Identification of Fkh1 and Fkh2 binding site variants associated with dynamically bound DNA elements including replication origins.

Forkhead Box (Fox) DNA binding proteins control multiple genome activities, including transcription, replication, and repair. These activities are organized spatially and temporally in the nucleus, and Fox proteins Fkh1 and Fkh2 have emerged as regulators of long-range chromosomal interactions involved with these activities, such as the clustering of replication origins programmed for early initiation. Fkh1 and Fkh2 bind a subset of replication origins and are thought to dimerize to mediate long-range chromosomal contacts between these origins. The binding of Fkh1 and/or Fkh2 (Fkh1/2) to replication origins and the recombination enhancer (RE), which is involved in DNA repair required for mating-type switching, is cell cycle-regulated and thus appears to be more dynamic than Fkh1/2 binding at regulated target genes. Here we report the identification of Fkh1/2 binding sequence variants at replication origins and the RE compared with Fkh1/2 binding sequences found at target genes of the CLB2 group. These different binding sequences have previously been characterized as weak and strong, respectively, suggesting that the presence of weak sites contributes to more dynamic interactions at replication origins and RE, possibly facilitated by Fkh1/2 dimerization and cooperative interactions with accessory proteins. We discuss the wealth of regulatory potential imbued in these features of the DNA and its binding proteins.

Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.

Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.

ChIP-Seq to Analyze the Binding of Replication Proteins to Chromatin.

Chromatin immunoprecipitation (ChIP) is a widely used method to study interactions between proteins and discrete chromosomal loci in vivo. ChIP was originally developed for in vivo analysis of protein associations with candidate DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays enabled the unbiased, genome-scale identification of all DNA sequences enriched by ChIP, providing a genomic map of a protein's chromatin binding. This method, termed ChIP-chip, is broadly applicable and has been particularly valuable in DNA replication studies to map potential replication origins in Saccharomyces cerevisiae and other organisms based on the specific association of certain replication proteins with these chromosomal elements, which are distributed throughout the genome. More recently, high-throughput sequencing (HTS) technologies have replaced microarrays as the preferred method for genomic analysis of ChIP experiments, and this combination is termed ChIP-Seq. We present a detailed ChIP-Seq protocol for S. cerevisiae that can be adapted for different HTS platforms and for different organisms. We also outline general schemes for data analysis; however, HTS data analyses usually must be tailored specifically for individual studies, depending on the experimental design, data characteristics, and the genome being analyzed.

Fkh1 and Fkh2 bind multiple chromosomal elements in the S. cerevisiae genome with distinct specificities and cell cycle dynamics.

Forkhead box (FOX) transcription factors regulate a wide variety of cellular functions in higher eukaryotes, including cell cycle control and developmental regulation. In Saccharomyces cerevisiae, Forkhead proteins Fkh1 and Fkh2 perform analogous functions, regulating genes involved in cell cycle control, while also regulating mating-type silencing and switching involved in gamete development. Recently, we revealed a novel role for Fkh1 and Fkh2 in the regulation of replication origin initiation timing, which, like donor preference in mating-type switching, appears to involve long-range chromosomal interactions, suggesting roles for Fkh1 and Fkh2 in chromatin architecture and organization. To elucidate how Fkh1 and Fkh2 regulate their target DNA elements and potentially regulate the spatial organization of the genome, we undertook a genome-wide analysis of Fkh1 and Fkh2 chromatin binding by ChIP-chip using tiling DNA microarrays. Our results confirm and extend previous findings showing that Fkh1 and Fkh2 control the expression of cell cycle-regulated genes. In addition, the data reveal hundreds of novel loci that bind Fkh1 only and exhibit a distinct chromatin structure from loci that bind both Fkh1 and Fkh2. The findings also show that Fkh1 plays the predominant role in the regulation of a subset of replication origins that initiate replication early, and that Fkh1/2 binding to these loci is cell cycle-regulated. Finally, we demonstrate that Fkh1 and Fkh2 bind proximally to a variety of genetic elements, including centromeres and Pol III-transcribed snoRNAs and tRNAs, greatly expanding their potential repertoire of functional targets, consistent with their recently suggested role in mediating the spatial organization of the genome.

Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae.

The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1 phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization.

Droperidol for acute migraine headache.

The use of intramuscular droperidol to treat acute migraine headache has not been previously reported in the emergency medicine literature. It is a promising therapy for migraine. The authors performed a pilot review of all patients receiving droperidol for migraine in our emergency department (ED) to evaluate its efficacy. We used a retrospective case series, in a suburban ED with an annual patient census of 48,000. All patients with a discharge diagnosis of migraine headache who were treated with i.m. droperidol during a consecutive 5-month period in our ED were identified. All patients received droperidol 2.5 mg intramuscular. As per ED protocol, their clinical progress was closely followed and documented at 30 minutes after drug administration (t30). Demographic and clinical variables were recorded on a standardized, closed-question, data collection instrument. The primary outcome measurement was relief of symptoms at t30 to the point that the patient felt well enough to go home without further ED intervention (symptomatic relief). Thirty-seven patients were treated (84% female), with an ED diagnosis of acute migraine with droperidol during the study period. The mean age was 36 +/- 12 years. Analgesics had been used within 24 hours before ED presentation by 62% of patients. At t30, 30 (81%) patients had symptomatic relief, 2 (5%) felt partial relief but required rescue medication, and 5 (14%) had no relief of symptoms. Drowsiness (14%) and mild akathisia (8%) were the only adverse reactions observed following drug administration. Droperidol 2.5 mg intramuscular may be a safe and effective therapy for the ED management of acute migraine headache. Randomized, controlled trials are warranted to further validate the findings of this preliminary study.

The age grading of physical activity among children.

To test the hypothesis that children view participation in physical activity as less appropriate among adults of increasing chronological age, pre-school children (N = 51 males; N = 51 females) were shown six facial pictures of two adults who had been aged fictitiously through make-up, and were asked to rate the competence of these adults on six physical activities. The most significant finding of the study was that these children's assessments of the competence of adult participation in physical activity was, to a large extent, dictated by their perceptions of the age of the adult. Adults perceived as more advanced in chronological age were viewed as less proficient at each physical activity. The children's chronological age, gender, or motor skill level did not interact with their responses to the pictures.