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CBP and development A Caenorhabditis elegans gene closely related to CBP/p300, referred to as cbp-1, is required during early embryogenesis to specify several major differentiation pathways. There is extensive sequence conservation throughout the whole protein between CBP/p300 and CBP-1. The largest block of a highly conserved region between human CBP/p300 and CBP-1 spans amino acids 861-1757 of CBP-1. This region contains several important functional domains defined in CBP/p300 including the histone acetyltransferase (HAT) domain, the region that is critical for binding to E1A and the HAT, P/CAF, as well as the bromodomain and the C/H 2 domain that mediate p300 interaction with ATF-2. Between CBP and CBP-1, sequence comparison shows a 51% amino acid identity in the HAT domain and 70% in the region important for E1A and P/CAF interactions. The bromodomain and the C/H2 domain show 61% and 50% amino acid identity, respectively. Another functional domain that is highly conserved is the KIX domain of CBP, which binds transcription factor CREB; it shares 70% amino acid identity with CBP-1 (Shi, 1998).
CBP-1 protein is first detectable in nuclei beginning at the two-cell stage; this ubiquitous nuclear staining persists at least through the 100-cell stage of embryogenesis. The fact that CBP-1 protein is present at the two-cell stage, before the earliest detected zygotic transcription, suggests that CBP-1 is likely to be a maternally expressed protein in C. elegans (Shi, 1998).
A reverse genetic assay, termed RNAi, was used to determine the phenotypic consequences of blocking CBP-1 expression. Several recent studies have shown that an RNA injection procedure induces phenotypes that appear identical to those that are known to result from strong or null genetic mutations in the genes tested. These RNA-induced phenotypes have been correlated with a lack of protein expression from the targeted genes. For the RNAi assay, in vitro transcribed RNA from a cbp-1 cDNA clone was injected into wild-type adult hermaphrodites. Beginning a few hours after injection, wild-type hermaphrodites that receive cbp-1 RNA produce exclusively inviable embryos. Strikingly, these terminal embryos arrest development with nearly twice the normal complement of cells, and most embryos completely lack evidence of muscle, intestinal, and hypodermal differentiation. In contrast, the germ cells seem unaffected and appear morphologically normal, showing proper localizations for the germ-line-specific P-granules. The numerous small cells produced in these embryos most closely resemble neurons, which led to the hypothesis that many of the cells that would have differentiated into other tissues in normal embryos have instead adopted neuronal fates. Analysis of this phenotype suggests a critical role for CBP-1 in promoting all non-neuronal pathways of somatic differentiation in the C. elegans embryo (Shi, 1998).
Interfering with expression of HDAC1and RbAp48-related genes can partially suppress the differentiation defects of worms subject to the RNAi treatment. C. elegans hda-1 is related to the mammalian HDAC1 component of histone deacetylase. Two open reading frames were identified that share extensive sequence homology with RbAp48, RbAp46, an RbAp48-related protein in mammalian cells, and with p55, the RbAp48 homolog in Drosophila (known as Chromatin assembly factor 1 subunit). These two C. elegans genes are referred to as rba-1 and rba-2, for RbAp48-related genes. RBA-1 shares 53% amino acid identity and 63% amino acid similarity with RbAp48, 52% and 63% with RbAp46, and 53% and 62% with Drosophila Chromatin assembly factor 1 subunit. RBA-2 shares 72% amino acid identity and 79% amino acid similarity with RbAp48, 71% and 80% with RbAp46, and 72% and 81% with Drosophila Chromatin assembly factor 1 subunit (Shi, 1998).
The endoderm differentiation defects observed in embryos subject to cbp-1 RNAi treatment are similar to those caused by mutations in the transcription factor SKN-1, a maternally expressed gene required to specify the fate of ventral blastomeres in C. elegans embryos. This suggests that CBP-1 may function with SKN-1 or downstream transcription factors to specify endoderm differentiation. Such a hypothesis would predict that inhibiting HDA-1 expression in the skn-1 mutant background might suppress the skn-1 mutant phenotype. Endoderm differentiation is restored completely by inhibition of hda-1 expression in skn-1 mutant embryos. Similarly, inhibiting the expression of either rba-1 or rba-2 also completely restored endoderm differentiation in the skn-1 mutant. These findings suggest a model in which CBP-1 may activate transcription and differentiation in C. elegans by directly or indirectly antagonizing a repressive effect for histone deacetylase (Shi, 1998).
Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of
both muscle-specific and ubiquitous genes. Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding
protein (CBP)/p300, are crucial to this process. While both possess histone acetyl-transferase (HAT) activity, previous studies have
failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. This issue has been addressed directly using a
chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program (Polesskaya, 2001).
The coactivators CBP and its paralog p300 are thought to supply adaptor molecule and protein acetyltransferase functions to many transcription factors that regulate gene expression. Normal development requires CBP and p300, and mutations in these genes are found in hematopoietic and epithelial tumors. It is unclear, however, which functions of CBP and p300 are essential in vivo. The protein-binding KIX domains of CBP and p300 have nonredundant functions in mice. In mice homozygous for point mutations in the KIX domain of p300 designed to disrupt the binding surface for the transcription factors c-Myb and CREB, multilineage defects occur in hematopoiesis, including anaemia, B-cell deficiency, thymic hypoplasia, megakaryocytosis and thrombocytosis. By contrast, age-matched mice homozygous for identical mutations in the KIX domain of CBP are essentially normal. There is a synergistic genetic interaction between mutations in c-Myb and mutations in the KIX domain of p300, which suggests that the binding of c-Myb to this domain of p300 is crucial for the development and function of megakaryocytes. Thus, conserved domains in two highly related coactivators have contrasting roles in hematopoiesis (Kasper, 2002).
Impaired terminal differentiation is found in interfollicular keratinocytes of p107/p130-double-null mice epidermis. A decreased number of hair follicles and a clear developmental delay is seen in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice show altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, are also observed in the p107/p130 skin transplants, in addition to normal hair formation. Finally, the hair abnormalities in p107/p130-null skin are associated with altered Bmp4-dependent signaling, including decreased DeltaNp63 expression, an ectodermal specific Bmp4 target. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions (Ruiz, 2003).
The stabilization of learned information into long-term memories requires new gene expression. CREB binding protein (CBP) is a coactivator of transcription that can be independently regulated in neurons. CBP functions both as a platform for recruiting other required components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. To dissect the chromatin remodeling versus platform function of CBP or the developmental versus adult role of this gene, transgenic mice were generated that express CBP in which HAT activity is eliminated. Acquisition of new information and short-term memory still occurs in these mice, while the stabilization of short-term memory into long-term memory is impaired. The behavioral phenotype is due to an acute requirement for CBP HAT activity in the adult; it is rescued by both suppression of transgene expression or by administration of the histone deacetylase inhibitor Trichostatin A (TSA) in adult animals (Korzus, 2004).
It is suggested that CBP acetyltransferase activity is critical for activation of genes controlling memory consolidation. It is well known that the transcription factor CREB is required for long-term memory consolidation and that CREB phosphorylation at S133 is necessary to recruit CBP and for subsequent transcriptional activation. However, behaviorally induced CREB phosphorylation is transient and does not correlate with peak c-fos induction. Moreover, S133 phosphorylation of CREB alone is not sufficient to induce transcription. NMDA-dependent phosphorylation of S301 on CBP has been also shown to be required for CBP-dependent gene expression. This suggests that a separate signaling pathway directly to CBP must be activated in neurons to allow normal CREB-mediated gene activation, as well as other non-CREB-dependent genes that use CBP as a coactivator. The current results suggest that this CBP pathway is also critical for memory consolidation by recruitment of functional CBP histone acetyltransferase activity and possible alteration of local chromatin structure. In addition to its chromatin remodeling function, it is possible that nuclear nonhistone substrates for CBP acetyltransferase activity might be critical during memory consolidation. This activation of CBP-mediated acetylation at specific gene targets could serve to alter the subsequent requirements for transcriptional activation of those genes in response to future cellular signals. This sort of the acetylation-mediated covalent modification would open up a temporal window in which cellular signals, which did not recruit an active acetyltransferase to the promoter, would nevertheless stimulate transcription as shown in Figure 6C. It is possible that after initial CBP-mediated integration of short-lasting neuronal signals leading to acetylation-dependent alteration of local chromatin structure, CBP HAT or even CBP platform function may become dispensable for subsequent steps. In fact, CBP-independent activation of CREB (or CREM, a CREB family member), which is mediated by other coactivators and bypasses the classical requirements for phosphorylation of CREB/CREM and interaction with CBP, has been described. This could allow for prolonged elevation of transcription in response to an initial learning event by maintaining transcription even after signals to CREB and CBP were no longer present (Korzus, 2004).
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