We previously established that the phage C31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.For the past 25 years, it has been possible to construct precisely designed DNA molecules in the test tube thanks to the techniques of recombinant DNA. In contrast, the ability to make controlled and efficient alterations in the genomes of living higher cells has been limited. The use of site-specific recombinases such as Cre and FLP provided an important advance (17), but because of the reversibility of these enzyme reactions, their main utility has been for creating deletions. For the integration of new material into the genome, fortuitous integration of transfected DNA is most often used, and it produces integration at random locations at low frequency. Homologous recombination provides site specificity, but at very low efficiency (26).We began working with another site-specific recombinase, the phage C31 integrase, because it offered the potential for unidirectional integration that would therefore occur at higher net frequencies than the reversible integration directed by recombinases, such as Cre. Cre recombines two identical loxP sites, recreating two identical sites after recombination that can undergo a subsequent round of recombination. In contrast, the attB and attP recognition sites recognized by the C31 integrase are dissimilar in sequence (15). After reaction, the recombined att sites differ from attB and attP and are refractory to further synapsis by the integrase, thus locking in integration reactions (23,24). We demonstrated that this enzyme, derived from a Streptomyces phage (9), worked well in the human cell environment (7), consistent with its lack of cofactor requirements (23). This feature distinguishes it from better known phage integrases, such as that of phage , which does require cofactors (10). The integrase is in the family of recombinases that includes Cre and FLP and carries out a tyrosine-mediated strand exchange (4, 13). The C31 integrase is in the other major family of site-specific recombinases that includes many resolvases and invertases and uses a serine-catalyzed reaction mechanism (20). The two site-specific recombinase families are unrelated. The C31 integrase is a member of a recently discovered subclass of the serine recombinase family whose members are especia...

Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are co-transcriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing co-transcriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is a key determinant of nucleosome eviction.

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.Nucleosomes inhibit transcription initiation by RNA polymerase II (Pol II) by impeding assembly of the preinitiation complex (PIC) at the promoter. Transcriptional activators bind to upstream activation sequence (UAS) elements and recruit ATP-dependent chromatin remodeling complexes to displace or evict nucleosomes from promoter regions as a means of stimulating PIC assembly. Neutralization of the positive charges on Lys residues by acetylation weakens histone interactions with DNA. Moreover, acetylation provides recognition sites for subunits of remodeling complexes that harbor bromodomains (BDs). This enables KAT complexes to enhance nucleosome displacement by remodeling complexes in vitro, and it may underlie their roles in histone eviction from promoters in vivo (4,22,29,53).Nucleosomes also impede elongation by Pol II, and histones are evicted from coding sequences in a manner directly correlated with the transcription rate (51, 60). The mechanism of cotranscriptional nucleosome eviction is not well understood. The histone chaperone Asf1 and the chromatin remodeling complex SWI/SNF have been implicated in nucleosome disassembly during elongation in Saccharomyces cerevisiae cells (50, 52). As during initiation, histone acetylation by KAT complexes could stimulate the passage of elongating Pol II by altering DNA-histone contacts (42) or by enhancing recruitment of chromatin remodeling complexes, as demonstrated for RSC and its ability to facilitate elongation through a reconstituted mononucleosome in vitro (9). This elongation-promoting activity of histone acetylation is not well documented in vivo, however, and until recently it was thought that KAT complexes are confined to the UAS and promoter regions.We and others have found that the H3 KAT complex SAGA occupies the coding sequences of tra...

We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.Prokaryotic enzymes have supplied us with abundant tools for engineering DNA. For example, restriction enzymes and ligases, largely derived from bacterial and phage genomes, provided the tools for recombinant DNA. This technology has allowed construction of molecules at will in vitro, causing a wholesale transformation of biomedical science over the past 25 years. More recently, in vivo engineering of the genomes of living higher eukaryotic cells is becoming possible, often through the agency of prokaryotic enzymes such as Cre, an autonomous, site-specific, tyrosine-catalyzed recombinase from phage P1 (1). Recombinases such as Cre and FLP require no host-specific cofactors and perform well in higher eukaryotic cells, carrying out efficient site-specific recombination between two identical recognition sites (18,22). These enzymes are useful for carrying out deletion and translocation-type recombination reactions in living cells (21).Another useful reaction is integration for the purpose of creating knockin and knockout alterations of the genome, such as those desirable in gene therapy, creation of transgenic organisms, and modification of cells in culture. For integration, a unidirectional recombinase such as a phage integrase is ideal, because there is no reverse reaction that could depress net integration frequency (9). Phage integrases mediate recombination between nonidentical phage attP and bacterial attB recognition sites (13). The well-studied lambda integrase is, like Cre and FLP, a member of the tyrosine-catalyzed recombinase family (17). However, the integrases from lambda phage and the closely related phage HK022 have cofactor requirements that hamper their use in eukaryotic cells (11,15).Some phage integrases are members of the unrelated serinecatalyzed family of recombinases (24) and are autonomous with no cofactor requirements, which makes them potentially ideal for use in foreign host environments, such as mammalia...

Background: Histone H3 methylation on lysines 4 and 36 stimulates interaction between histone deacetylase complexes and chromatin. Results: The NuA4 lysine acetyltransferase complex also binds to methylated H3 and stimulates nucleosomal binding and H3 acetylation by SAGA. Conclusion: Histone H3 methylation stimulates both nucleosomal acetylation and deacetylation. Significance: H3 methylation is key in properly regulating the level of acetylation at a transcribed gene.

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NuA4 is the only essential lysine acetyltransferase complex in Saccharomyces cerevisiae, where it has been shown to stimulate transcription initiation and elongation. Interaction with nucleosomes is stimulated by histone H3 Lys-4 and Lys-36 methylation, but the mechanism of this interaction is unknown. Eaf3, Eaf5, and Eaf7 form a subcomplex within NuA4 that may also function independently of the lysine acetyltransferase complex. The Eaf3/5/7 complex and the Rpd3C(S) histone deacetylase complex have both been shown to bind di- and trimethylated histone H3 Lys-36 stimulated by Eaf3. We investigated the role of the Eaf3/5/7 subcomplex in NuA4 binding to nucleosomes. Different phenotypes of eaf3/5/7Δ mutants support functions for the complex as both part of and independent of NuA4. Further evidence for Eaf3/5/7 within NuA4 came from mutations in the subcomplex leading to ∼40% reductions in H4 acetylation in bulk histones, probably caused by binding defects to both nucleosomes and RNA polymerase II. In vitro binding assays showed that Eaf3/5/7 specifically stimulates NuA4 binding to di- and trimethylated histone H3 Lys-36 and that this binding is important for NuA4 occupancy in transcribed ORFs. Consistent with the role of NuA4 in stimulating transcription elongation, loss of EAF5 or EAF7 resulted in a processivity defect. Overall, these results reveal the function of Eaf3/5/7 within NuA4 to be important for both NuA4 and RNA polymerase II binding.

Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.