Fos-related antigen
Serum response element binding protein (SRE BP) is a novel binding factor present in nuclear extracts of avian and NIH 3T3 fibroblasts that specifically bind to the cfos SRE within a region overlapping and immediately 3' to the CArG box. Binding of both Serum response factor (See Drosophila SRF) and SRE BP is necessary for maximal serum induction of the SRE. Homodimers and heterodimers of p35C/EBP (a transactivator) and p20C/EBP (a repressor) (See Drosophila Slow border cells) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBP is dependent on SRF binding but not on ternary complex factor (TCF) formation. Both p35C/EBP and p20C/EBP bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. SRE mutants that retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with an affinity nearly identical to that of the wild-type SRE, whereas another mutant, one that is not responsive to the TCF-independent pathway, has a nearly 10-fold lower affinity for SRE BP. It is proposed that C/EBP may play a role in conjunction with SRF in the TCF-independent signaling pathway for SRE activation (Sealy, 1997). A study was made of the promoter of the c-fos gene, specifically the serum response element. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2-10 min transiently increases binding of the two complexes; binding returns to control values within 30 min. The two GH-stimulated complexes are supershifted by antibodies against the serum response factor (SRF), indicating that they contain SRF or an antigenically related protein. One of the GH-stimulated complexes is supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH is lost when the SRF binding site in the SRE is mutated, and mutation of either the SRF or TCF binding site alters the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibits the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression is mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulates the serine phosphorylation of Elk-1. The increase is evident within 10 min and subsides after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1, contributing to GH-promoted transcriptional activation via the SRE (Liao, 1997).
The rapid and transient induction of the human proto-oncogene c-fos in response to a variety of stimuli depends on the serum response element (SRE). In vivo footprinting experiments show that this promoter element is bound by a multicomponent complex, including the serum response factor (SRF) and a ternary complex factor such as Elk-1. SRF is thought to recruit a ternary complex factor monomer into an asymmetric complex. A quaternary complex forms over the SRE which, in addition to an SRF dimer, contains two Elk-1 molecules. Its formation at the SRE is strictly dependent on phosphorylation of S-383 in the Elk-1 regulatory domain and appears to involve a weak intermolecular association between the two Elk-1 molecules. The influence of mutations in Elk-1 on quaternary complex formation in vitro correlates with their effects on the induction of c-fos reporter expression in response to mitogenic stimuli in vivo (Gille, 1996).
Transcriptional induction of the c-fos proto-oncogene in response to serum growth factors is mediated in part by a ternary complex that forms on the serum response element (SRE) within c-fos's promoter. This complex consists of Elk-1, serum response factor (SRF) and the SRE. Elk-1 is phosphorylated by MAP kinase, which correlates with the induction of c-fos transcription. Circular permutation analysis demonstrates that the minimal DNA-binding domain of SRF, which contains the MADS box, is sufficient to induce flexibility into the center of SRF's binding site within the SRE. Phasing analysis indicates that at least part of this flexibility results in the production of a directional bend toward the minor groove. The isolated ETS domains from Elk-1 and SAP-1 induce neither DNA bending nor increased DNA flexibility. Formation of ternary complexes by binding of Elk-1 to the binary SRF:SRE complex results in a change in the flexibility of the SRE. Phosphorylation of Elk-1 by MAP kinase (p42/ERK2) induces further minor changes in this DNA flexibility. However, phasing analysis reveals that the recruitment of Elk-1 to form the ternary complex affects the SRF-induced directional DNA bend in the SRE (Sharrocks, 1995).
