Just the FACTs: histone H2B ubiquitylation and nucleosome dynamics.

In this issue of Molecular Cell, Fleming et al. (2008) show that histone H2B ubiquitylation and FACT function interdependently to facilitate nucleosome reassembly during transcription elongation, thereby demonstrating that histone posttranslational modifications can provide important but transient transcriptional signaling cues.

A key role for Chd1 in histone H3 dynamics at the 3' ends of long genes in yeast.

Chd proteins are ATP-dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5' and 3' ends of genes, accelerating H3 replacement at the 5' ends of genes while protecting the 3' ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3' ends of genes.

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle.

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.

Protein interacting with C-kinase 1/protein kinase Calpha-mediated endocytosis converts netrin-1-mediated repulsion to attraction.

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.

Structural determination of NSC 670224, synthesis of analogues and biological evaluation.

Follow my lead! NSC 670224, previously shown to be toxic to Saccharomyces cerevisiae at low micromolar concentrations, potentially acts via a mechanism of action related to that of tamoxifen (NSC 180973), breast cancer drug. The structure of NSC 670224, previously thought to be a 2,4-dichloro arene, was established as the 3,4-dichloro arene, and a focused library of analogues were synthesized and biologically evaluated.

Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5.

Pre-mRNA binding to the yeast U2 small nuclear ribonucleoprotein (snRNP) during prespliceosome formation requires ATP hydrolysis, the highly conserved UACUAAC box of the branch point region of the pre-mRNA, and several factors. Here we analyzed the binding of a radiolabeled 2'-O-methyl oligonucleotide complementary to U2 small nuclear RNA to study interactions between the UACUAAC box, U2 snRNP, and Prp5p, a DEAD box protein necessary for prespliceosome formation. Binding of the 2'-O-methyl oligonucleotide to the U2 snRNP in yeast cell extract was assayed by gel electrophoresis. Binding was rapid, enhanced by ATP, and dependent on the integrity and conformation of the U2 snRNP. It was also stimulated by Prp5p that was found to associate physically with U2 snRNP. In vitro heat inactivation of the temperature-sensitive prp5-1 mutant extract decreased oligonucleotide binding to U2 and the ATP enhancement of binding by 3-fold. Furthermore, the temperature-sensitive prp5-1 mutation maps to the ATP-binding motif I within the helicase-like domain. Thus the catalytic activity of Prp5p likely promotes a conformational change in the U2 snRNP.