(Ronald Margolis, "Conference: Complex Complexes: Report of an NIDDK Workshop on Coactivators and Corepressors, Bethesda, USA, 15-16 December 1998", Trends in Endocrinology and Metabolism 10(7):286-289, 1999)

Progress in understanding gene transcription has provided an essential framework of the transcriptional machinery¹. Subsequent studies have focused on understanding the cellular and nuclear factors that facilitate regulated changes in gene transcription, particularly in response to signal transduction². This is especially true for nuclear hormone receptors in their role as nuclear signals for regulated expression of target genes³. At the same time, studies of signaling through cell surface receptors also revealed downstream components which were able to affect gene transcription⁴. Exciting recent discoveries have focused attention on classes of nuclear accessory, and other, proteins which confer the ability to bridge the steroid and cell surface receptor signals and the hormone response element to the transcriptional machinery in ways that foster either repression or activation of transcription^5-8. Moreover, it has become evident that factors associated with cell surface receptor action interact with steroid receptors, and vice versa⁹, suggesting that a diversity of signaling pathways may be operating under a common set of rules mediated by nuclear accessory proteins. Thus, rapidly emerging observations about the regulation of transcription in response to external signals has opened a window onto the entire complex process of gene transcription. With that in mind the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), at the NIH, convened a workshop ("Co-activators and Co-repressors in Gene Expression") designed to take a snapshot of this remarkably accelerating field and reveal insights that would help us to see not just from where we've come, but also where we are going.

Gene transcription is a complex, highly conserved, process requiring several classes of DNA-associated, nuclear accessory, and signaling molecules in addition to the genes, themselves. The basal transcriptional machinery comprising the DNA, nucleosomal histones, transcriptional activation factors (TAFs), the RNA polymerase (RNApol-II) complex, and other factors, mediates baseline transcription¹⁰. Activated, or regulated, transcription requires additional factors, including activators or co-activators, as well as enhancers, often interacting by reciprocal displacement of repressor or co-repressor activit(ies)y. One of the early steps in transcriptional activation is the formation of a preinitiation complex (PIC), the stabilization of which may depend on the presence or recruitment of appropriate upstream activators¹¹. One such factor, PC4, can act as a suppressor during formation of the early PIC, but when other factors are present it functions as a coactivator. One of these additional accessory factors may include a nuclear receptor interacting protein, thyroid hormone receptor-associated protein (TRAP)-220, which mediates ligand-dependent gene activation in response to external signals in a multi-step process with PC4¹². The interactions between various nuclear accessory proteins and the nascent PIC can be modulated at many levels, including suppressors, which act to prevent other factors from binding to and stabilizing the complex. One such factor, known as RBP¹³, blocks interaction of the TAF TFII with other TAFs, thus blocking gene activation by interfering with activator components of the basal transcription complex, itself.

For the complex process of regulated gene expression to occur, chromatin must be accessed. Sequences in the promoter region must be available to the various TAFs, to the RNApolII complex, and other factors, for effective transcription to proceed. In addition, upstream sequences, which include enhancer binding sites, often reflected as hormone response elements (HRE), and other regulatory domains, must also be accessed. Certain minimal factors for basal transcription include RSF (remodeling and spacing factor) and FACT (facilitates chromatin transcription), both of which are chromatin remodeling factors, which help to allow access¹⁴. Related to these are factors found initially in Drosophila, such as Nucleosome Remodeling Factor (NURF) which also have the effect of "unlocking" chromatin, to facilitate transcription¹⁵. NURF functions in an ATP-dependent fashion to remodel chromatin, and also recruits other factors, which in aggregate serve to allow co-activators, receptors, and other factors, access to enhancer/promoter regions. Chromatin in the basal state may be silenced by the state of methylation of the DNA^15,16. Nuclear accessory proteins, like MeCP2 (methyl-CpG-binding protein), a transcriptional repressor found initially in Xenopus, attract other proteins, including nuclear accessory proteins such as mSin3 and HDAC (see below) to maintain this silenced state of chromatin through methylation¹⁶.\

Transcriptionally inactive chromatin exists primarily in a hypoacetylated state, with the lysine residues of core histones "tucked" into the DNA. In this structural configuration, accessibility of factors required for gene transcription is reduced¹. When acetylated on lysine residues, the arms of core histones project out from the DNA, providing access for the factors necessary for regulated transcription. Studies in yeast, and other lower organisms, have revealed that several nuclear accessory proteins with histone acetyltransferase (HAT) or deacetylase (HDAC) activities are associated with coactivator or corepressor complexes and with activation or suppression of gene transcription, respectively¹⁷. One such HAT from yeast, Gcn5, implicated in vitro, but now found to regulate transcription in vivo¹⁸, appears to work in a promoter-specific fashion to relieve repression of transcription through histone acetylation, perhaps also involving phosphorylation of histone(s) by nuclear protein kinases¹⁹. Gcn5, as a coactivator, appears also to affect chromatin remodeling²⁰. HAT activity is not confined to lower organisms, but is found in higher eukaryotes as well. In addition, HAT activity has been found associated with other coactivators, such as p300/CBP(CREB binding protein)²¹, with formation of higher order complexes involving proteins such as PCAF (p300/CBP associated factor) mediating the ability of p300/CBP to function²¹. The mechanism of action is through acetylation of histones, and possibly other proteins (e.g. p53), in promoter-specific contexts and at least partially through displacement of corepressor complexes²². The latter consist of nuclear accessory proteins, such as mSin3, HDAC, and other factors, which bind in a competitive fashion to receptors on HREs, suppressing transcription (see below). Thus, a dynamic balance between histone acetylation/deacetylation serves as a focal point for activation or suppression of gene expression, respectively.

