Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing - Supplemental data.

Replication of the genome before mitotic cell division is a highly regulated process that ensures the fidelity of DNA duplication. DNA replication initiates at specific locations, termed origins of replication, and progresses in a defined temporal order during the S phase of the cell cycle. The relationship between replication timing and gene expression has been the subject of some speculation. A recent genome-wide analysis in Saccharomyces cerevisiae showed no association between replication timing and gene expression. In higher eukaryotes, the limited number of genomic loci analyzed has not permitted a firm conclusion regarding this association. To explore the relationship between DNA replication and gene expression in higher eukaryotes, we developed a strategy to measure the timing of DNA replication for thousands of genes in a single DNA array hybridization experiment. Using this approach, we generated a genome-wide map of replication timing for Drosophila melanogaster. Moreover, by surveying over 40% of all D. melanogaster genes, we found a strong correlation between DNA replication early in S phase and transcriptional activity. As this correlation does not exist in S. cerevisiae, this interplay between DNA replication and transcription may be a unique characteristic of higher eukaryotes

Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts

We have investigated the molecular basis of human immunodeficiency virus type 1 (HIV-1) latency in a tissue culture model and in HIV-infected people. We show that increased levels of Tat, but not Rev, can release the proviruses from latency in U1 cells. The absence of Tat in these cells is manifested by the accumulation of promoter-proximal viral transcripts, whereas the presence of Tat correlates with increased expression of viral proteins and an increase in promoter-distal transcripts. The presence of promoter-proximal transcripts also serves as a marker for latency in humans. We observed the exclusive presence of promoter-proximal viral transcripts in peripheral mononuclear cells from the majority (10/11) of asymptomatic HIV-infected individuals examined. Activation of these cells in vitro, and viremia in vivo, correlated with a switch from promoter-proximal transcription to promoter-distal transcription. These results suggest that the control between latency and replication of HIV in vivo is at the level of transcription elongation

Networking the nucleus

The nuclei of differentiating cells exhibit several fundamental principles of self-organization. They are composed of many dynamical units connected physically and functionally to each other—a complex network—and the different parts of the system are mutually adapted and produce a characteristic end state. A unique cell-specific signature emerges over time from complex interactions among constituent elements that delineate coordinate gene expression and chromosome topology. Each element itself consists of many interacting components, all dynamical in nature. Self-organizing systems can be simplified while retaining complex information using approaches that examine the relationship between elements, such as spatial relationships and transcriptional information. These relationships can be represented using well-defined networks. We hypothesize that during the process of differentiation, networks within the cell nucleus rewire according to simple rules, from which a higher level of order emerges. Studying the interaction within and among networks provides a useful framework for investigating the complex organization and dynamic function of the nucleus

Coordinate Gene Regulation during Hematopoiesis Is Related to Genomic Organization

Gene loci are found in nuclear subcompartments that are related to their expression status. For instance, silent genes are often localized to heterochromatin and the nuclear periphery, whereas active genes tend to be found in the nuclear center. Evidence also suggests that chromosomes may be specifically positioned within the nucleus; however, the nature of this organization and how it is achieved are not yet fully understood. To examine whether gene regulation is related to a discernible pattern of genomic organization, we analyzed the linear arrangement of co-regulated genes along chromosomes and determined the organization of chromosomes during the differentiation of a hematopoietic progenitor to erythroid and neutrophil cell types. Our analysis reveals that there is a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. We suggest that proximity in the form of chromosomal gene distribution and homolog association may be the basis for organizing the genome for coordinate gene regulation during cellular differentiation

Variant Histone H2A.Z Is Globally Localized to the Promoters of Inactive Yeast Genes and Regulates Nucleosome Positioning

H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions—typically one or two nucleosomes wide—decorated with H2A.Z. Those “Z loci” are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed

An expansive human regulatory lexicon encoded in transcription factor footprints.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency

An Acetyl-Methyl Switch Drives a Conformational Change in p53

Individual posttranslational modifications (PTMs) of p53 mediate diverse p53-dependent responses, however much less is known about the combinatorial action of adjacent modifications. Here, we describe crosstalk between the early DNA damage response mark p53K382me2 and the surrounding PTMs that modulate binding of p53 co-factors, including 53BP1 and p300. The 1.8 Å resolution crystal structure of the tandem Tudor domain (TTD) of 53BP1 in complex with p53 peptide acetylated at K381 and dimethylated at K382 (p53K381acK382me2) reveals that the dual PTM induces a conformational change in p53. The α-helical fold of p53K381acK382me2 positions the side chains of R379, K381ac, and K382me2 to interact with TTD concurrently, reinforcing a modular design of double PTM mimetics. Biochemical and NMR analyses show that other surrounding PTMs, including phosphorylation of serine/threonine residues of p53, affect association with TTD. Our findings suggest a novel PTM-driven conformation switch-like mechanism that may regulate p53 interactions with binding partners