Laura Schramm

Bio: Laura Schramm is an academic researcher from St. John's University. The author has contributed to research in topics: RNA polymerase III & Transcription Factor TFIIIB. The author has an hindex of 18, co-authored 23 publications receiving 1469 citations. Previous affiliations of Laura Schramm include Memorial Sloan Kettering Cancer Center & Stony Brook University.

Recruitment of RNA polymerase III to its target promoters

Abstract: A key step in retrieving the information stored in the complex genomes of eukaryotes involves the identification of transcription units and, more specifically, the recognition of promoter sequences by RNA polymerase. In eukaryotes, the task of recognizing nuclear gene promoters and then transcribing the genes is divided among three highly related enzymes, RNA polymerases I, II, and III. Each of these RNA polymerases is dedicated to the transcription of specific sets of genes, and each depends on accessory factors, the so-called transcription factors, to recognize its cognate promoter sequences.

Different human TFIIIB activities direct RNA polymerase III transcription from TATA-containing and TATA-less promoters

Abstract: Correct initiation of transcription by RNA polymerase III requires a number of factors. Of particular interest is the transcription factor IIIB (TFIIIB), because TFIIIB directly contacts RNA polymerase III, and in yeast, once recruited to the promoter, TFIIIB is sufficient to support several rounds of RNA polymerase III transcription (reviewed in Paule and White 2000). Yeast TFIIIB is well defined and consists of three subunits, the TATA box–binding protein TBP (Kassavetis et al. 1992), the TFIIB-related factor BRF (TDS4/PCF4) (Buratowski and Zhou 1992; Colbert and Hahn 1992; Lopez-De-Leon et al. 1992), and the B′′ protein (TFIIIB90/TFC5/TFC7) (Kassavetis et al. 1995; Roberts et al. 1996; Ruth et al. 1996). In various RNA polymerase III promoters, the mode of TFIIIB recruitment varies. In the gene-internal 5S and transfer RNA (tRNA) promoters, the promoter elements are first recognized by TFIIIA and TFIIIC or directly by TFIIIC, respectively, and this allows the subsequent recruitment of TFIIIB (reviewed in Paule and White 2000). On the yeast U6 promoter, which contains a TATA box ∼25 nucleotides upstream of the transcription start site, TFIIIB is also recruited in a TFIIIC-dependent manner (Brow and Guthrie 1990; Burnol et al. 1993; Eschenlauer et al. 1993), but on naked DNA templates in vitro, it can be recruited by direct binding of the TBP subunit to the TATA box (Moenne et al. 1990; Margottin et al. 1991). In all these cases, however, the same TFIIIB complex, containing the same three subunits, is used (Joazeiro et al. 1994). In mammalian cells, the situation appears more complicated. Like yeast TFIIIB, mammalian TFIIIB contains TBP (Lobo et al. 1992; Taggart et al. 1992; White and Jackson 1992) and a homolog of yeast BRF, human BRF (hBRF) (Mital et al. 1996), also called TFIIIB90 (Wang and Roeder 1995). These two subunits associate strongly with each other (Wang and Roeder 1995; Mital et al. 1996). A homolog of yeast B′′ has not been identified in mammals or any other organism. In addition, unlike in yeast, there appear to be variants of the TFIIIB complex. In particular, the human U6 promoter, which exemplifies a class of RNA polymerase III promoters whose essential elements are all located within the 5′ flanking sequence of the