Transcription factors: bound to activate or repress

Research Update

TRENDS in Biochemical Sciences Vol.26 No.4 April 2001

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Transcription factors: bound to activate or repress David S. Latchman The complex processes of eukaryotic gene expression are controlled by a relatively small number of transcription factors whose activities are modulated by a diverse set of regulatory mechanisms. A recent paper describes the molecular basis for one such mechanism in which the Pit-1 transcription factor activates or represses transcription depending on the sequence of the DNA to which it binds. The mechanism by which the binding-site sequence regulates the activity of Pit-1 is discussed in relation to that of other transcription factors.

Early studies of eukaryotic gene regulation identified specific, short DNA sequences that were present in all genes activated by a common stimulus but absent from genes that were not regulated by this stimulus (reviewed in Ref. 1). Subsequently, it was shown that these sequences (e.g. the heat shock element or the glucocorticoid response element) acted by binding positively acting transcription factors, which were activated by the stimulus. The activation of these transcription factors resulted in the stimulation of gene transcription (reviewed in Refs 2,3). These early studies led to the idea that transcription factors regulated gene expression in a positive manner in response to specific stimuli, or in particular cell types. Subsequently, however, it became clear that many transcription factors could act by inhibiting, as opposed to activating, gene expression. Such repression can occur either via a negatively acting factor interfering with the action of a positively acting factor, or by a direct interaction between the negative factor and the basal transcriptional complex that reduces the activity of this complex (reviewed in Refs 2,4). In turn, these studies led to the idea that the transcription rate of a particular gene, either in a specific cell type or following exposure to a particular stimulus, was regulated by the relative balance of activating and inhibiting transcription factors.

particular, it is now becoming increasingly recognized that although some factors are pure activators or pure repressors, many can both activate and repress transcription in a manner that is dependent on the particular situation. Steroid and thyroid hormone receptors

An early example of such a ‘dual’-acting transcription factor was provided by the thyroid hormone receptor, which regulates gene expression in response to thyroid hormone and is a member of the nuclear receptor gene superfamily (i.e. the steroid hormone receptor family) of transcription factors (reviewed in Ref. 5). In the absence of thyroid hormone, the thyroid hormone receptor binds to its DNA-binding site in thyroid hormone-responsive genes, albeit in a conformation that enables it to bind co-repressor molecules such as N-CoR and mSIN3; consequently, transcription is repressed (Fig. 1a). However, following binding of thyroid hormone, the receptor changes its conformation so that it can no longer bind co-repressor molecules. Instead, the receptor binds coactivator molecules such as CREB binding protein (CBP), resulting in the activation of transcription (Fig. 1a). (a)

In the case of the thyroid hormone receptor, therefore, a single transcription factor can act as either an activator or a repressor depending on the presence or absence of thyroid hormone, respectively. Interestingly however, in the case of the glucocorticoid receptor (another member of the nuclear receptor family of transcription factors), the ability of the receptor to activate or repress transcription depends on the nature of the DNA sequence to which it is bound. Unlike the thyroid hormone receptor, the glucocorticoid receptor binds to its DNA target sites only following hormone treatment. Initial studies focused on genes that were activated by this steroid hormone and that contained a glucocorticoid response element (GRE). Following hormone binding, the glucocorticoid receptor binds as a dimer to the GRE and activates transcription (Fig. 1b; reviewed in Refs 6,7). However, subsequent studies indicated that there were other genes (e.g. that encoding proopiomelanocortin) that are repressed by the glucocorticoid receptor following hormone treatment and that contain a sequence distinct from, but related to, the GRE, known as an nGRE. Interestingly, it has been demonstrated that the receptor binds to this nGRE as a trimer

–T

+T T

TR TRE

(b)

TR

–

TRE

No transcription

GRE sequence

+ Transcription

nGRE sequence

– GR

GR

GR

GR –

+ GRE

Transcription

GR

nGRE

No transcription

Ti BS

A single factor can both activate and repress transcription

Although this idea is basically correct, the situation has become more complex as further studies have been conducted. In

