Julia Zeitlinger

Polycomb complexes repress developmental regulators in murine embryonic stem cells.

The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.

Transcriptional regulatory code of a eukaryotic genome

DNA-binding transcriptional regulators interpret the genomes regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeasts transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeasts transcriptional regulators.

Genome-wide Map of Nucleosome Acetylation and Methylation in Yeast

Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.

RNA polymerase is poised for activation across the genome

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues.

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the ∼10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development.

Serial Regulation of Transcriptional Regulators in the Yeast Cell Cycle

Genome-wide location analysis was used to determine how the yeast cell cycle gene expression program is regulated by each of the nine known cell cycle transcriptional activators. We found that cell cycle transcriptional activators that function during one stage of the cell cycle regulate transcriptional activators that function during the next stage. This serial regulation of transcriptional activators forms a connected regulatory network that is itself a cycle. Our results also reveal how the nine transcriptional regulators coordinately regulate global gene expression and diverse stage-specific functions to produce a continuous cycle of cellular events. This information forms the foundation for a complete map of the transcriptional regulatory network that controls the cell cycle.

Program-Specific Distribution of a Transcription Factor Dependent on Partner Transcription Factor and MAPK Signaling

Specialized gene expression programs are induced by signaling pathways that act on transcription factors. Whether these transcription factors can function in multiple developmental programs through a global switch in promoter selection is not known. We have used genome-wide location analysis to show that the yeast Ste12 transcription factor, which regulates mating and filamentous growth, is bound to distinct program-specific target genes dependent on the developmental condition. This condition-dependent distribution of Ste12 requires concurrent binding of the transcription factor Tec1 during filamentation and is differentially regulated by the MAP kinases Fus3 and Kss1. Program-specific distribution across the genome may be a general mechanism by which transcription factors regulate distinct gene expression programs in response to signaling.In the unactivated Limulus sperm, a 60-µm-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus. At fertilization, the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process. This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymerization1. In a 9.5-Å electron cryomicroscopic structure of the extended bundle, we show that twist, tilt and rotation of actin–scruin subunits deviate widely from a ‘standard’ F-actin filament. This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly.

Promoter elements associated with RNA Pol II stalling in the Drosophila embryo

RNA Polymerase II (Pol II) is bound to the promoter regions of many or most developmental control genes before their activation during Drosophila embryogenesis. It has been suggested that Pol II stalling is used to produce dynamic and rapid responses of developmental patterning genes to transient cues such as extracellular signaling molecules. Here, we present a combined computational and experimental analysis of stalled promoters to determine how they come to bind Pol II in the early Drosophila embryo. At least one-fourth of the stalled promoters contain a shared sequence motif, the “pause button” (PB): KCGRWCG. The PB motif is sometimes located in the position of the DPE, and over one-fifth of the stalled promoters contain the following arrangement of core elements: GAGA, Inr, PB, and/or DPE. This arrangement was used to identify additional stalled promoters in the Drosophila genome, and permanganate footprint assays were used to confirm that the segmentation gene engrailed contains paused Pol II as seen for heat-shock genes. We discuss different models for Pol II binding and gene activation in the early embryo.

High conservation of transcription factor binding and evidence for combinatorial regulation across six Drosophila species

The binding of some transcription factors has been shown to diverge substantially between closely related species. Here we show that the binding of the developmental transcription factor Twist is highly conserved across six Drosophila species, revealing strong functional constraints at its enhancers. Conserved binding correlates with sequence motifs for Twist and its partners, permitting the de novo discovery of their combinatorial binding. It also includes over 10,000 low-occupancy sites near the detection limit, which tend to mark enhancers of later developmental stages. These results suggest that developmental enhancers can be highly evolutionarily constrained, presumably because of their complex combinatorial nature.

High-resolution computational models of genome binding events

Direct physical information that describes where transcription factors, nucleosomes, modified histones, RNA polymerase II and other key proteins interact with the genome provides an invaluable mechanistic foundation for understanding complex programs of gene regulation. We present a method, joint binding deconvolution (JBD), which uses additional easily obtainable experimental data about chromatin immunoprecipitation (ChIP) to improve the spatial resolution of the transcription factor binding locations inferred from ChIP followed by DNA microarray hybridization (ChIP-Chip) data. Based on this probabilistic model of binding data, we further pursue improved spatial resolution by using sequence information. We produce positional priors that link ChIP-Chip data to sequence data by guiding motif discovery to inferred protein-DNA binding sites. We present results on the yeast transcription factors Gcn4 and Mig2 to demonstrate JBDs spatial resolution capabilities and show that positional priors allow computational discovery of the Mig2 motif when a standard approach fails.