Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease. +To whom correspondence should be addressed: oettinger@frodo.mgh.harvard.edu; ogozani@stanford.edu. * These authors contributed equally to the work Note added in proof: While this work was under review, another study also reported that the RAG2 PHD finger binds to methylated H3K4 30 .Atomic coordinates and structure factors of the RAG2 PHD -H3K4me3 peptide complex have been deposited in the Protein Data Bank with the accession code of 2v89. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Since RAG2 contains a noncanonical plant homeodomain (PHD) finger 6,7 -a module that can mediate interactions with chromatin 8-10 -we asked whether a polypeptide encompassing the RAG2 PHD finger (RAG2 PHD : aa 414-527) can recognize modified histone proteins. In an in vitro screen of peptide microarrays containing ~70 distinct modified histone peptides, we found that RAG2 PHD specifically binds to histone H3 trimethylated at lysine 4 (H3K4me3) ( Fig. 1a ; Fig. S1; data not shown). The specificity of this interaction was confirmed by peptide pulldown assays ( Fig. 1b ; Fig. S2; Fig. S3). RAG2 has a C-terminal extension of 40 aa that is essential for phosphoinositide (PtdInsP)-binding 7 (aa 488-527), but this region is dispensable for H3K4me3-binding as the minimal PHD finger alone (aa 414-487) is sufficient for H3K4me3-recognition (Fig. 1c). In addition, the acidic hinge region of RAG2 (aa 388-412), previously implicated in...

Cohesion between sister chromatids is established during S phase and maintained through G2 phase until it is resolved in anaphase (for review, see [1-3]). In Saccharomyces cerevisiae, a complex consisting of Scc1, Smc1, Smc3, and Scc3 proteins, called "cohesin," mediates the connection between sister chromatids. The evolutionary conserved yeast protein Eco1 is required for establishment of sister chromatid cohesion during S phase but not for its further maintenance during G2 or M phases or for loading the cohesin complex onto DNA. We address the molecular functions of Eco1 with sensitive sequence analytic techniques, including hidden Markov model domain fragment searches. We found a two-domain architecture with an N-terminal C2H2 Zn finger-like domain and an approximately 150 residue C-terminal domain with an apparent acetyl coenzyme A binding motif (http://mendel.imp.univie.ac.at/SEQUENCES/ECO1/). Biochemical tests confirm that Eco1 has the acetyltransferase activity in vitro. In vitro Eco1 acetylates itself and components of the cohesin complex but not histones. Thus, the establishment of cohesion between sister chromatids appears to be regulated, directly or indirectly, by a specific acetyltransferase.

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential proteinprotein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cellfree assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.

SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.Regulation of transcriptional elongation is a critical process in the control of viral and cellular gene expression (reviewed in references 3 and 28). A number of cellular factors that regulate transcriptional elongation have been defined using both biochemical and genetic techniques. These factors include the general transcription factors TFIIF and TFIIS, as well as other factors including the elongin and ELL proteins (20,41,48).In addition, cellular kinases play an important role in the control of transcriptional elongation based on their ability to phosphorylate the RNA polymerase II C-terminal domain (CTD) (27). One of these kinases, CDK-activating kinase (CAK), is composed of the CDK7 kinase in addition to cyclin H and MAT1. CAK is contained in the multiprotein TFIIH complex and is involved in modulating promoter clearance of specific promoters (13,45,47). A second kinase complex, PTEFb, is composed of cyclin T1 and CDK9 and also phosphorylates the RNA polymerase II CTD and stimulates transcriptional elongation (18,32,33,36,64). The Tat protein, which is a potent stimulator of transcriptional elongation, interacts with P-TEFb to stimulate human immunodeficiency virus type 1 (HIV-1) gene expression (4, 7, 17-19, 25, 26, 30, 31, 55, 56, 62, 64).SPT4 and SPT5 are highly conserved proteins which are present in a variety of species from yeast to humans and are involved in the regulation of transcriptional elongation (23,53,58,60,61). Genetic assays in yeast demonstrate that SPT5 conditional mutants can be suppressed by mutations in the genes encoding two largest subunits of RNA polymerase II (23). Furthermore, SPT5 interacts directly with RNA polymerase II via a domain in SPT5 that has homology to the Escherichia coli transcription elongation factor NusG (23, 53, 61). The human homologues of the SPT4 and SPT5 proteins have also been character...