The Ras signaling pathway targets transcription factors such as the ternary complex factors that are recruited by the serum response factor to form complexes on the serum response element (SRE) of the fos promoter. A new ternary complex factor, Net-b, has been identified. The net gene produces at least two splice variants: net-b and net-c. net-b RNA and protein are expressed in a variety of tissues and cell lines. net-c RNA is expressed at low levels, and the protein is not detected, raising the possibility that it is a cryptic splice variant. A study was performed of the composition of ternary complexes that form on the SRE of the fos promoter with extracts from fibroblasts (NIH 3T3) cultured under various conditions and pre-B cells (70Z/3) before and after differentiation with lipopolysaccharide (LPS). The fibroblast complexes contain mainly Net-b followed by Sap1 and Elk1. Net-b complexes, as well as Sap1 and Elk1, are induced by epidermal growth factor (EGF) stimulation of cells cultured in low serum. Pre-B-cell complexes contain mainly Sap1, with less of Net-b and little of Elk1. There is little change upon LPS-induced differentiation compared to the increase with EGF in fibroblasts. Net-b is a nuclear protein that constitutively represses transcription. Net-b is not activated by Ras signaling, in contrast to Net, Sap1a, and Elk1. Down-regulation of Net proteins with antisense RNA increases SRE activity. The increase in SRE activity is observed at low serum levels and is even greater after serum stimulation, showing that the SRE is under negative regulation by Net proteins and that the level of repression increases during induction. Net-b, the predominant factor in ternary complexes in fibroblasts, may both keep the activity of the SRE low in the absence of strong inducing conditions and rapidly shut the activity off after stimulation (Giovane, 1997).
TFII-I is a transcription factor that was initially characterized as a factor that binds to the initiator sites of various promoters. It has been implicated in the initiation of transcription of TATA-less promoters and in cell-type-specific transcription as well. Deletions of TFII-I are closely associated with neurodevelopmental Williams-Beuren syndrome in humans. TFII-I can also bind to E-box elements and can interact with upstream regulatory factors, including USF1 and c-myc. In addition, TFII-I can associate with Bruton's tyrosine kinase, and TfII-I's phosphorylation on tyrosine is stimulated by BTK. The activity of TFII-I is regulated by phosphorylation, and one of the potential phosphorylation sites is a mitogen-activated protein (MAP) kinase phosphorylation site. These observations suggest that TFII-I may play a role in signal transduction as well as in transcriptional initiation. In addition, TFII-I associates with the serum response factor (SRF) and the Phox1 protein, which are both involved in the regulation of the c-fos promoter (Kim, 1998 and references).
Overexpression of TFII-I can enhance the response of the wild-type c-fos promoter to a variety of stimuli. This effect depends on the c-fos c-sis-platelet-derived growth factor-inducible factor binding element (SIE) and serum response element (SRE). There is no effect of cotransfected TFII-I on the TATA box containing the c-fos basal promoter. Three TFII-I binding sites can be found in the c-fos promoter. Two of these overlap the c-fos SIE and SRE, and another is located just upstream of the TATA box. Mutations that distinguish between serum response factor (SRF), STAT, and TFII-I binding to the c-fos SIE and SRE suggest that the binding of TFII-I to these elements is important for c-fos induction in conjunction with the SRF and STAT transcription factors. Moreover, TFII-I can form in vivo protein-protein complexes with the c-fos upstream activators SRF, STAT1, and STAT3. These results suggest that TFII-I may mediate the functional interdependence of the c-fos SIE and SRE elements. In addition, the ras pathway is required for TFII-I to exert its effects on the c-fos promoter; growth factor stimulation enhances tyrosine phosphorylation of TFII-I. These results indicate that TFII-I is involved in signal transduction as well as transcriptional activation of the c-fos promoter (Kim, 1998).
Several studies have characterized the upstream regulatory region of c-fos, and identified cis-acting elements, termed the cyclic AMP (cAMP) response elements (CREs), which are critical for c-fos transcription in response to a variety of extracellular stimuli. Although several transcription factors can bind to CREs in vitro, the identity of the transcription factor(s) that activates the c-fos promoter via the CRE in vivo remains unclear. To help identify the trans-acting factors that regulate stimulus-dependent transcription of c-fos via the CREs, there have been developed dominant-negative (D-N) inhibitor proteins that function by preventing DNA binding of B-ZIP proteins in a dimerization domain-dependent fashion. A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain. The acidic extension of A-CREB interacts with the basic region of CREB, forming a coiled-coil extension of the leucine zipper and thus preventing the basic region of wild-type CREB from binding to DNA. Other D-N inhibitors generated in a similar manner with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP do not block CREB DNA binding activity, nor do they inhibit transcriptional activation of a minimal promoter containing a single CRE in PC12 cells. A-CREB inhibits activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular cAMP; membrane depolarization, which promotes Ca2+ influx, and nerve growth factor (NGF). A-CREB completely inhibits cAMP-mediated transcription of a reporter gene containing 750 bp of the native c-fos promoter, but A-CREB only partially inhibits Ca2+- and NGF-mediated transcription of the same reporter gene. Glutamate induction of c-fos expression in primary cortical neurons is dependent on CREB. In contrast, induction of c-fos transcription by UV light is not inhibited by A-CREB. A-CREB also attenuates NGF induction of morphological differentiation in PC12 cells. These results suggest that CREB or its closely related family members are general mediators of stimulus-dependent transcription of c-fos and are required for at least some of the long-term actions of NGF (Ahn, 1998).