gene, appears to use another form of TFIIIB than the tRNA-type Ad2 VAI promoter (Lobo et al. 1992; Teichmann and Seifart 1995; Mital et al. 1996; Henry et al. 1998a), which contains a gene-internal promoter. The core U6 promoter, which is sufficient to direct basal levels of transcription in vitro, consists of a proximal sequence element (PSE) centered around position −56 relative to the start site and a TATA box centered around position −27. The U6 promoter is highly similar to the RNA polymerase II small nuclear RNA (snRNA) core promoters, which consist of only the PSE (see Henry et al. 1998a and references therein). The PSE recruits a multisubunit complex called SNAPc (Henry et al. 1995, 1998b) or PTF (Yoon et al. 1995), and the U6 TATA box recruits TBP (Lobo et al. 1991; Simmen et al. 1991). It is not clear which other TFIIIB components aside from TBP are involved in U6 transcription. Wang and Roeder (1995) showed that depletion of extracts with anti-hBRF antibodies debilitated U6 transcription, but addition of recombinant hBRF did not restore activity. They concluded that hBRF was required for U6 transcription but as part of a complex that somehow differed from the TFIIIB complex required for VAI transcription. On the other hand, we showed that depletion of extracts with antibodies directed against the C-terminal half of hBRF debilitated transcription from the VAI promoter but not from the U6 promoter. Because the antibody treatment removed >95% of the endogenous hBRF present in extracts, the results suggest that hBRF is in fact not required for U6 transcription (Mital et al. 1996). Indeed, we were able also show that upon depletion of extracts with anti-TBP and anti-hBRF antibodies, VAI transcription was only restored by addition of a combination of both TBP and hBRF, whereas U6 transcription could be restored by addition of only recombinant TBP. In fact, U6 transcription was diminished by addition of hBRF (Henry et al. 1998a). Thus, two main questions remain concerning mammalian TFIIIB. First, it is currently unclear whether a mammalian homolog of yeast B′′ exists, and whether it is required for RNA polymerase III transcription of genes with internal promoters, such as the VAI gene, and genes with external promoters that recruit SNAPc, such as the human U6 gene. Second, the apparent lack of requirement for hBRF in U6 transcription raises the possibility that the mammalian snRNA-type RNA polymerase III promoters use a factor different from, but related to, hBRF. Here, we report the characterization of two new transcription factors, human B′′ (hB′′) and a BRF-related factor we refer to as hBRFU. hB′′ is required for transcription of both the VAI and the U6 genes. hBRFU is a protein with an N-terminal domain related to both hBRF and TFIIB, and a divergent C-terminal domain. The protein is required for U6 but not VAI transcription. These results show that there are two forms of the basal RNA polymerase III transcription factor IIIB in mammalian cells. They also identify the first transcription factors uniquely required for transcription of RNA polymerase III but not RNA polymerase II—snRNA promoters. Together with our previous results indicating that TFIIB is required for transcription of the human RNA polymerase II snRNA genes (Kuhlman et al. 1999), these results suggest that the key event in the determination of RNA polymerase specificity in the human snRNA promoters is the recruitment of hBRFU versus TFIIB.