Fig. 1. Activation and repression by members of the nuclear receptor family: (a) In the absence of thyroid hormone (T), the thyroid hormone receptor (TR) binds to the thyroid hormone response element (TRE) in a conformation that binds co-repressor molecules and thereby inhibits transcription. Following binding of thyroid hormone, the structure of the receptor changes so that it binds coactivator molecules and activates transcription. (b) The glucocorticoid receptor (GR) binds as a dimer to the glucocorticoid response element (GRE) and activates transcription. By contrast, the receptor binds as a trimer to the related but distinct negative GRE (nGRE) sequence and represses rather than activates transcription.

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rather than as a dimer (Fig.1b), and this receptor trimer represses rather than activates transcription8 (reviewed in Ref. 6). Pit-1

A recent paper by Scully et al.9 has extended this idea to the cell type-specific transcription factor Pit-1, demonstrating the precise structural basis for the ability of this factor to act as either an activator or repressor of cell-type specific genes depending on the nature of its binding site. Pit-1 is a founding member of the POU (Pit, Oct, Unc) family of transcription factors, which play key roles in regulating gene expression during development (reviewed in Ref. 10). These proteins have a bipartite DNA-binding domain consisting of a POU-specific domain and a POU-homeodomain related to the homeobox found in other transcription factors. Pit-1 plays a key role in (a)

TRENDS in Biochemical Sciences Vol.26 No.4 April 2001

development of the pituitary gland and is required for activation of the genes encoding growth hormone, prolactin and thyrotropin in somatotrope, lactotrope and thyrotrope cell types, respectively. A key question in understanding this system is how Pit-1 activates only the appropriate gene in each cell type. The study by Scully et al.9 throws light on this question. The authors focus their attention on the fact that the binding site for Pit-1 in the gene encoding growth hormone, contains an extra two bases in the centre of the site compared to the Pit-1-binding site in the prolactin (PRL) gene. They therefore prepared a construct in which the Pit-1 binding site from the growth hormone (GH1) promoter was substituted with that from the PRL gene in an otherwise intact GH1 promoter. When this construct was introduced into transgenic animals, the GH1 promoter was Growth hormone

Prolactin

N-CoR Pit-1

Pit-1

+

Pit-1

–

Pit-1

TATACATTTATTCATG

TATATATATTCATG

No transcription

Transcription

(b)

HSV immediate-early genes

Cellular genes

VP16 Oct-1

+

+

+

Oct-1 ATGCAAAT

TAATGARAT

Weak transcription

Strong transcription

(c)

MORE sequence

Oct-1

Oct-1

PORE sequence

+

OBF-1

OBF-1

Oct-1

Oct-1

+

+

Oct-1

ATTTGAAATGCAAAT

ATGCATATGCAT Weak transcription

Strong transcription Ti BS

Fig. 2. (a) Following binding to its DNA site in the promoter of the PRL gene, the Pit-1 dimer activates transcription. By contrast, the two extra T bases (shown in bold) in the centre of the binding site for Pit-1 in the GH1 promoter results in a different conformation following binding of transcription factor to DNA, which results in the recruitment of the N-CoR transcriptional co-repressor and consequent transcriptional repression. (b) Binding of Oct-1 to the TAATGARAT sequence in the HSV immediate-early gene promoters results in a change in the conformation of this transcription factor, allowing it to bind the strong transcriptional activator VP16. Recruitment of VP16 does not occur upon binding of Oct-1 to the ATGCAAAT sequence in cellular genes and, consequently, transcriptional activation is only weak. (c) Dimerization of Oct-1 on the MORE sequence results in the masking of the amino acid residues (red line) that are normally used to interact with the transcriptional coactivator OBF-1 and hence only weak activation of transcription occurs. By contrast, following dimerization on the PORE sequence, these amino acids are exposed and can be used to recruit OBF-1, leading to strong activation of transcription. Abbreviations: GH1, gene encoding growth hormone; HSV, herpes simplex virus; MORE, more PORE; OBF–1, Oct-binding factor 1; PORE, palendromic Oct factor recognition element; PRL, gene encoding prolactin.

