Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data.

Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein-DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a "sensor" for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2.

DNA binding specificity of all four Saccharomyces cerevisiae forkhead transcription factors.

Quantifying the nucleotide preferences of DNA binding proteins is essential to understanding how transcription factors (TFs) interact with their targets in the genome. High-throughput in vitro binding assays have been used to identify the inherent DNA binding preferences of TFs in a controlled environment isolated from confounding factors such as genome accessibility, DNA methylation, and TF binding cooperativity. Unfortunately, many of the most common approaches for measuring binding preferences are not sensitive enough for the study of moderate-to-low affinity binding sites, and are unable to detect small-scale differences between closely related homologs.

Dbf4 Zn-Finger Motif Is Specifically Required for Stimulation of Ctf19-Activated Origins in .

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system , two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins.

Rpd3 regulates single-copy origins independently of the rDNA array by opposing Fkh1-mediated origin stimulation.

Eukaryotic chromosomes are organized into structural and functional domains with characteristic replication timings, which are thought to contribute to epigenetic programming and genome stability. Differential replication timing results from epigenetic mechanisms that positively and negatively regulate the competition for limiting replication initiation factors. Histone deacetylase Sir2 negatively regulates initiation of the multicopy (∼150) rDNA origins, while Rpd3 histone deacetylase negatively regulates firing of single-copy origins.

Multimodality approach to the nipple-areolar complex: a pictorial review and diagnostic algorithm.

The anatomic and histologic characteristics of the nipple-areolar complex make this breast region special. The nipple-areolar complex can be affected by abnormal development and a wide spectrum of pathological conditions, many of which have unspecific clinical and radiological presentations that can present a challenge for radiologists. The nipple-areolar complex requires a specific imaging workup in which a multimodal approach is essential.

Dynamic relocalization of replication origins by Fkh1 requires execution of DDK function and Cdc45 loading at origins.

Chromosomal DNA elements are organized into spatial domains within the eukaryotic nucleus. Sites undergoing DNA replication, high-level transcription, and repair of double-strand breaks coalesce into foci, although the significance and mechanisms giving rise to these dynamic structures are poorly understood. In , replication origins occupy characteristic subnuclear localizations that anticipate their initiation timing during S phase.

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.

Quantitative Bromodeoxyuridine Immunoprecipitation Analyzed by High-Throughput Sequencing (qBrdU-Seq or QBU).

Incorporation into DNA of nucleoside analogs like 5-bromo-2'-deoxyuridine (BrdU) is a powerful tool for in vivo studies of DNA synthesis during replication and repair. Immunoprecipitation of BrdU-labeled DNA analyzed by DNA sequencing (BrdU-IP-seq) allows for genome-wide, sequence-specific tracking of replication origin and replication fork dynamics under different conditions, such as DNA damage and replication stress, and in mutant strains. We have recently developed a quantitative method for BrdU-IP-seq (qBrdU-seq) involving DNA barcoding to enable quantitative analysis of multiple experimental samples subjected to BrdU-IP-seq.

Reconstructing cell cycle pseudo time-series via single-cell transcriptome data.

Single-cell mRNA sequencing, which permits whole transcriptional profiling of individual cells, has been widely applied to study growth and development of tissues and tumors. Resolving cell cycle for such groups of cells is significant, but may not be adequately achieved by commonly used approaches. Here we develop a traveling salesman problem and hidden Markov model-based computational method named reCAT, to recover cell cycle along time for unsynchronized single-cell transcriptome data.

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

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 Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G 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.

Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.

The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions.

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.

Two-dimensional agarose gel electrophoresis for analysis of DNA replication.

The initiation, elongation, and termination of DNA replication are each associated with distinct, nonlinear DNA structures that can be resolved and identified by two-dimensional (2D) agarose gel electrophoresis. This method involves: isolation of genomic DNA while preserving fragile replication structures, digestion of the DNA with a restriction enzyme, separation of DNA by size and shape through two distinct stages of agarose gel electrophoresis, and Southern blotting to probe for the specific sequence(s) of interest. The method has been most commonly used to determine the activity level of putative replication origin-containing sequences, and has also been used to analyze replication timing, fork progression, fork pausing, fork stalling and collapse, termination, and recombinational repair.

Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome.

Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors.

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.

The level of origin firing inversely affects the rate of replication fork progression.

DNA damage slows DNA synthesis at replication forks; however, the mechanisms remain unclear. Cdc7 kinase is required for replication origin activation, is a target of the intra-S checkpoint, and is implicated in the response to replication fork stress. Remarkably, we found that replication forks proceed more rapidly in cells lacking Cdc7 function than in wild-type cells.

Location, location, location: it's all in the timing for replication origins.

The differential replication timing of eukaryotic replication origins has long been linked with epigenetic regulation of gene expression and more recently with genome stability and mutation rates; however, the mechanism has remained obscure. Recent studies have shed new light by identifying novel factors that determine origin timing in yeasts and mammalian cells and implicate the spatial organization of origins within nuclear territories in the mechanism. These new insights, along with recent findings that several initiation factors are limiting relative to licensed origins, support and shape an emerging model for replication timing control.

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.

Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair.

Adenovirus virus-associated (VA) RNAs are processed to functional viral miRNAs or mivaRNAs. mivaRNAs are important for virus production, suggesting that they may target cellular or viral genes that affect the virus cell cycle. To look for cellular targets of mivaRNAs, we first identified genes downregulated in the presence of VA RNAs by microarray analysis.

Strategies for analyzing highly enriched IP-chip datasets.

Background: Chromatin immunoprecipitation on tiling arrays (ChIP-chip) has been employed to examine features such as protein binding and histone modifications on a genome-wide scale in a variety of cell types. Array data from the latter studies typically have a high proportion of enriched probes whose signals vary considerably (due to heterogeneity in the cell population), and this makes their normalization and downstream analysis difficult.

Results: Here we present strategies for analyzing such experiments, focusing our discussion on the analysis of Bromodeoxyruridine (BrdU) immunoprecipitation on tiling array (BrdU-IP-chip) datasets.

To promote and protect: coordinating DNA replication and transcription for genome stability.

Duplication of the eukaryotic genome occurs in the context of a chromatin template that is actively engaged in regulating gene expression and other protein-DNA interactions. Chromatin structures influence the dynamics of DNA replication by regulating the selection of replication origin sites as well as the initiation timing of the selected origins. Recent studies indicate that active origins frequently localize with active or potentially active gene promoters, suggesting coordination of these fundamental DNA transactions.

DNA polymerase epsilon, acetylases and remodellers cooperate to form a specialized chromatin structure at a tRNA insulator.

Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA.

ChIP-chip to analyze the binding of replication proteins to chromatin using oligonucleotide DNA microarrays.

Chromatin immunoprecipitation (ChIP) is a widely used method to study the interactions between proteins and discrete chromosomal loci in vivo. Originally, ChIP was developed for analysis of protein associations with DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays has enabled the identification of all DNA sequences enriched by ChIP, providing a genomic view of protein binding.