BRCA1 Inhibition of Telomerase Activity in Cultured Cells

Abstract: Telomerase, an enzyme that maintains telomere length, plays major roles in cellular immortalization and cancer progression. We found that an exogenous BRCA1 gene strongly inhibited telomerase enzymatic activity in human prostate and breast cancer cell lines and caused telomere shortening in cell lines expressing wild-type BRCA1 (wtBRCA1) but not a tumor-associated mutant BRCA1 (T300G). wtBRCA1 inhibited the expression of the catalytic subunit (telomerase reverse transcriptase [TERT]) but had no effect on the expression of a subset of other components of the telomerase holoenzyme or on the expression of c-Myc, a transcriptional activator of TERT. However, endogenous BRCA1 associated and partially colocalized with c-Myc; exogenous wtBRCA1 strongly suppressed TERT promoter activity in various cell lines. The TERT inhibition was due, in part, to suppression of c-Myc E-box-mediated transcriptional activity. Suppression of TERT promoter and c-Myc activity required the amino terminus of BRCA1 but not the carboxyl terminus. Finally, endogenous BRCA1 and c-Myc were detected on transfected mouse and human TERT promoter segments in vivo. We postulate that inhibition of telomerase may contribute to the BRCA1 tumor suppressor activity.

CK2 Forms a Stable Complex with TFIIIB and Activates RNA Polymerase III Transcription in Human Cells

Abstract: CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

p53--an acrobat in tumorigenesis.

Abstract: Thep53tumorsuppressor protein plays acentral role inmaintaining genomic integrity. Itdoessobyoccupying anodal point intheDNAdamagecontrol pathway. Whencells aresubject toionizing radiation orother mutagenic events, p53 mediates cell cycle arrest orprogrammed cell death (apoptosis). Furthermore, someevidence suggests that p53plays arole intherecognition andrepair ofdamaged DNA.Biochemically, p53isasequence-specific transcriptional stimulator andanon- specific transcriptional repressor butalso engages inmultiple protein-protein interactions. Conversely, disruption ofthep53 response pathway strongly correlates withtumorigenesis. p53isfunctionally inactivated bystructural mutations, neutraliza- tion byviral products, andnon-mutational cellular mechanisms inthemajority ofhumancancers. p53-deficient micehavea highly penetrant tumorphenotype, withover90%tumorincidence within ninemonths. Insomecancers, direct physical evi- dence exists identifying thep53geneasatarget ofknownenvironmental carcinogens suchasUVlight andbenzojalpyrene in cancers oftheskin andlung. Whenp53loss occurs, cells donotgetrepaired oreliminated butrather proceed toreplicate dam- agedDNA,which results inmorerandom mutations, geneamplifications, chromosomal re-arrangements, andaneuploidy. In someexperimental models, loss ofp53confers resistance toanticancer therapy duetoloss ofapoptotic competence. The translational potential ofthese discoveries isbeginning tobetested innovel p53-based therapies.

Identification of microRNAs of the herpesvirus family

Abstract: Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.

The RNA polymerase II core promoter.

Abstract: ▪ Abstract The events leading to transcription of eukaryotic protein-coding genes culminate in the positioning of RNA polymerase II at the correct initiation site. The core promoter, which can extend ∼35 bp upstream and/or downstream of this site, plays a central role in regulating initiation. Specific DNA elements within the core promoter bind the factors that nucleate the assembly of a functional preinitiation complex and integrate stimulatory and repressive signals from factors bound at distal sites. Although core promoter structure was originally thought to be invariant, a remarkable degree of diversity has become apparent. This article reviews the structural and functional diversity of the RNA polymerase II core promoter.

Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.

Abstract: O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

p53 and human cancer: the first ten thousand mutations.

Abstract: Publisher Summary The p53 protein is a tight, hydrophobic central globule containing the DNA binding domain, flanked by accessible N- and C-terminal regions This protein is expressed in all cell types but has a rapid turnover and is latent under normal conditions p53 is mutated in most common human malignancies and behaves as a multifunctional transcription factor involved in the control of cell cycle, programmed cell death, senescence, differentiation and development, transcription, DNA replication, DNA repair, and maintenance of genomic stability p53 is converted to an active form in response to a number of physical or chemical DNA-damaging agents such as X or gamma irradiation, UV rays, oxidizing agents, cytotoxic drugs, and cancer-causing chemicals Induction of p53 implies nuclear retention, accumulation of the protein as a result of post-translational stabilization, and allosteric conversion to a form with high sequence-specific DNA-binding capacity p53 is activated in response to DNA damage, thus acting as a “guardian of the genome” against genotoxic stress The chapter describes a three-step model of pS3 activation by stress signals The downstream pS3 signaling is mediated by transcriptional activation of specific genes and by complex formation between p53 and heterologous proteins The mutations and variations in the p53 gene are due to p53 polymorphisms, somatic mutations, and germline mutations in p53 The chapter also accounts for p53 mutations in sporadic cancers focussing on host-environment interactions The chapter concludes with the potential clinical applications of the detection of p53 mutations in human tissues