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expressed not only in somatotropes but also in lactotropes, where normally only the PRL promoter is active. Hence, in lactotropes, Pit-1 can bind to its binding sites in the PRL and GH1 promoters, and activate or repress transcription of the corresponding genes, respectively (Fig. 2a). In further studies, Scully et al.9 cocrystallized Pit-1 with its two different binding sites and showed that the additional two-base pair sequence in the growth hormone site results in the POUspecific and POU-homeodomain portions of the POU domain being accommodated on the same face of the DNA, whereas on the prolactin element, they are bound to perpendicular faces of the DNA. Hence, although both DNA sites bind a Pit-1 dimer, this dimer is in distinct conformations in the two situations. In addition, Scully et al.9 were able to demonstrate that, in lactotropes, the corepressor N-CoR is able to bind to the Pit-1 dimer that is bound to the promoter of the GH1 gene, resulting in the repression of expression. By contrast, this repressor molecule does not bind to the Pit-1 dimer bound to the prolactin-binding site and, therefore, Pit-1 activates expression from this promoter in lactotropes. Although this study helps explain how Pit-1 can selectively activate and/or repress gene expression in a particular cell type, it does not provide the complete story because Pit-1 must also be able to activate growth hormone expression in the distinct somatotrope cell type. The nature of the ‘override’ mechanism by which the repressive activity of Pit-1 on the GH1 promoter is reversed in this cell type, awaits further study. Nonetheless, the work of Scully et al.9 advances our understanding of the role of DNA sequences in regulating the activity of transcription factors.

Interestingly, the POU family of transcription factors to which Pit-1 belongs, has been subject to a variety of different studies that have previously elucidated the key role of DNA-binding sites in other aspects of these factors. For example, the Oct-1 transcription factor acts as a weak transactivator, stimulating the expression of several cellular genes that contain its specific binding site ATGCAAAT. However, following binding of Oct-1 to the related sequence TAATGARAT (R = purine) in the immediate-early gene

Research Update

promoters of herpes simplex virus (HSV), transcription is strongly activated. This differential binding affinity results from a conformational change in Oct-1, induced upon binding to the TAATGARAT sequence, that allows it to bind the HSV VP16 (Vmw65) protein (a strong transactivator), thereby increasing the transactivation potency of Oct-1 (Ref. 11) (Fig. 2b). A recent study12 has extended this to the binding of a cellular transcriptional coactivator, OBF-1 (Oct-binding factor 1), to dimers of Oct-1 that are bound to two distinct sites with different sequences. Thus, when Oct-1 binds as a dimer to a sequence known as the palendromic Oct factor recognition element (PORE), it can then bind OBF-1, resulting in strong activation of transcription. By contrast, when Oct-1 binds as a dimer to the site known as MORE (More PORE), the residues in Oct-1 that interact with OBF-1 on the PORE site are used instead to form the dimer interface between the Oct-1 monomers. Hence, when bound to the MORE site, Oct-1 cannot recruit OBF-1, and strong activation of transcription is precluded (Fig. 2c).

TRENDS in Biochemical Sciences Vol.26 No.4 April 2001

Conclusion: the key role of the DNA-binding site

A variety of studies into transcriptional regulation indicate that the DNA binding site is not simply a passive partner that is merely recognized by a particular transcription factor. Rather, when transcription factors bind to different sites they assume different protein structures. In turn, these structures determine whether the bound transcription factor can interact with particular coactivator or corepressor proteins. Hence, protein changes, which occur on DNA binding, provide an additional facet to the complexity of transcription factors, allowing them to activate transcription to varying degrees, to have no effect, or to inhibit transcription. This mechanism is one of many that enable transcription factors to control the inducible and cell type-specific gene expression that is central to the complexity of the multicellular eukaryotic organism. References 1 Latchman, D.S. (1998) Gene Regulation – a eukaryotic perspective (3rd edn), Stanley Thorne Publishers 2 Latchman, D.S. (1998) Eukaryotic transcription factors (3rd edn), Academic Press

