Kevin Struhl

An Epigenetic Switch Involving NF-κB, Lin28, Let-7 MicroRNA, and IL6 Links Inflammation to Cell Transformation

Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. We show that transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal.

The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α Helices: Crystal structure of the protein-DNA complex

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission

The cancer stem cell hypothesis suggests that, unlike most cancer cells within a tumor, cancer stem cells resist chemotherapeutic drugs and can regenerate the various cell types in the tumor, thereby causing relapse of the disease. Thus, drugs that selectively target cancer stem cells offer great promise for cancer treatment, particularly in combination with chemotherapy. Here, we show that low doses of metformin, a standard drug for diabetes, inhibits cellular transformation and selectively kills cancer stem cells in four genetically different types of breast cancer. The combination of metformin and a well-defined chemotherapeutic agent, doxorubicin, kills both cancer stem cells and non-stem cancer cells in culture. Furthermore, this combinatorial therapy reduces tumor mass and prevents relapse much more effectively than either drug alone in a xenograft mouse model. Mice seem to remain tumor-free for at least 2 months after combinatorial therapy with metformin and doxorubicin is ended. These results provide further evidence supporting the cancer stem cell hypothesis, and they provide a rationale and experimental basis for using the combination of metformin and chemotherapeutic drugs to improve treatment of patients with breast (and possibly other) cancers.

TREX is a conserved complex coupling transcription with messenger RNA export.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)+ RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX (‘transcription/export’) complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.

Sterile host yeasts (SHY): A eukaryotic system of biological containment for recombinant DNA experiments

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewers/Bakers yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.

Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of Yeast

Yeast GCN4 protein binds specifically to the promoters of amino acid biosynthetic genes and coordinately induces their transcription. Serially deleted GCN4 and hybrid LexA-GCN4 proteins were assayed for specific DNA binding activity in vitro, and for stimulation of transcription in vivo. The specific DNA binding activity resides in the 60 C-terminal amino acids, a basic region of GCN4. However, certain deletions containing the entire DNA binding region are unable to activate transcription and instead act as repressors in vivo. The activation function appears to critically involve just 19 amino acids that are centrally located in an acidic region of GCN4. In addition to their functional separation, the DNA binding and transcriptional activation regions of the protein can be separated physically by elastase cleavage. The implications of these results for the mechanisms of DNA sequence recognition and transcription activation are discussed.

Methylation of H3-Lysine 79 Is Mediated by a New Family of HMTases without a SET Domain

The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.

The gradient morphogen bicoid is a concentration-dependent transcriptional activator.

The bicoid (bcd) protein is expressed in an anteroposterior gradient in early Drosophila embryos and controls the zygotic activation of the segmentation gene hunchback (hb) in a broad but precisely bounded anterior domain. Here we show that the hb gene contains multiple regulatory elements that mediate transcriptional activation in response to bcd protein. Further, we demonstrate that the resulting patterns of expression in vivo depend critically on both the bcd gradient profile and the number and quality of these hb elements. Finally, we show that these same elements mediate bcd-dependent transcriptional activation in yeast and that this interaction requires distinct DNA binding and activating regions in the bcd protein. Our results argue that bcd protein normally binds and activates the hb gene in a concentration-dependent fashion, thereby allowing the gradient of bcd protein to dictate where the hb gene is initially turned on in early embryos. They also suggest that the bcd gradient has the instructive capacity to activate other subordinate control genes by the same mechanism, each in a distinct spatial domain according to its affinity for bcd protein.

STAT3 Activation of miR-21 and miR-181b-1 via PTEN and CYLD Are Part of the Epigenetic Switch Linking Inflammation to Cancer

A transient inflammatory signal can initiate an epigenetic switch from nontransformed to cancer cells via a positive feedback loop involving NF-kappaB, Lin28, let-7, and IL-6. We identify differentially regulated microRNAs important for this switch and putative transcription factor binding sites in their promoters. STAT3, a transcription factor activated by IL-6, directly activates miR-21 and miR-181b-1. Remarkably, transient expression of either microRNA induces the epigenetic switch. MiR-21 and miR-181b-1, respectively, inhibit PTEN and CYLD tumor suppressors, leading to increased NF-kappaB activity required to maintain the transformed state. These STAT3-mediated regulatory circuits are required for the transformed state in diverse cell lines and tumor growth in xenografts, and their transcriptional signatures are observed in colon adenocarcinomas. Thus, STAT3 is not only a downstream target of IL-6 but, with miR-21, miR-181b-1, PTEN, and CYLD, is part of the positive feedback loop that underlies the epigenetic switch that links inflammation to cancer.

Repression by Ume6 Involves Recruitment of a Complex Containing Sin3 Corepressor and Rpd3 Histone Deacetylase to Target Promoters

Sin3 and Rpd3 negatively regulate a diverse set of yeast genes. A mouse Sin3-related protein is a transcriptional corepressor, and a human Rpd3 homolog is a histone deacetylase. Here, we show that Sin3 and Rpd3 are specifically required for transcriptional repression by Ume6, a DNA-binding protein that regulates genes involved in meiosis. A short region of Ume6 is sufficient to repress transcription, and this repression domain mediates a two-hybrid and physical interaction with Sin3. Coimmunoprecipitation and two-hybrid experiments indicate that Sin3 and Rpd3 are associated in a complex distinct from TFIID and Pol II holoenzyme. Rpd3 is specifically required for repression by Sin3, and artificial recruitment of Rpd3 results in repression. These results suggest that repression by Ume6 involves recruitment of a Sin3-Rpd3 complex and targeted histone deacetylation.