The c-fos proto-oncogene is activated by a plethora of signals via the transcription factors Sap-1a and CREB. Recently, the coactivator CBP has been demonstrated to act in concert with CREB when CREB is phosphorylated by protein kinase A. CBP also binds directly to Sap-1a. While phosphorylation of Sap-1a by mitogen-activated protein kinases is not necessary for CBP/Sap-1a interaction, functional cooperation between these two proteins requires Sap-1a to become phosphorylated. CBP-antagonists impair Sap-1a-mediated transactivation. Similarly, the CBP antagonist E1A suppresses c-fos upregulation by phosphorylated CREB, indicating that CBP is a central component of c-fos regulation. CBP is phosphorylated by protein kinase A in vitro and the transactivation potential of the carboxy-terminal region of CBP is enhanced in the presence of active protein kinase A in vivo. Thus, CBP, in addition to CREB, is a target for cAMP-dependent signaling. However, combined phosphorylation of CBP by protein kinase A and mitogen-activated protein kinases appears to be non-cooperative, suggesting that CBP functions as a dampening integrator for two different signaling pathways (Janknecht, 1996).
The human paired class homeodomain protein Phox1 (which has no known Drosophila homolog) can impart serum-responsive transcriptional activity to the c-fos serum response element (SRE) by interacting with serum response factor (SRF). This activity is shared with other paired class homeodomains but not with more distantly related homeodomains. To understand the mechanism of Phox1 action at the SRE and the basis for the selective activity of paired class homeodomains in this context, a detailed mutagenesis was performed of the Phox1 homeodomain. Homeodomain amino acid residues that contact the major groove of the DNA are required for SRE activation in vivo. In contrast, substitution of a lysine residue in the N-terminal arm of the Phox1 homeodomain appears to abolish DNA binding without affecting activity in vivo. Certain substitutions on the exposed surfaces of helices 1 and 2, not required for DNA binding, abolish activity in vivo, suggesting that these surfaces contact an accessory protein(s) required for this activity. Transfer of a single amino acid residue from the surface of Phox1 helix 1 to the corresponding position in the distantly related Deformed (Dfd) homeodomain imparts to Dfd the ability to activate the SRE in vivo. It is proposed that Phox1 interacts with one or more factors at the SRE, in addition to SRF, and that the specificity of this interaction is determined by residues on the surfaces of helices 1 and 2 (Simon, 1997).
The human homeodomain protein Phox1 interacts functionally with Serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. This study reports the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. This protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro. It is proposed that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery (Grueneberg, 1997).
cAMP-responsive-element (CRE)-binding factor's interaction with nucleosomal DNA has been investigated in vitro on the human c-fos promoter. Analysis of nucleosome reconstitution of this promoter shows a preferential nucleosome positioning on the proximal promoter sequences, including the CRE centered at -60 relative to the start site of transcription. CRE-binding protein (CREB) and modulator protein (CREM) are unable to interact with their recognition site incorporated in a nucleosome. However, competition between transcription factor binding and nucleosome assembly allows CREM binding and induces important modifications in the nucleosomal structure suggesting the displacement of nucleosomes. These findings imply that binding of transcription factors to the CRE prior to cAMP induction might be required to prevent the incorporation of this element in a nucleosome (Schild-Poulter, 1996).
Many parathyroid hormone (PTH)-mediated events in osteoblasts are thought to require immediate early gene expression. PTH induces the immediate early gene, c-fos, in this cell type through a cAMP-dependent pathway. The present work investigated the nuclear mechanisms involved in PTH regulation of c-fos in the osteoblastic cell line, UMR 106-01. By transiently transfecting c-fos promoter 5' deletion constructs into UMR cells, it has been demonstrated that PTH induction of the c-fos promoter requires the major cAMP response element (CRE). Point mutations created in the major CRE within the largest construct inhibit both PTH-stimulated and basal expression. This element, therefore, performs concerted basal and PTH-responsive cis-acting functions. CRE-binding protein (CREB) constitutively binds the major CRE but becomes phosphorylated at its cAMP-dependent protein kinase consensus recognition site following PTH treatment. CREB is functionally implicated in c-fos regulation by coexpressing a dominant CREB repressor, KCREB (killer CREB), with the c-fos promoter constructs. KCREB suppresses both basal and PTH-mediated c-fos induction. It is concluded that PTH activates c-fos in osteoblasts through cAMP-dependent protein kinase-phosphorylated CREB interaction with the major CRE in the promoter region of the c-fos gene (Pearman, 1996).
Changes in environmental conditions, such as the addition of growth factors or irradiation of cells in culture, first affect immediate response genes. Short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF, which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), it is found that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signaling pathway to CREB and ATF-1, as it is not p38/RK/HOG-1 dependent, CREB being a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to suramin-inhibitable growth factor receptors and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route for UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway (Iordanov, 1997).
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that has been shown to support cell proliferation in murine fibroblasts engineered to stably express both chains of the human GM-CSF receptor (NIH-GMR). Because the proto-oncogene c-fos is believed to provide a link between short-term signals elicited at the membrane and long-term cellular response, the mechanism of GM-CSF-dependent cell regulation was studied using c-fos promoter activity as a molecular marker in both NIH-GMR transfectants and in the CD34+ cell line TF-1. The importance of c-fos and related AP-1 activity in GM-CSF signaling is suggested by a tight correlation between GM-CSF-dependent activation of the c-fos promoter and cell proliferation and by the inhibitory effect of a trans-dominant c-fos mutant on cell growth. To evaluate the contribution of the serum response factor (SRF) associated with the ternary complex factor (TCF) and of STAT proteins to c-fos promoter activation in response to GM-CSF, the SRF binding site (SRE) and/or the STAT binding site (SIE) were inactivated. In serum-free medium, both SRE and SIE are essential to c-fos promoter activation by GM-CSF in NIH-GMR transfectants and in TF-1 cells. No response to GM-CSF is observed when both sites are mutated. The nature of the STAT family member was further investigated by Wester blots and DNA retardation assays using an SIE probe. GM-CSF induces DNA binding of both STAT1 and STAT3 in NIH-GMR and mainly of STAT3 in TF-1 cells. STAT5 tyrosine phosphorylation is also observed in TF-1 cells. Finally, expression of a dominant negative MAPK mutant, ERK192A, results in a decrease of both SRE- and SIE-dependent activation of c-fos promoter by GM-CSF, suggesting that STAT1/3 are regulated not only by tyrosine kinases, but also partially by MAPK (Rajotte, 1996).
The expression of the high mobility group I (HMGI)-C chromatin component is essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, the AP-1 complex was analyzed in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. Neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation (Vallone, 1997).
Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure. Human SW-13
adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for
SWI-SNF function. BRG1 specifically represses transcription from a transfected c-fos promoter and
correspondingly blocks transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent
ATPase domain of BRG1 significantly reduces its ability to repress c-fos transcription. Repression by BRG1 requires the cyclic AMP response element of the c-fos
promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Repression of c-fos by BRG1, a known Rb binding protein, depends on Rb. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb
protein, transcriptional repression by BRG1 is weak unless wild-type Rb is also supplied. Interestingly, Rb-dependent repression by BRG1 is found to take
place through a pathway that is independent of transcription factor E2F (Murphy, 1999).
Considering the -76 to +10 region of the c-fos promoter in isolation, mutation of the cyclic AMP response element (CRE) results in complete loss of sensitivity to BRG1 in these
assays. A CRE is also located in the corresponding location of the human promoter. Thus, a possible model for the effect of BRG1 on c-fos could involve
nucleosomal rearrangement of the c-fos promoter (already occupied by ATF/CREB) and repression of ATF/CREB-dependent transcription by an Rb-associated
histone deacetylase. Interestingly, the CRE is known to bind transcription factor ATF-2, which was reported to interact directly with Rb (in that
case, the ATF-2-Rb interaction correlated with transcriptional activation, not repression). ATF-2 has been detected in extracts of SW-13 cells by Western blot
analysis using a specific antibody against ATF-2, and experiments are now in progress to examine its role in BRG1-mediated repression (Murphy, 1999).
Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that activates serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, its role in serum induction of c-fos and other SRE-regulated genes was assessed with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). DN-MKL1 and RNAi was found to specifically block SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi requires the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduces the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, is nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia has a markedly increased ability to activate SRE reporter genes, suggesting that fusion protein activation of SRF target genes may contribute to leukemogenesis (Cen, 2003).
The immediate-early (IE) genes Fos, Egr1 and Egr2 have been identified as transcriptional targets of brain derived neurotrophic factor (BDNF)/TrkB signaling in primary cortical neurons; the Fos serum response element area responds to BDNF/TrkB in a manner dependent on a combined C/EBP-Ebox element. The Egr1 and Egr2 promoters contain homologous regulatory elements. C/EBPα/β and NeuroD formed complexes in vitro and in vivo, and are recruited to all three homologous promoter regions. C/EBPα and NeuroD co-operatively activated the Fos promoter in transfection assays. Genetic depletion of Trk receptors led to impaired recruitment of C/EBPs and NeuroD in vivo, and elimination of Cebpa and Cebpb alleles reduced BDNF induction of Fos, Egr1 and Egr2 in primary neurons. Finally, defective differentiation of cortical dendrites, as measured by MAP2 staining, was observed in both compound Cebp and Ntrk knockout mice. Therefore this study identifed three IE genes as targets for BDNF/TrkB signaling, shows that C/EBPα and -β are recruited along with NeuroD to target promoters, and that C/EBPs are essential mediators of Trk signaling in cortical neurons. C/EBPs and Trks are required for cortical dendrite differentiation, consistent with Trks regulating dendritic differentiation via a C/EBP-dependent mechanism. Finally, this study indicates that BDNF induction of IE genes important for neuronal function depends on transcription factors (C/EBP, NeuroD) up-regulated during neuronal development, thereby coupling the functional competence of the neuronal cells to their differentiation (Calella, 2007).
Histone modifications are reported to show different behaviors, associations, and functions in different genomic niches and organisms. This study shows that rapid, continuous turnover of acetylation specifically targeted to all K4-trimethylated H3 tails (H3K4me3), but not to bulk histone H3 or H3 carrying other methylated lysines, is a common uniform characteristic of chromatin biology in higher eukaryotes, being precisely conserved in human, mouse, and Drosophila. Furthermore, dynamic acetylation targeted to H3K4me3 is mediated by the same lysine acetyltransferase, p300/cAMP response element binding (CREB)-binding protein (CBP), in both mouse and fly cells. RNA interference or chemical inhibition of p300/CBP using a newly discovered small molecule inhibitor, C646, blocks dynamic acetylation of H3K4me3 globally in mouse and fly cells, and locally across the promoter and start-site of inducible genes in the mouse, thereby disrupting RNA polymerase II association and the activation of these genes. Thus, rapid dynamic acetylation of all H3K4me3 mediated by p300/CBP is a general, evolutionarily conserved phenomenon playing an essential role in the induction of immediate-early (IE) genes. These studies indicate a more global function of p300/CBP in mediating rapid turnover of acetylation of all H3K4me3 across the nuclei of higher eukaryotes, rather than a tight promoter-restricted function targeted by complex formation with specific transcription factors (Crump, 2011).
Dynamic acetylation of all H3K4me3 mediated by CBP is evolutionarily conserved, observed before the divergence of the single Drosophila enzyme dCBP into the paralogs p300 and CBP in mammals. CBP was discovered as a transcriptional coactivator that binds to CREB and p300 complements this activity. Whereas p300 and CBP are often considered functionally redundant, some studies support unique roles. The majority of genes bound by one also show high levels of the other, suggesting common targeting. They interact with many transcription factors and coactivators, initially suggesting a structural role in promoter complexes; p300 and CBP have been localized to c-fos and c-jun by ChIP and through interactions with other proteins. A purely structural role was challenged following discovery of their acetyltransferase activity with the catalytic domain required for transcription from chromatinized promoter constructs in vitro and in vivo. Recent in vitro studies with reconstituted nucleosome arrays show a requirement for p300 KAT activity to allow decompaction of 30 nm chromatin, nucleosome remodelling, and transcription factor binding (Crump, 2011).