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3 Latchman, D.S., ed., (1997) Landmarks in Gene Regulation, Portland Press 4 Hanna-Rose, W. and Hansen, U. (1996) Active repression mechanisms of eukaryotic transcription repressors. Trends Genet. 12, 229–234 5 Mangelsdorf, D.J. et al. (1995) The nuclear receptor super family; the second decade. Cell 83, 835–839 6 Lefstin, J.A. and Yamamoto, K.R. (1998) Allosteric effects of DNA on transcriptional regulators. Nature 392, 885–888 7 Beato, M. et al. (1995) Steroid hormone receptors: many actors in search of a plot. Cell 83, 851–857 8 Drouin, J. et al. (1993) Novel glucocorticoid receptor complex with DNA element of the hormone repressed POMC gene. EMBO J. 12, 145–156 9 Scully, K.M. et al. (2000) Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification. Science 290, 1127–1131 10 Ryan, A.K. and Rosenfeld, M.G. (1997) POU domain family values: flexibility, partnerships and developmental codes. Genes Dev. 11, 1207–1225 11 Walker, S. et al. (1994) Site-specific conformational alteration of the Oct-1 POU domain–DNA complex as the basis for differential recognition by Vmw65 (VP16). Cell 741, 841–852 12 Tomilin, A. et al. (2000) Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration. Cell 103, 853–864

David S. Latchman Institute of Child Health, University College London, 30 Guilford Street, London, UK WC1N 1EH. e-mail: [email protected]

Protein Sequence Motif

Nicastrin, a presenilin-interacting protein, contains an aminopeptidase/transferrin receptor superfamily domain Richard Fagan, Mark Swindells, John Overington and Malcolm Weir Nicastrin, a protein implicated in Alzheimer’s disease, has a domain that is found in the aminopeptidase/transferrin receptor superfamily. In nicastrin, this domain might possess catalytic activity (as observed with aminopeptidases) or it could serve merely as a binding domain (with analogy to the transferrin receptors) for the β-amyloid precursor protein.

Nicastrin is a 709 amino acid type I transmembrane glycoprotein that has been recently identified1 as a key component of the Alzheimer-linked multiprotein complex formed with the proteases presenilin 1 and presenilin 2. The formation of this complex is the final step in the production of the neurotoxic β-amyloid peptide (also known as the amyloid), which is observed in brain

plaques of familial Alzheimer’s disease patients. The amyloid protein is produced from the membrane-bound β-amyloid precursor protein, β-APP, in two distinct sequential steps. First, β-APP is cleaved by the protease β-secretase (BACE-2) and second, the amyloid protein is liberated by further γ-secretase processing. Current opinion suggests that presenilin 1 and presenilin 2 possess the protease catalytic activity that is necessary for the production of the neurotoxic β-amyloid peptide (amyloid protein)1,2. It was shown recently that nicastrin binds to β-APP (and its α- and β-cleaved versions) and is able to modulate the production of β-amyloid peptide1. This implicates a direct role for nicastrin in the pathogenesis of Alzheimer’s disease and

suggests that it could be a suitable target for therapeutic intervention. It was speculated that the function of nicastrin might be to bind substrates of presenilin–γ-secretase complexes or, alternatively, to modulate γ-secretase activity. However, no significant amino acid sequence similarity with known proteases, nor indeed with any other functionally annotated proteins, was found. The molecular basis for biological function of this protein therefore remained unclear. We show through the use of Genome Threader (Ref. 3; Fig. 1) that the central region of nicastrin is a new member of the aminopeptidase superfamily, which also includes the non-protease transferrin receptor (TfR). Furthermore, these

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