The discovery of a class of corepressors, such as NCoR (nuclear receptor corepressor)⁵, and SMRT (silencing mediator of retinoid and thyroid receptor)⁶, spurred studies to develop an understanding of their role in the regulation of transcription²². Yeast two-hybrid, and other, studies helped to define complexes formed between corepressors and adapter proteins, such as mSin3, and HDAC⁹. Targeted deacetylation of core nucleosomal histones represses transcription. Another means of repression occurs via DNA methylation, through proteins such as the MeCP2^16,23. The ability of MeCP2 to recruit and bind mSin3A and HDAC, suggests a functional connection between histone deacetylation and DNA methylation to effect repression. The recruitment of corepressor-HDAC complexes appears to be a common theme for repression, as suggested by studies of leukemogenesis. Chromosomal translocations forming fusion proteins lie at the basis of acute myeloid leukemia (AML)²⁴ and acute promyelocytic leukemia (APL)²⁵. Since the normal factors (e.g. AML1B) are transcription factors regulating hematopoietic differentiation, disruption causes blocks at various stages of hematopoiesis. For AML the chromosome 8:21 (ETO) translocation forms an AML-ETO fusion protein which represses transcription. The mechanism of action is via recruitment of corepressors, including NCoR, SMRT, and HDAC^24,26. The APL-RAR (retinoic acid receptor) fusion protein also recruits high levels of SMRT. For APL, treatment with derivatives of retinoic acid has had positive effects, but addition of the HDAC inhibitor trichostatin A may produce additional therapeutic benefit.

The initial discovery of nuclear receptor coactivators, such as NCoA/SRC-1, GRIP1 (nuclear receptor coactivator, steroid receptor coactivator and glucocorticoid receptor interacting protein-1, respectively), and others^7,27, significantly enhanced our understanding of how nuclear receptors, bound at the HRE, were able to foster ligand-dependent transcription. Specificity in coactivator usage and recruitment to specific nuclear receptors appears to depend on specific interaction domains. A common, highly conserved, LXXLL interaction domain is found in the nuclear receptor interaction domain (NR) of many coactivators²². Three such domains in this class of coactivator allow for interaction with the C-terminal AF(activation function)2 domain of nuclear receptors. Contributing to specificity appears to be the spacing between LXXLL domain -1, -2, or -3, as well as with the presence/absence of specific amino acid residues in the near vicinity of specified domains. Additional, C-terminal, LXXLL domains (-4 and –5) in coactivators have been linked to interaction with CBP/p300, another coactivator²², providing the means for integration of multiple signaling pathways in the regulation of gene transcription. In some contexts the latter interaction may provide a stronger coactivator function than NCoA/SRC-1²². That other activities are also recruited comes from data with GRIP1 suggesting that a coactivator associated arginine methyltransferase (CARM)-1 activity binds to GRIP1 and SRC-1 to further enhance function, as a secondary co-activator (Stallcup, M., pers. commun.), thus adding change in methylation of DNA as another focal point for coactivator action. Finally, novel factors, such as SRA (steroid receptor activator) may provide coactivator activity for the N-terminal AF (activation factor)1 domain of nuclear receptors, and reflect novel structure and function as a bridging factor with SRC-1 (O’Malley, B.W., pers. commun.).

CBP (CREB binding protein) was first studied in the context of a coactivator in the cAMP pathway leading to the CREB (cyclic AMP response element binding protein) transcription factor⁴. Structural studies have now defined the domains on each, KIX for CBP, and KID for CREB, as well as key residue phosphorylation, that mediate the interaction of coactivator and transcription factor²⁸. CBP is highly related to p300, another coactivator, both of which are targets of the E1A adenovirus⁸. While each has LXXLL domains that mediate interaction with nuclear receptors, both CBP and p300 have roles as coactivators of CREB²², with each implicated as important factors in development. In Caenorhabditis elegans cbp-1, a gene closely related to CBP/p300, regulates several major differentiation pathways, particularly during early embryogenesis²⁹. In Drosophila CBP has a key role in signal transduction in the hedgehog pathway and is involved in all aspects of development³⁰. When transgenic mice lacking p300 are generated, there is universal embryonic lethality³¹. Moreover, heterozygotes have significant lethality, suggesting a gene dosage effect. Similar results are found in CBP -/- and +/- mice. Effects include impaired embryogenesis, differentiation, and cell proliferation. While there appears to be a clear role for the HAT activity of CBP/p300 in effecting chromatin remodeling and activation of transcription, other, equally important, coactivators are also recruited. These include members of the p270 family, homologous to the yeast SWI/SNF complexes that express ATPase activity and have a role in chromatin remodeling³². Thus coactivators, initially thought to have restricted roles in signal transduction, appear to have broader roles within the context of developmental function.

With the first demonstration of the role of coactivators and corepressors in the regulation of gene expression came a flood of new findings expanding on the numbers of such factors and the function(s) they subserve. Localization and characterization of coactivators and corepressors from species as diverse as yeast, C. elegans, Drosophila, mouse, and human makes a strong statement about the evolutionary conservation of a basic principle of gene expression: cell signaling requires key adapter proteins in the nucleus to couple external (and internal) signals to changes in transcription. Emerging evidence on the effects of mutations or alterations in levels of selected coactivators and/or corepressors and subsequent roles in producing developmental defects and disease, suggest that these factors also represent important new targets for therapeutic intervention. Finally, from this workshop has developed a keen understanding of the important questions that remain: How is specificity among the many, sometimes overlapping, classes of nuclear accessory proteins determined? How are diverse signaling pathways sorted out with regard to usage of individual coactivators or corepressors? How do coactivators and corepressors recruit other factors? And finally, how do defects or alterations in function translate into disease?

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