Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity.

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G(+4) base pair for the wild-type A:T(+4) base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T(+4) were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T(+4) or the C:G(+4) base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G(+4) recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T(+4) target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G(+4) target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G(+4) substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity.

The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high-specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3'-overhangs of 4 bp. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially. A novel hairpin substrate was used to demonstrate that although wild-type I-SceI cleaves either the top or bottom DNA strand first to generate two nicked DNA intermediates, the enzyme has a preference for cleaving the bottom strand. The kinetics data are consistent with a parallel sequential reaction mechanism. Substitution of two pseudo-symmetric residues, Lys122 and Lys223, markedly reduces top and bottom-strand cleavage, respectively, to generate enzymes with significant strand- and sequence-specific nicking activity. The two active sites are partially interdependent, since alterations to one site affect the second. The kinetics analysis is consistent with X-ray crystal structures of I-SceI/DNA complexes that reveal a role for the lysines in establishing important solvent networks that include nucleophilic water molecules thought to attack the scissile phosphodiester bonds.

Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling.

The Rtf1 subunit of the Paf1 complex is required for proper monoubiquitination of histone H2B and methylation of histone H3 on lysines 4 (H3K4) and 79 in yeast Saccharomyces cerevisiae. Using RNAi, we examined the role of Rtf1 in histone methylation and gene expression in Drosophila melanogaster. We show that Drosophila Rtf1 (dRtf1) is required for proper gene expression and development. Furthermore, we show that RNAi-mediated reduction of dRtf1 results in a reduction in histone H3K4 trimethylation levels on bulk histones and chromosomes in vivo, indicating that the histone modification pathway via Rtf1 is conserved among yeast, Drosophila, and human. Recently, it was demonstrated that histone H3K4 methylation mediated via the E3 ligase Bre1 is critical for transcription of Notch target genes in Drosophila. Here we demonstrate that the dRtf1 component of the Paf1 complex functions in Notch signaling.

A COMPASS in the voyage of defining the role of trithorax/MLL-containing complexes: linking leukemogensis to covalent modifications of chromatin.

Chromosomal rearrangements and translocations play a major role in the pathogenesis of hematological malignancies. The trithorax-related mixed lineage leukemia (Mll) gene located on chromosome 11 is rearranged in a variety of aggressive human B and T lymphoid tumors as well as acute myeloid leukemia (AML) in both children and adults. It was first demonstrated for the yeast MLL homolog complex, Set1/COMPASS, and now for the MLL complex itself, that these complexes are histone methyltransferases capable of methylating the fourth lysine of histone H3. The post-translational modifications of histones by methylation have emerged as a key regulatory mechanism for both repression and activation of gene expression. Studies from several laboratories during the past few years have brought about a watershed of information defining the molecular machinery and factors involved in the recognition and modification of nucleosomal histones by methylation. In this review, we will discuss the recent findings regarding the molecular mechanism and consequences of histone modification by the MLL related protein containing complex COMPASS.

Regulation of heat shock gene expression by RNA polymerase II elongation factor, Elongin A.

The elongation stage of transcription by RNA polymerase II (Pol II) has emerged as an essential regulated step. Elongin A (EloA) is the largest subunit of the Elongin complex that can increase the catalytic rate of mRNA synthesis by Pol II. We recently demonstrated that the Elongin A homologue in Drosophila, dEloA, is essential and has properties consistent with those of a Pol II elongation factor in vivo. The goal of this study was to test whether dEloA is required for heat shock gene transcription, since heat shock gene expression is thought to be controlled at the level of Pol II elongation. Here, we demonstrate that dEloA is rapidly recruited to heat shock loci with Pol II in response to heat shock. Furthermore, through the use of RNA interference in vivo, we show that dEloA is required for the proper expression of one of these genes, HSP70, and that its requirement for heat shock gene expression is exerted after the initiation of transcription at heat shock loci. Our data represent the first demonstration of an essential role for an RNA polymerase II elongation factor in the regulation of heat shock gene expression in an animal model.

In vivo requirement of the RNA polymerase II elongation factor elongin A for proper gene expression and development.

A number of transcription factors that increase the catalytic rate of mRNA synthesis by RNA polymerase II (Pol II) have been purified from higher eukaryotes. Among these are the ELL family, DSIF, and the heterotrimeric elongin complex. Elongin A, the largest subunit of the elongin complex, is the transcriptionally active subunit, while the smaller elongin B and C subunits appear to act as regulatory subunits. While much is known about the in vitro properties of elongin A and other members of this class of elongation factors, the physiological role(s) of these proteins remain largely unclear. To elucidate in vivo functions of elongin A, we have characterized its Drosophila homologue (dEloA). dEloA associates with transcriptionally active puff sites within Drosophila polytene chromosomes and exhibits many of the expected biochemical and cytological properties consistent with a Pol II-associated elongation factor. RNA interference-mediated depletion of dEloA demonstrated that elongin A is an essential factor that is required for proper metamorphosis. Consistent with this observation, dEloA expression peaks during the larval stages of development, suggesting that this factor may be important for proper regulation of developmental events during these stages. The discovery of the role of elongin A in an in vivo model system defines the novel contribution played by RNA polymerase II elongation machinery in regulation of gene expression that is required for proper development.