Consistent with previous mass spectrometry and biochemical work, this study shows that H3K4me3 tails are dynamically modified up to the pentaacetylated state, including at lysines 9, 14, and 18, This suggests that the enzyme responsible, p300/CBP, targets specific H3 tails but no specific lysine. In p300/CBP double-knockout mouse fibroblasts, forskolin-induced acetylation of lysine 5, 8, 12, and 16 of histone H4 at c-fos is inhibited, further suggesting no specific targeting of residues. A high level of acetylation is insufficient for efficient gene expression in vivo; treatment of cells with TSA enhances acetylation but interferes with c-fos and c-jun induction. Further, loss of gene expression and Pol II localization caused by p300/CBP inhibition cannot be relieved by preacetylating nucleosomes before inhibition. This indicates a more dynamic role for acetylation in gene expression, suggesting that turnover may be the important factor. Analyses of quiescent cells in which c-fos and c-jun are poised but inactive and inhibition of transcription with DRB both indicate that transcription is not required for dynamic acetylation (Crump, 2011).
The finding that dynamic acetylation and H3K4me3 colocalize on the same nucleosomes across the promoter and start site of c-fos and c-jun raises the question of their cotargeting. Even when the KAT-HDAC enzyme balance is drastically forced in favor of acetylation by HDAC inhibitors, strict targeting to H3K4me3 does not break down. Numerous ChIP studies have established the presence of H3K4me3, H3K9ac, p300/CBP, and HDACs at the promoter and 5' end of many genes , suggesting widespread colocalization (Crump, 2011).
There are two classes of model by which cotargeting of H3K4me3 and rapid dynamic acetylation may occur. The first involves independent targeting to the same loci and H3 tails, as previously shown for serine-10 phosphorylation and lysine-9 acetylation. This suggests that the relevant enzymes may be part of a common process, and cotargeting may arise from independent DNA sequence recognition or unique interactions with the machinery of signal transduction and transcriptional regulation; p300 and CBP have been isolated in complexes containing TATA-binding protein (TBP) and RNA polymerase II (Crump, 2011).
A second class of model is based on dependence of one modification on the other. For example, p300/CBP may mediate dynamic acetylation through direct or indirect recognition of trimethylated lysine 4, which may provide a binding platform or enhance KAT activity. In support of this mechanism WDR5 knockdown, which depletes H3K4 methylation, attenuates the TSA-induced increase in H3K9ac levels at promoters. Other KATs are known to be recruited to H3K4me3 to induce histone acetylation; yeast Yng1 and Yng2, which recognize H3K4me3 via their PHD fingers, form part of the NuA3 and NuA4 KAT complexes, respectively, and mammalian ING4 links HBO1 acetyltransferase activity to H3 lysine-4 trimethylated nucleosomes (Crump, 2011).
A sequential targeting mechanism is conceivable. Unmethylated CpG dinucleotides within CpG islands may primarily recruit CXXC motif-containing proteins, including the H3K4 methyltransferase MLL1 (56) and CGBP/Cfp1, which associates with H3K4 methyltransferases. DNA binding by Cfp1 has recently been shown to restrict Setd1A and H3K4me3 to euchromatic nonmethylated CpG regions. Similarly, ChIP-seq analysis has shown a tight association between Cfp1 and H3K4me3 at CpG islands, and Cfp1 knockdown depletes H3K4me3 levels at nonmethylated CpGs. This provides a plausible mechanism to target H3K4me3 to these regions, which could then recruit p300/CBP for dynamic histone acetylation (Crump, 2011).
Transcription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)-especially in rapidly inducible systems-is largely unknown. Using fluorescence in situ hybridization and computational models, this study quantified the transcriptional activity of the proto-oncogene c-Fos with single mRNA accuracy at individual endogenous alleles. It was shown that, during MAPK induction, the TF concentration modulates the burst frequency of c-Fos, whereas other bursting parameters remain mostly unchanged. By using synthetic TFs with Transcription Activator-Like Effector (TALE) DNA-binding domains, different aspects of these bursts were systematically altered. Specifically, the polymerase initiation frequency was linked to the strength of the transactivation domain and the burst duration to the TF lifetime on the promoter. These results show how TFs and promoter binding domains collectively act to regulate different bursting parameters, offering a vast, evolutionarily tunable regulatory range for individual genes (Senecal, 2014. PubMed ID: 24981864).
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