Pamela A. Silver

Genome-wide analysis of estrogen receptor binding sites

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

Chromosome-Wide Mapping of Estrogen Receptor Binding Reveals Long-Range Regulation Requiring the Forkhead Protein FoxA1

Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.

Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization

The association of genes with the nuclear pore complex (NPC) and nuclear transport factors has been implicated in transcriptional regulation. We therefore examined the association of components of the nuclear transport machinery including karyopherins, nucleoporins, and the Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome. We find that most nucleoporins and karyopherins preferentially associate with a subset of highly transcribed genes and with genes that possess Rap1 binding sites whereas the RanGEF preferentially associates with transcriptionally inactive genes. Consistent with coupling of transcription to the nuclear pore, we show that transcriptional activation of the GAL genes results in their association with nuclear pore proteins, relocation to the nuclear periphery, and loss of RanGEF association. Taken together, these results indicate that the organization of the genome is coupled via transcriptional state to the nuclear transport machinery.

How proteins enter the nucleus

Nuclear protein import is a selective process. Proteins destined for the nucleus contain NLSs. These short stretches of amino acids interact with proteins located in the cytoplasm, on the nuclear envelope, and/or at the nuclear pore complex. Following binding at the pore complex, proteins are translocated through the pore into the nucleus in a manner requiring ATP. The biochemical dissection of the nuclear pore complex has begun. Alteration of protein import into the nucleus is emerging as a new and complex form of regulation. However, we are left with the following problems: How do proteins move through the cytoplasm to reach the nuclear pore? How does the nuclear pore complex open and close in a selective manner? How is ATP utilized during import? And finally, how is bi-directional traffic of both proteins and RNA through the pore regulated?

Organization of Intracellular Reactions with Rationally Designed RNA Assemblies

Multidimensional RNA structures can act as scaffolds for the spatial organization of bacterial metabolism. The rules of nucleic acid base-pairing have been used to construct nanoscale architectures and organize biomolecules, but little has been done to apply this technology in vivo. We designed and assembled multidimensional RNA structures and used them as scaffolds for the spatial organization of bacterial metabolism. Engineered RNA modules were assembled into discrete, one-dimensional, and two-dimensional scaffolds with distinct protein-docking sites and used to control the spatial organization of a hydrogen-producing pathway. We increased hydrogen output as a function of scaffold architecture. Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo.

State of the Arg: Protein Methylation at Arginine Comes of Age

(see below), the most striking characteristic of this Posttranslational modification of proteins allows the cell emerging family of enzymes is the presence of an S-adento expand its repertoire beyond the constraints imposed osyl methionine (AdoMet) binding motif (Figure 2A), by the twenty encoded amino acids. Methylation at argiwhich is closely related to the motif found in nucleic nines, although discovered over 30 years ago, has only acid and small molecule methyltransferases that use recently come to the attention of cell biologists through AdoMet as a methyl donor. In addition to the AdoMet a combination of genetic and molecular biology experibinding motif, four of five putative mammalian arginine ments that have implicated arginine methylation in promethyltransferases studied to date also share a less cesses from signaling and transcription activation to proconserved C-terminal domain, which is presumably intein sorting. The panoply of arginine methylated substrates volved in arginine substrate interactions. suggests that this specifically eukaryotic modification The majority of arginine methylation in eukaryotic cells may parallel phosphorylation in its level of complexity. appears to be performed by a specific methyltransferase We will summarize much of the recent information about subfamily, which includes mammalian PRMT1 and its the function of methylation and the methyltransferase functional homolog, yeast Hmt1/Rmt1. The enzymes in enzymes that modify arginines. this subfamily contain few residues outside the core Many Proteins Are Targets of Arginine Methylation region. In contrast, Carm1/PRMT4 contains both Nand Three main forms of methylarginine have been identified C-terminal extensions to the methyltransferase core rein eukaryotes: N-monomethylarginine (MMA), NN gion. Other family members have N-terminal extensions, (asymmetric) dimethylarginine (aDMA), and NN G (symseveral of which contain additional motifs such as an metric) dimethylarginine (sDMA), all of which involve SH3 domain (PRMT2) and a zinc finger motif (PRMT3). modification of guanidino nitrogen atoms (Figure 1). AlWhereas all arginine methyltransferase activities identhough early purification of mammalian protein arginine tified to date can monomethylate arginine in the context methyltransferases used methylation of histones to of a protein substrate, methyltransferases have been track activity, the majority of nuclear asymmetric diclassified as type I or type II enzymes according to methylarginine residues are found in heterogeneous nuwhether further dimethylation is asymmetric (type I) or clear ribonucleoproteins (hnRNPs), which play roles in symmetric (type II). Most PRMT genes discovered to pre-mRNA processing and nucleocytoplasmic RNA date encode type I enzymes, but recent data have retransport. Subsequent work on numerous hnRNPs and vealed that PRMT5/JBP1 (Janus kinase binding protein other RNA binding proteins has revealed that they are 1) is a type II methyltransferase (Branscombe et al., methylated on arginine residues, frequently in the con2001). Although in vivo substrates for the type I PRMT1/ text of RGG tripeptides. Notably, all methylarginine resiHmt1 enzymes have been defined, the substrate specidues within RGG motifs have been shown to be MMA ficity of the majority of arginine methyltransferases reor aDMA rather than sDMA. Proteins have also been mains mysterious. In the case of PRMT3, however, its identified that are asymmetrically dimethylated at RXR N-terminal zinc finger domain has been shown to influand RG motifs. ence its substrate specificity (Frankel and Clarke, 2000). Myelin basic protein, which was one of the first argiThe three-dimensional structures of the core regions nine-methylated proteins identified, stands in contrast of yeast Hmt1 and human PRMT3 have been determined to most methylated RNA binding proteins in that it conby X-ray crystallography and a comparison of these tains symmetrically dimethylated arginine residues in structures underscores the structural similarity between addition to monomethylarginine. Recently, however, these enzymes (Figure 2B) (Weiss et al., 2000; Zhang et two RNA binding proteins, spliceosomal snRNP proteins al., 2000). Whereas the AdoMet binding domains are

REGULATED NUCLEO/CYTOPLASMIC EXCHANGE OF HOG1 MAPK REQUIRES THE IMPORTIN BETA HOMOLOGS NMD5 AND XPO1

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild‐type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran–GSP1, but not the NLS‐binding importin α/β heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin β homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress‐activated MAP kinase.

Nuclear transport and cancer: from mechanism to intervention

Nuclear-cytoplasmic transport, which occurs through special structures called nuclear pores, is an important aspect of normal cell function, and defects in this process have been detected in many different types of cancer cells. These defects can occur in the signal-transduction pathways that regulate the transfer of factors such as p53 and β-catenin in and out of the nucleus, or in the general nuclear import and export machinery itself. In some cases, nuclear transport factors are overproduced, whereas in others, chromosomal translocations disrupt the structural proteins that make up the nuclear pore, leading to cell transformation. How does disruption of nuclear-cytoplasmic transport promote transformation, and is this process a viable therapeutic target?

A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells

The PI3K/PTEN/Akt signal transduction pathway plays a key role in many tumors. Downstream targets of this pathway include the Forkhead family of transcription factors (FOXO1a, FOXO3a, FOXO4). In PTEN null cells, FOXO1a is inactivated by PI3K-dependent phosphorylation and mislocalization to the cytoplasm, yet still undergoes nucleocytoplasmic shuttling. Since forcible localization of FOXO1a to the nucleus can reverse tumorigenicity of PTEN null cells, a high-content, chemical genetic screen for inhibitors of FOXO1a nuclear export was performed. The compounds detected in the primary screen were retested in secondary assays, and structure-function relationships were identified. Novel general export inhibitors were found that react with CRM1 as well as a number of compounds that inhibit PI3K/Akt signaling, among which are included multiple antagonists of calmodulin signaling.

Elimination of Replication Block Protein Fob1 Extends the Life Span of Yeast Mother Cells

A cause of aging in yeast is the accumulation of circular species of ribosomal DNA (rDNA) arising from the 100-200 tandemly repeated copies in the genome. We show here that mutation of the FOB1 gene slows the generation of these circles and thus extends life span. Fob1p is known to create a unidirectional block to replication forks in the rDNA. We show that Fob1p is a nucleolar protein, suggesting a direct involvement in the replication fork block. We propose that this block can trigger aging by causing chromosomal breaks, the repair of which results in the generation of rDNA circles. These findings may provide a novel link between metabolic rate and aging in yeast and, perhaps, higher organisms.