Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein α gene

During adipocyte differentiation, the expression of C/EBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPα gene led to the identification of a potential repressive element within the proximal 5′ flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoter-luciferase constructs containing both the proximal 5′ flanking and the entire 5′ untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5′ untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5′ untranslated region. Although mutation of either CUP element in constructs containing both the 5′ flanking and 5′ untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPα gene prior to induction of the adipocyte differentiation program.

Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells

Methylation of cytosines in the dinucleotide CpG has been shown to suppress transcription of a number of tissue-specific genes, yet the precise mechanism is not fully understood. The vertebrate globin genes were among the first examples in which an inverse correlation was shown between CpG methylation and transcription. We studied the methylation pattern of the 235-bp ρ-globin gene promoter in genomic DNA from primary chicken erythroid cells using the sodium bisulfite conversion technique and found all CpGs in the promoter to be methylated in erythroid cells from adult chickens in which the ρ-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the gene is transcribed. To elucidate further the mechanism of methylation-induced silencing, an expression construct consisting of 235 bp of 5′ promoter sequence of the ρ-globin gene along with a strong 5′ erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, ρ-CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp ρ-globin gene promoter fragment at every CpG resulted in a 20- to 30-fold inhibition of transcription, and this effect was not overridden by the presence of potent erythroid-specific enhancers. The ability of the 235-bp ρ-globin gene promoter to bind to a DNA Methyl Cytosine binding Protein Complex (MeCPC) was tested in electrophoretic mobility shift assays utilizing primary avian erythroid cell nuclear extract. The results were that fully methylated but not unmethylated 235-bp ρ-globin gene promoter fragment could compete efficiently for MeCPC binding. These results are a direct demonstration that site-specific methylation of a globin gene promoter at the exact CpGs that are methylated in vivo can silence transcription in homologous primary erythroid cells. Further, these data implicate binding of MeCPC to the promoter in the mechanism of silencing.

Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in Schizosaccharomyces pombe

Homologous recombination hotspots increase the frequency of recombination in nearby DNA. The M26 hotspot in the ade6 gene of Schizosaccharomyces pombe is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5′-ATGACGT-3′) defined by extensive mutagenesis. A heterodimeric M26 DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold. Cloning, disruption, and genetic analyses of the mts genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity. This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot. Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses. However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities. Furthermore, ade6 mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in mts mutants, indicating that hotspot activation is not a consequence of elevated transcription levels. These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination. Other studies have recently shown that the Mts1/Mts2 protein and M26 sites are involved in meiotic recombination elsewhere in the S. pombe genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.

Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1–1, mab2–1, and mab3–1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1–1, mab2–1, and mab3–1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1–1 and mab2–1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3–1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.

Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library

We have developed a strategy for the identification of peptides able to functionally replace a zinc finger domain in a transcription factor. This strategy could have important ramifications for basic research on gene regulation and for the development of therapeutic agents. In this study in yeast, we expressed chimeric proteins that included a random peptide combinatorial library in association with two zinc finger domains and a transactivating domain. The library was screened for chimeric proteins capable of activating transcription from a target sequence in the upstream regulatory regions of selectable or reporter genes. In a screen of approximately 1.5 × 107 transformants we identified 30 chimeric proteins that exhibited transcriptional activation, some of which were able to discriminate between wild-type and mutant DNA targets. Chimeric library proteins expressed as glutathione S-transferase fusions bound to double-stranded oligonucleotides containing the target sequence, suggesting that the chimeras bind directly to DNA. Surprisingly, none of the peptides identified resembled a zinc finger or other well-known transcription factor DNA binding domain.

Ash1p is a site-specific DNA-binding protein that actively represses transcription

ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known “repression domain” of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Herpes simplex virus 1 α regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1

The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a promiscuous transactivator shown to enhance the expression of gene introduced into cells by infection or transfection. At the molecular level, ICP0 is a 775-aa ring finger protein localized initially in the nucleus and late in infection in the cytoplasm and mediates the degradation of several proteins and stabilization of others. None of the known functions at the molecular level account for the apparent activity of ICP0 as a transactivator. Here we report that ICP0 functionally interacts with cellular transcription factor BMAL1, a member of the basic helix–loop–helix PER-ARNT-SIM (PAS) super family of transcriptional regulators. Specifically, sequences mapped to the exon II of ICP0 interacted with BMAL1 in the yeast two-hybrid system and in reciprocal pull-down experiments in vitro. Moreover, the enhancement of transcription of a luciferase reporter construct whose promoter contained multiple BMAL1-binding sites by ICP0 and BMAL1 was significantly greater than that observed by ICP0 or BMAL1 alone. Although the level of BMAL1 present in nuclei of infected cells remained unchanged between 3 and 8 h after infection, the level of cytoplasmic BMAL1 was reduced at 8 h after infection. The reduction of cytoplasmic BMAL1 was significantly greater in cells infected with the ICP0-null mutant than in the wild-type virus-infected cells, suggesting that ICP0 mediates partial stabilization of the protein. These results indicate that ICP0 interacts physically and functionally with at least one cellular transcription-regulatory factor.

Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix–turn–helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Quantitative analysis of mRNA amplification by in vitro transcription

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

The N- and C-terminal regions of RBP-J interact with the ankyrin repeats of Notch1 RAMIC to activate transcription

The evolutionarily-conserved DNA-binding protein RBP-J directly interacts with the RAM domain and the ankyrin (ANK) repeats of the Notch intracellular region (RAMIC), and activates transcription of downstream target genes that regulate cell differentiation. In vitro binding assays demonstrate that the truncated N- and C-terminal regions of RBP-J bind to the ANK repeats but not to the RAM domain. Using an OT11 mouse cell line, in which the RBP-J locus is disrupted, we showed that RBP-J constructs mutated in the N- and C-terminal regions were defective in their transcriptional activation induced by either RAMIC or IC (the Notch intracellular region without the RAM domain) although they had normal levels of binding activity to DNA and the RAM domain. The studies using chimeric molecules between RBP-J and its homolog RBP-L showed that the N- and C-terminal regions of RBP-J conferred the IC- as well as RAMIC-induced transactivation potential on RBP-L, which binds to the same DNA sequence as RBP-J but fails to interact with RAMIC. Taken together, these results indicate that the interactions between the N- and C-terminal regions of RBP-J and the ANK repeats of RAMIC are important for transactivation of RBP-J by RAMIC.

rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations

rSNP_Guide is a novel curated database system for analysis of transcription factor (TF) binding to target sequences in regulatory gene regions altered by mutations. It accumulates experimental data on naturally occurring site variants in regulatory gene regions and site-directed mutations. This database system also contains the web tools for SNP analysis, i.e., active applet applying weight matrices to predict the regulatory site candidates altered by a mutation. The current version of the rSNP_Guide is supplemented by six sub-databases: (i) rSNP_DB, on DNA–protein interaction caused by mutation; (ii) SYSTEM, on experimental systems; (iii) rSNP_BIB, on citations to original publications; (iv) SAMPLES, on experimentally identified sequences of known regulatory sites; (v) MATRIX, on weight matrices of known TF sites; (vi) rSNP_Report, on characteristic examples of successful rSNP_Tools implementation. These databases are useful for the analysis of natural SNPs and site-directed mutations. The databases are available through the Web, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/.