assemble on particular cell-specific TA factors, - there are at least two additional transcriptional co-activator complexes common to most cells. One of these is the Mediator complex, which functions as an "adaptor" complex that bridges between the tissue-specific co-activator complexes assembled in the proximal promoter (or distal enhancers). The human Mediator complex has been shown to contain at least 19 protein distinct components. Different combinations of these co-activator proteins are also found to be components of specific transcription Co-Activator complexes, such as the DRIP, TRAP and ARC complexes described below.

TBP/TAF complex: Another large Co-A complex is the "TBP-associated factors" (TAFs) that assemble on TBP (TATA-Binding Protein), which is bound to the TATA box present in many promoters. There are at least 23 human TAF proteins that have been identified. Many of these are ubiquitously expressed, but TAFs can also be expressed in a cell or tissue-specific pattern.

Specific Coactivator Complexes for DNA-binding Transcription Factors.

A number of specific co-activator complexes for DNA-binding transcription factors have been identified, including DRIP, TRAP, and ARC (reviewed in Bourbon, 2004, Blazek, 2005, Conaway, 2005, and Malik, 2005). The DRIP co-activator complex was originally identified and named as a specific complex associated with the Vitamin D Receptor member of the nuclear receptor family of transcription factors (Rachez, 1998). Similarly, the TRAP co-activator complex was originally identified as a complex that associates with the thyroid receptor (Yuan, 1998). It was later determined that all of the components of the DRIP complex are also present in the TRAP complex, and the ARC complex (discussed further below). For example, the DRIP205 and TRAP220 proteins were show to be identical, as were specific pairs of the other components of these complexes (Rachez, 1999).

In addition, these various transcription co-activator proteins identified in mammalian cells were found to be the orthologues or homologues of the Mediator ("adaptor") complex proteins (reviewed in Bourbon, 2004). The Mediator proteins were originally identified in yeast by Kornberg and colleagues, as complexes associated with DNA polymerase (Kelleher, 1990). In higher organisms, Adapter complexes bridge between the basal transcription factors (including Pol II) and tissue-specific transcription factors (TFs) bound to sites within upstream Proximal Promoter regions or distal Enhancer regions (Figure 1). However, many of the Mediator homologues can also be found in complexes associated with specific transcription factors in higher organisms. A unified nomenclature system for these adapter / co-activator proteins now labels them Mediator 1 through Mediator 31 (Bourbon, 2004). For example, the DRIP205 / TRAP220 proteins are now identified as Mediator 1 (Rachez, 1999), based on homology with yeast Mediator 1.

Example Pathway: Specific Regulation of Target Genes During Notch Signaling:

One well-studied example of cell-specific regulation of gene transcription is selective regulation of target genes during Notch signaling. Notch signaling was first identified in Drosophila, where it has been studied in detail at the genetic, molecular, biochemical and cellular levels (reviewed in Justice, 2002; Bray, 2006; Schweisguth, 2004; Louvri, 2006). In Drosophila, Notch signaling to the nucleus is thought always to be mediated by one specific DNA binding transcription factor, Suppressor of Hairless. In mammals, the homologous genes are called CBF1 (or RBPJkappa), while in worms they are called Lag-1, so that the acronym "CSL" has been given to this conserved transcription factor family. There are at least two human CSL homologues, which are now named RBPJ and RBPJL.

In Drosophila, Su(H) is known to be bifunctional, in that it represses target gene transcription in the absence of Notch signaling, but activates target genes during Notch signaling. At least some of the mammalian CSL homologues are believed also to be bifunctional, and to mediate target gene repression in the absence of Notch signaling, and activation in the presence of Notch signaling.

Notch Co-Activator and Co-Repressor complexes: This repression is mediated by at least one specific co-repressor complexes (Co-R) bound to CSL in the absence of Notch signaling. In Drosophila, this co-repressor complex consists of at least three distinct co-repressor proteins: Hairless, Groucho, and dCtBP (Drosophila C-terminal Binding Protein). Hairless has been show to bind directly to Su(H), and Groucho and dCtBP have been shown to bind directly to Hairless (Barolo, 2002). All three of the co-repressor proteins have been shown to be necessary for proper gene regulation during Notch signaling in vivo (Nagel, 2005).

In mammals, the same general pathway and mechanisms are observed, where CSL proteins are bifunctional DNA binding transcription factors (TFs), that bind to Co-Repressor complexes to mediate repression in the absence of Notch signaling, and bind to Co-Activator complexes to mediate activation in the presence of Notch signaling. However, in mammals, there may be multiple co-repressor complexes, rather than the single Hairless co-repressor complex that has been observed in Drosophila.

During Notch signaling in all systems, the Notch transmembrane receptor is cleaved and the Notch intracellular domain (NICD) translocates to the nucleus, where it there functions as a specific transcription co-activator for CSL proteins. In the nucleus, NICD replaces the Co-R complex bound to CSL, thus resulting in de-repression of Notch target genes in the nucleus (Figure 2). Once bound to CSL, NICD and CSL proteins recruit an additional co-activator protein, Mastermind, to form a CSL-NICD-Mam ternary co-activator (Co-A) complex. This Co-R complex was initially thought to be sufficient to mediate activation of at least some Notch target genes. However, there now is evidence that still other co-activators and additional DNA-binding transcription factors are required in at least some contexts (reviewed in Barolo, 2002).

Thus, CSL is a good example of a bifunctional DNA-binding transcription factor that mediates repression of specific targets genes in one context, but activation of the same targets in another context. This bifunctionality is mediated by the association of specific Co-Repressor complexes vs. specific Co-Activator complexes in different contexts, namely in the absence or presence of Notch signaling.