Nancy M. Hannett

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.

Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells

MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

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.

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.

Redundant roles for the TFIID and SAGA complexes in global transcription

The transcription factors TFIID and SAGA are multi-subunit complexes involved in transcription by RNA polymerase II. TFIID and SAGA contain common TATA-binding protein (TBP)-associated factor (TAFII) subunits and each complex contains a subunit with histone acetyltransferase activity. These observations have raised questions about whether the functions of the two complexes in vivo are unique or overlapping. Here we use genome-wide expression analysis to investigate how expression of the yeast genome depends on both shared and unique subunits of these two complexes. We find that expression of most genes requires one or more of the common TAF II subunits, indicating that the functions of TFIID and SAGA are widely required for gene expression. Among the subunits shared by TFIID and SAGA are three histone-like TAFIIs, which have been proposed to form a sub-complex and mediate a common function in global transcription. Unexpectedly, we find that the histone-like TAFIIs have distinct roles in expression of the yeast genome. Most importantly, we show that the histone acetylase components of TFIID and SAGA (TAFII145 and Gcn5) are functionally redundant, indicating that expression of a large fraction of yeast genes can be regulated through the action of either complex.

Exchange of RNA Polymerase II Initiation and Elongation Factors during Gene Expression In Vivo

We have systematically explored the in vivo occupancy of promoters and open reading frames by components of the RNA polymerase II transcription initiation and elongation apparatuses in yeast. RNA polymerase II, Mediator, and the general transcription factors (GTFs) were recruited to all promoters tested upon gene activation. RNA polymerase II, TFIIS, Spt5, and, unexpectedly, the Paf1/Cdc73 complex, were found associated with open reading frames. The presence of the Paf1/Cdc73 complex on ORFs in vivo suggests a novel function for this complex in elongation. Elongator was not detected under any conditions tested, and further analysis revealed that the majority of elongator is cytoplasmic. These results suggest a revised model for transcription initiation and elongation apparatuses in living cells.

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.

Transcription factor trapping by RNA in gene regulatory elements.

Noncoding RNA helps protein binding Besides reading the coding regions of genes, RNA polymerase generates RNA at promoter-proximal and -distal DNA elements, but the function of these molecules is largely unknown. Sigova et al. show that these RNAs facilitate interactions between gene regulators and the regulatory elements they occupy. Nascent RNA associates with the transcription factor YY1 and increases its ability to bind DNA. Thus, transcription at active regulatory elements may provide a positive feedback loop that reinforces regulatory elements contributing to the stability of gene expression programs. Science, this issue p. 978 Nascent RNAs facilitate interactions between gene regulators and regulatory elements. Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.

YY1 Is a Structural Regulator of Enhancer-Promoter Loops

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.