Generic transcription pathway (Homo sapiens)
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Description
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.
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The DRIP co-activator complex is a subset of 14 proteins from the set of at least 31 Mediator proteins that, in different combinations, form "Adapter" complexes. 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 (reviewed in Maston, 2006 and Naar, 2001).
The DRIP complex was originally identified and named as a co-activator complex associated with the Vitamin D Receptor member of the nuclear receptor family of transcription factors (Rachez, 1998). It was later determined that all of the components of the DRIP complex were also in the TRAP complex, and the ARC complex.
The DRIP complex contains the following 14 proteins, which also are common to the ARC and TRAP complexes: MED1, MED4, MED6, MED7, MED10, MED12, MED13, MED14, MED16, MED17, MED23, MED24, CDK8, CycC.
All of the DRIP adapter complex components are present in the ARC adapter complex, but the ARC complex also has 4 additional components (Rachez, 1999). These ARC-specific components are now called: MED8, MED15, MED25, and MED 26 in the unified nomenclature scheme (Bourbon, 2004).
Similarly, all 14 of the DRIP adapter complex components are present in the TRAP adapter complex, but the TRAP complex also has 4 additional components (Bourbon, 2004), These TRAP-specific components are now called: MED20, MED27, MED30, and MED 31 in the unified nomenclature scheme.
In addition, these various transcription co-activator proteins identified in mammalian cells were found to be the orthologues or homologues of the Mediator complex identified in yeast, first identified by Kornberg and colleagues (Kelleher, 1990).
activity of SMAD2/SMAD3:SMAD4
heterotrimer(TAZ)-stimulated
gene expressionAnnotated Interactions
The ARC co-activator complex is a subset of 18 proteins from the set of at least 31 Mediator proteins that, in different combinations, form "Adapter" complexes in human cells. 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 (reviewed in Maston, 2006 and Naar, 2001).
The ARC complex was originally identified and named as a co-activator complex associated with transcription activator proteins (reviewed in Malik, 2005 and references therein). It was subsequently determined that many of the components of the ARC complex are also in the DRIP complex, and in the TRAP complex..
The ARC complex contains the following 14 proteins, which also are common to the DRIP and TRAP complexes: MED1, MED4, MED6, MED7, MED10, MED12, MED13, MED14, MED16, MED17, MED23, MED24, CDK8, CycC.
The ARC complex also contains 4 additional, ARC-specific components, which are now called: MED8, MED15, MED25, and MED 26 in the unified nomenclature scheme (Bourbon, 2004).
In addition, these various transcription co-activator proteins identified in mammalian cells were found to be the orthologues or homologues of the Mediator complex proteins in yeast, first identified by Kornberg and colleagues (Kelleher, 1990). The unified nomenclature system for these adapter / co-activator proteins now labels them Mediator 1 through Mediator 31 (Bourbon, 2004).
The order of addition of the ARC proteins during complex assembly is not fully determined, and may vary in different cell contexts. Therefore, ARC complex assembly is represented as a single reaction event, in which all 19 components assemble simultaneously into the ARC co-activator complex.
The TRAP co-activator complex is a subset of 18 proteins from the set of at least 31 Mediator proteins that, in different combinations and in different contexts, form specific co-activator or "Adapter" complexes in human cells. These 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 (reviewed in Maston, 2006 and Naar, 2001).
The TRAP complex was originally identified and named as a co-activator complex associated with the Thyroid Hormone Receptor member of the nuclear receptor family of transcription factors (Yuan, 1998). It was later determined that many of the components of the TRAP complex are also in the DRIP complex, and in the ARC complex.
The TRAP complex contains the following 14 proteins, which also are common to the DRIP and ARC complexes: MED1, MED4, MED6, MED7, MED10, MED12, MED13, MED14, MED16, MED17, MED23, MED24, CDK8, CycC.
The TRAP complex also contains 4 additional components, which are now called: MED20, MED27, MED30, and MED 31 in the unified nomenclature scheme (Bourbon, 2004).
In addition, these various transcription co-activator proteins identified in mammalian cells were found to be the orthologues or homologues of the Mediator complex proteins in yeast, first identified by Kornberg and colleagues (Kelleher, 1990). The unified nomenclature system for these adapter / co-activator proteins now labels them Mediator 1 through Mediator 31 (Bourbon, 2004).
