The mixed lineage leukemia 4 (MLL4) methyltransferase complex is involved in transforming growth factor beta (TGF-β)-activated gene transcription.

Sma and Mad related (SMAD)-mediated Transforming Growth Factor β (TGF-β) and Bone Morphogenetic Protein (BMP) signaling is required for various cellular processes. The activated heterotrimeric SMAD protein complexes associate with nuclear proteins such as the histone acetyltransferases p300, PCAF and the Mixed Lineage Leukemia 4 (MLL4) subunit Pax Transactivation domain-Interacting Protein (PTIP) to regulate gene transcription. We investigated the functional role of PTIP and PTIP Interacting protein 1 (PA1) in relation to TGF-β-activated SMAD signaling.

Quantitative Proteomics of the SMAD (Suppressor of Mothers against Decapentaplegic) Transcription Factor Family Identifies Importin 5 as a Bone Morphogenic Protein Receptor SMAD-specific Importin.

Gene-specific transcription factors (GSTFs) control gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. Mutations in the GSTFs Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4 are frequently associated with colon and rectal carcinomas. These proteins play an important role in bone morphogenic protein (BMP) and transforming growth factor β (TGF-β) signaling pathways controlling cell fate and proliferation.

A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.

Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion.

Cell cycle regulation by the PRMT6 arginine methyltransferase through repression of cyclin-dependent kinase inhibitors.

PRMT6 belongs to the family of Protein Arginine Methyltransferase (PRMT) enzymes that catalyze the methylation of guanidino nitrogens of arginine residues. PRMT6 has been shown to modify the tail of histone H3, but the in vivo function of PRMT6 is largely unknown. Here, we show that PRMT6 regulates cell cycle progression.

Distinct promoter dynamics of the basal transcription factor TBP across the yeast genome.

Transcription regulation in eukaryotes involves rapid recruitment and proper assembly of transcription factors at gene promoters. To determine the dynamics of the transcription machinery on DNA, we used a differential chromatin immunoprecipitation procedure coupled to whole-genome microarray detection in Saccharomyces cerevisiae. We find that TATA-binding protein (TBP) turnover is low at RNA polymerase I (Pol I) promoters.

Cooperative action of NC2 and Mot1p to regulate TATA-binding protein function across the genome.

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Mot1p and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Mot1p, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)-chip for cells in exponential growth and during reprogramming of transcription.

Snf1p-dependent Spt-Ada-Gcn5-acetyltransferase (SAGA) recruitment and chromatin remodeling activities on the HXT2 and HXT4 promoters.

We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p.

Differential requirement of SAGA subunits for Mot1p and Taf1p recruitment in gene activation.

Transcription activation in yeast (Saccharomyces cerevisiae) involves ordered recruitment of transcription factor complexes, such as TFIID, SAGA, and Mot1p. Previously, we showed that both Mot1p and Taf1p are recruited to the HXT2 and HXT4 genes, which encode hexose transporter proteins. Here, we show that SAGA also binds to the HXT2 and HXT4 promoters and plays a pivotal role in the recruitment of Mot1p and Taf1p.

Mot1p is essential for TBP recruitment to selected promoters during in vivo gene activation.

Recruitment of TATA-binding protein (TBP) is central to activation of transcription by RNA polymerase II (pol II). This depends upon co-activator proteins including TBP-associated factors (TAFs). Yeast Mot1p was identified as a general transcriptional repressor in genetic screens and is also found associated with TBP.

Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways.

Mitogenic signaling of insulin-like growth factor I in MCF-7 human breast cancer cells requires phosphatidylinositol 3-kinase and is independent of mitogen-activated protein kinase.

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation.

Strong promoter activity of human and rat islet amyloid polypeptide/amylin gene constructs in mouse beta cells (beta TC 3).

Islet amyloid polypeptide (IAPP)('amylin') is co-produced with insulin in pancreatic beta cells and is the formative polypeptide of pancreatic amyloid in patients with type 2 (non-insulin-dependent) diabetes mellitus. Islet amyloid and type 2 diabetes occur in man, but not in rat. To study transcription regulation of IAPP gene expression in man and rat, luciferase reporter constructs containing different portions of the upstream region of both IAPP genes were expressed in transfected cells.

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene.

Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction.

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown.

Two juxtaposed tyrosyl-OH groups participate in phi X174 gene A protein catalysed cleavage and ligation of DNA.

Bacteriophage phi X174 encoded gene A protein is an enzyme required for initiation and termination of successive rounds of rolling circle phi X DNA replication. This enzyme catalyses cleavage and ligation of a phosphodiester bond between nucleotide residues G and A at the phi X origin. The cleavage reaction which occurs during initiation involves formation of a free GOH residue at one end and a covalent bond between tyrosine-OH of the gene A protein and 5' phosphate of the A residue, at the other end of the cleavage site.

Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein.

Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication. To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein. The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region.

The bond in the bacteriophage phi X174 gene A protein--DNA complex is a tyrosyl-5'-phosphate ester.

The bacteriophage phi X174 gene A protein cleaves the viral strand of the double-stranded replicative form (RF) DNA of the phage at a specific site, the origin. It leaves a free 3'-OH at nucleotide 4305 (G) of the phi X DNA sequence and binds covalently to the DNA. The nature and position of the covalent bond have been determined using the octadecadesoxyribonucleotide CAACTTG[32P]ATATTAATAAC.

A protein of bacteriophage phi X174 carries an oligonucleotide which it can transfer to the 3' -OH of a DNA chain.

The bacteriophage phi X174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single-stranded, DNA-specific endonuclease which remains covalently attached to the 5'-end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA.