The PHD Type Zinc Finger Is an Integral Part of the CBP Acetyltransferase Domain

Abstract

Histone acetyltransferases (HATs) such as CBP and p300 are regarded as key regulators of RNA polymerase II-mediated transcription, but the critical structural features of their HAT modules remain ill defined. The HAT domains of CBP and p300 are characterized by the presence of a highly conserved putative plant homeodomain (PHD) (C4HC3) type zinc finger, which is part of the functionally uncharacterized cysteinehistidine-rich region 2 (CH2). Here we show that this region conforms to the PHD type zinc finger consensus and that it is essential for in vitro acetylation of core histones and the basal transcription factor TFIIE34 as well as for CBP autoacetylation. PHD finger mutations also reduced the transcriptional activity of the full-length CBP protein when tested on transfected reporter genes. Importantly, similar results were obtained on integrated reporters, which reflect a more natural chromatinized state. Taken together, our results indicate that the PHD finger forms an integral part of the enzymatic core of the HAT domain of CBP.Regulation at the level of transcription is the central mechanism by which cells respond to developmental and environmental cues. RNA polymerase II-mediated transcription in eukaryotes is to a large extent regulated at the level of chromatin, which forms a physical barrier against the binding of proteins to the promoter region of a target gene. The basic unit of chromatin is the nucleosome, which consists of an octamer of histone proteins around which the DNA is wrapped. The nucleosomes form an array that is further organized into higher-order chromatin structures. The two major enzymatic activities which make the DNA more accessible for the transcription machinery are the ATP-dependent chromatin remodeling complexes and histone acetyltransferases (HATs) (15,32,39). The prime targets of the HATs in chromatin are the N-terminal tails of the core histones H2A, H2B, H3, and H4. Acetylation of these tails results in neutralization of the positively charged lysines, thereby influencing DNA-histone and histonehistone contacts. Furthermore, posttranslational histone modifications including acetylation can create specific docking sites for regulatory proteins (64). This so-called histone code may also be involved in the establishment of epigenetic inheritance (62). In addition to their ability to acetylate histones, some HATs have also been shown to acetylate nonhistone proteins, including several sequence-specific transcription factors and the basal transcription factors TFIIE and TFIIF (reviewed in reference 60).The acetyltransferases CREB binding protein (CBP) and the related protein p300 function as coactivators for a multitude of transcription factors (9, 23, 58, 63), including CREB (5,16,38). This coactivator function is not only due to their intrinsic HAT activity but is also the result of their ability to interact with other HAT proteins such as pCAF and SRC1 family members. Furthermore, CBP and p300 can stabilize the transcription complex by binding to several proteins simultaneous...

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice

“…Thus, the RAG2 C terminus might directly facilitate changes in chromatin structure or, more likely, facilitate assembly of the RAG1͞2 complex on RSSs wrapped in chromatin. The association of other PHD-containing proteins with chromatin remodeling and modification (21)(22)(23)(24) is consistent with such a role for RAG2 in influencing the accessibility of antigenreceptor loci. In this context, the PHD motif of RAG2 might serve a unique, undefined function in promoting accessibility of V H and V␤ segments.…”

“…Consistent with this notion, studies of Abelson murine leukemia virus-transformed pre-B cells have suggested that the noncore regions of RAG1 are important for IgH locus D H -to-J H rearrangement (17), whereas the C terminus of RAG2 is more important for V H -to-DJ H rearrangement than for D H -to-J H rearrangement (18). The C terminus of RAG2 is predicted to fold into a plant homeodomain (PHD) (19,20), a motif found in a number of chromatin-associated proteins including some that have chromatin-modifying activities (21)(22)(23)(24).…”

Sirtuins: critical regulators at the crossroads between cancer and aging

“…The balance between acetylation and deacetylation of specific factors is regulated at an additional level, since SIRT1 itself deacetylates several HATs and regulates their enzymatic activities (Kalkhoven et al, 2002;Thompson et al, 2004). SIRT1 directly interacts with PCAF and p300 to promote their deacetylation and enzymatic inactivation (Fulco et al, 2003;Motta et al, 2004;Cohen et al, 2004a).…”

FOXO4 Is Acetylated upon Peroxide Stress and Deacetylated by the Longevity Protein hSir2

“…pRc/RSV-CBP-HA and pRP265-GST-p300-HAT were kindly provided by R. Giles (20) and R. Vries (21), respectively. Gal4-DBD-CBP-HA constructs were a kind gift of E. Kalkhoven (22). pRL-TK (Tk-Renilla luciferase) was purchased from Promega.…”