The order of addition of the TRAP proteins during complex assembly is not fully determined, and may vary in different cell contexts. Therefore, TRAP co-activator complex assembly is represented as a single reaction event, in which all 18 components assemble simultaneously into the TRAP co-activator complex.
The DRIP co-activator complex is a subset of 14 proteins from the set of at least 31 Mediator proteins that, in different combinations, form "Adapter" complexes. 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 (reviewed in Maston, 2006 and Naar, 2001).
The DRIP complex was originally identified and named as a co-activator complex associated with the Vitamin D Receptor member of the nuclear receptor family of transcription factors (Rachez, 1998). It was later determined that all of the components of the DRIP complex were also in the TRAP complex, and the ARC complex.
The DRIP complex contains the following 14 proteins, which also are common to the ARC and TRAP complexes: MED1, MED4, MED6, MED7, MED10, MED12, MED13, MED14, MED16, MED17, MED23, MED24, CDK8, CycC.
All of the DRIP adapter complex components are present in the ARC adapter complex, but the ARC complex also has 4 additional components (Rachez, 1999). These ARC-specific components are now called: MED8, MED15, MED25, and MED 26 in the unified nomenclature scheme (Bourbon, 2004).
Similarly, all 14 of the DRIP adapter complex components are present in the TRAP adapter complex, but the TRAP complex also has 4 additional components (Bourbon, 2004), These TRAP-specific components are now called: MED20, MED27, MED30, and MED 31 in the unified nomenclature scheme.
In addition, these various transcription co-activator proteins identified in mammalian cells were found to be the orthologues or homologues of the Mediator complex identified in yeast, first identified by Kornberg and colleagues (Kelleher, 1990).
A general feature of the NR proteins is that they each contain a specific protein interaction domain (PID), or domains, that mediates the specific binding interactions with the MED1 proteins. In the ligand-bound state, NRs each take part in an NR-MED1 binding reaction to form an NR-MED1 complex. The bound MED1 then functions to nucleate the assembly of additional specific coactivator proteins, depending on the cell and DNA context, such as what specific target gene promoter or enhancer they are bound to, and in what cell type.
The formation of specific MED1-containing coactivator complexes on specific NR proteins has been well-characterized for a number of the human NR proteins. For example, binding of Vitamin D to the human Vitamin D3 Receptor was found to result in the recruitment of a specific complex of D Receptor Interacting Proteins - the DRIP coactivator complex (Rachez, 1998). Within the DRIP complex, the DRIP205 subunit was later renamed human "MED1", based on sequence similarities with yeast MED1 (reviewed in Bourbon, 2004).
Similarly, binding of thyroid hormone (TH) to the human TH Receptor (THRA or THRB) was found to result in the recruitment of a specific complex of Thyroid Receptor Associated Proteins - the TRAP coactivator complex (Yuan, 1998). The TRAP220 subunit was later identified to be the Mediator 1 (MED1) homologue (summarized in Bourbon, et al., 2004; Table 1).
The 48 human NR proteins each contain the PID(s) known to mediate interaction with the human MED1 protein. Direct NR-MED1 protein-protein interactions have been shown for a number of the NR proteins. The MED1-interacting PIDs are conserved in all of the human NRs. Therefore, each of the human NRs is known or expected to interact with MED1 in the appropriate cell context, depending on the cell type, the cell state, and the target gene regulatory region involved.Formation of the KRAB ZNF / KAP1 Corepressor Complex:
Transcription factors which contain tandem copies of the C2H2 zinc finger DNA binding motif (ZNFs) are the most abundant class of TFs in the human proteome, comprising more than 1000 members. The KRAB ZNF proteins are the largest subset of these (with 423 members) and are defined by having an additional conserved domain, the KRAB domain (Bellefroid,1991, Margolin, 1994, Urrutia, 2003, Huntley, 2006). The Kruppel Associated Box (KRAB) domain is a transcription repression domain (Margolin, 1994) which mediates the recruitment of a specific and dedicated co repressor protein for the KRAB-ZNF family - KAP1 - which is required for transcriptional repression and gene silencing (Friedman, 1996).
The larger family of ZNF transcription factors are present in almost all metazoans and generally their DNA binding specificities and transcription regulation functions are conserved from Drosophila to humans. Although the biological functions of most ZNF TFs is not known, they often function biochemically as sequence specific DNA binding proteins and can be activators, or more oftenly observed, repressors of transcription, depending on cellular context. Transcriptional repression is mediated via specific protein protein interaction surfaces in the ZNF that function as repression domains, by recruiting specific co repressors, such as KAP1 in humans (Friedman, 1996), and dCTBP in Drosophila (Nibu, 1998).
In contrast to the larger ZNF family, the KRAB-ZNFs only appear much later in vertebrate evolution: genes encoding the primordial KRAB ZNF subfamily first arose in tetrapods and the family has been greatly expanded in numbers and complexity in mammals. Interestingly,a large fraction of KRAB-ZNFs are found only in primates. In addition to their rapid and dynamic evolutionary history, comparative genomics and expression studies of primate KRAB-ZNFs suggest that these genes have played a significant role in shaping primate specific traits (Huntley, 2006, Nowick, 2009).
The biochemical pathway utilized by KRAB-ZNFs is well defined and probably nearly identical for each member: All KRAB-ZNF proteins which have been studied in detail are repressors and utilize the KRAB domain to bind the KAP1 co-repressor. This interaction is direct, of high affinity, and is obligate for the KRAB-ZNF to function as a repressor when bound to DNA in vivo (Peng, 2000a,b).. The KAP1co-repressor appears to function as a scaffold protein to assemble and coordinate multiple enzymes (histone de-acetylases, histone methyltransferases and heterochromatin proteins) which target and modify chromatin structure thus leading to a compacted, silent state (Lechner, 2000; Schultz, 2001 Schultz, 2002 , Ayyanathan, 2003). The post-translational modification of KAP1 by SUMO controls its ability to assemble the enzymatic apparatus in chromatin (Ivanov, 2007; Zeng, 2008). It is formally possible that some KRAB ZNF proteins may have additional functional domains that recruit coactivators in specific contexts, given that such bifunctionality is common for many classes of DNA binding transcription factors,. However, there is no experimental evidence for this yet.
There also is good evidence that the KRAB ZNF-KAP1 complex proteins can have long range gene silencing functions, by nucleating chromatin complexes that inactivate transcription of large numbers of genes over large distances by assembling silent heterochromatin (Ayyanathan, 2003). Although KAP1 was originally identified as a mediator of specific gene transcription repression, subsequent studies have shown that KAP1 also is involved in the recruitment of homologues of the HP1 protein family (Ryan, 1999, Ayyanathan, 2003; Lechner, 2000). These nonhistone heterochromatin associated proteins were first shown to have an epigenetic gene silencing function in Drosophila and more recently in mammalian cells . These studies suggest that KRAB ZNF proteins and KAP1 may also be involved in large scale chromatin regulation and gene silencing, not just in gene specific transcriptional repression. Whether this is a general property of most or all KRAB ZNF proteins will require additional studies.
Finally, several KRAB containing ZNFs in mammals also contain a conserved SCAN domain which, like the KRAB domain also functions as a protein protein interaction domain. (Edelstein, 2005, Peng, 2000a,b). The SCAN domain does not participate in KAP1 binding but rather functions to mediate homodimerization, or selective heterodimerization with other SCAN containing proteins. However, the biochemical and biological functions of the SCAN domain in KRAB-ZNF mediated repression are not known.
Remaining Questions: The single most important unanswered question for KRAB-ZNFDs is to determine their biological functions. While the mechanism utilized by the KRAB ZNF / KAP1 protein complex to mediate gene specific transcription repression is well understood , much less known about the specific biological pathways they control. Preliminary evidence from recent whole genome analysis of the target genes for the KRAB- ZNF263 protein suggest that it can have both positive and negative effects on transcriptional regulation of its target genes (Frietze, 2010). Presumably, each KRAB-ZNF, via its array of zinc fingers can bind to specific DNA recognition sequences in target promoters. This, combined with highly tissue specific expression of each gene, makes the potential transcriptome controlled by the 423 KRAB-ZNFs extremely large.