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PubMed1992

The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

Alleles↗
PubMed1992

HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae.

HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.

Coproporphyrinogen Oxidase↗
PubMed1994

The Drosophila dorsal morphogen represses the tolloid gene by interacting with a silencer element.

The dorsal protein (DL) regulates the transcriptional activity of several genes that determine cell fate along the dorsoventral axis of the Drosophila melanogaster embryo. DL is present at high levels in ventral nuclei, where it activates some genes (twi and sna) and represses others (zen, dpp, and tld). DL shows homology to the Rel family of proteins and interacts with specific DNA sequences in the regulatory regions of its target genes. The distal portion of the zen gene acts as a silencer that can mediate the repression of a heterologous promoter in ventral regions of the embryo. It contains four DL binding sites which alone are sufficient for activation but not repression. Here we analyze the interaction of DL with another one of its repressed targets, the tolloid (tld) gene. Approximately 800 bp of 5'-flanking sequences upstream of the tld coding region were shown to drive an expression pattern indistinguishable from the wild-type pattern. A 423-bp fragment located within these sequences contains two DL binding sites and was shown to act as a silencer to mediate ventral repression. Point mutations in the sites abolish not only DNA binding but also ventral repression. We discuss a comparison of the DNA sequences from the zen and tld promoters and the possible mechanisms of transcriptional silencing.

Animals↗
PubMed1994

Retinoic acid represses Oct-3/4 gene expression through several retinoic acid-responsive elements located in the promoter-enhancer region.

The Oct-3/4 gene product, which belongs to the POU family of transcription factors, is a good candidate for regulating initial differentiation decisions. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Retinoic acid (RA)-induced differentiation of embryonal carcinoma (EC) cells is accompanied by decreased expression of the Oct-3/4 gene. Previous findings show that sequences in the Oct-3/4 enhancer region (designated RARE1) are targets for RA-mediated repression (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Our present results demonstrate conclusively that the TATA-less Oct-3/4 promoter is also a target for RA-induced repression. We identified a novel cis element in the Oct-3/4 promoter harbors a putative Sp1 binding site and a RA-responsive element (designated RAREoct), which are juxtaposed to one another. Protein binding to the Sp1 site is independent of protein binding to the RAREoct sequence. Unlike the RARE1 situated in the Oct-3/4 enhancer which does not contain a typical RAR recognition site, the RAREoct identified in this study consists of three directly repeated motifs that exhibit extensive homology to RARE sequences located in RA-responsive genes. Moreover, the RAREoct shows different DNA-binding characteristics and DNase I footprint patterns with nuclear proteins isolated from undifferentiated versus RA-differentiated EC cells. This suggests that the RAREoct element binds different nuclear proteins in RA-treated and untreated EC cells which most probably belong to the RA receptor, retinoid X receptor, or orphan receptor families of transcription factors. Using site-directed mutagenesis, we show that the RAREoct contributes to the transcriptional activation of Oct-3/4 promoter in P19 cells and, most interestingly, mediates the RA-induced repression in RA-differentiated EC cells. Thus, the RAREoct element could be one of the points of integration of several signalling pathways influencing Oct-3/4 expression. In accordance with the suggestion that suppression of Oct-3/4 expression is a crucial step during embryogenesis, the Oct-3/4 upstream region contains multiple targets for RA-induced repression, probably to ensure accurate and prompt repression of Oct-3/4 expression. It is possible that these repressors are differentially used at specific stages of development in response to various signals.

Animals↗
PubMed1994

Repression of cyclin D1: a novel function of MYC.

Constitutive expression of human MYC represses mRNA levels of cyclin D1 in proliferating BALB/c-3T3 fibroblasts. We expressed a series of mutant alleles of MYC and found that downregulation of cyclin D1 is distinct from previously described properties of MYC. In particular, we found that association with Max is not required for repression of cyclin D1 by MYC in vivo. Conversely, the integrity of a small amino-terminal region (amino acids 92 to 106) of MYC is critical for repression of cyclin D1 but dispensable for transformation of established RAT1A cells. Runoff transcription assays showed that repression occurs at the level of transcription initiation. We cloned the promoter of the gene for human cyclin D1 and found that it lacks a canonical TATA element. Transcription starts at an initiator element similar to that of the adenovirus major late promoter; this element can be directly bound by USF in vitro. Expression of MYC represses the cyclin D1 promoter via core promoter elements and antagonizes USF-mediated transactivation. Taken together, our data define a new pathway for gene regulation by MYC and show that the cyclin D1 gene is a target gene for repression by MYC.

3T3 Cells↗
PubMed1994

Dependence of transcriptional repression on CpG methylation density.

CpG methylation is known to suppress transcription. This repression is generally thought to be related to alterations of chromatin structure that are specified by the methylation. The nature of these chromatin alterations is unknown. Moreover, it has not been clear if the methylation repression occurs in an all-or-none fashion at some critical methylation density, or if intermediate densities of methylation can give intermediate levels of repression. Here I report a stable episomal system which recapitulates many dynamic features of methylation observed in the genome. I have determined the extent of transcriptional repression as a function of four densities of CpG methylation. I find that the repression is a graded but exponential function of the CpG methylation density such that low levels of methylation yield a 67 to 90% inhibition of gene expression. Higher levels of methylation extinguished gene expression completely. Transcription from methylated minichromosomes can be increased by butyrate treatment, suggesting that histone acetylation can reverse some of the repression specified by the methylated state. Sites of preferential demethylation occurred and may have resulted from transcription factor binding or DNA looping.

5-Methylcytosine↗
PubMed1995

The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat.

Rev-Erb is an orphan nuclear receptor which binds as a monomer to the thyroid/retinoic acid receptor half-site AGGTCA flanked 5' by an A/T-rich sequence, referred to here as a Rev monomer site. Fusion of Rev-Erb to the DNA binding domain of yeast GAL4 strongly repressed basal transcription of a GAL4-luciferase reporter gene as a result of the presence of a C-terminal domain containing both the hinge and heptad repeat regions. Nevertheless, wild-type Rev-Erb did not repress basal transcription from the Rev monomer binding site. Therefore, a DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb may function on a different site as a dimer. This approach identified sequences containing two Rev monomer sites arranged as direct repeats with the AGGTCA motifs separated by 2 bp (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. The DNA binding domain contained all of the determinants for Rev-DR2-specific homodimerization. Rev-Erb bound cooperatively as a homodimer to Rev-DR2, and this interaction was 5 to 10 times more stable than Rev-Erb monomer binding to the Rev monomer site. Functionally, Rev-Erb markedly repressed the basal activity of a variety of promoters with a strong Rev-DR2 specificity. The C terminus was required for this repression, consistent with the GAL4 results. However, the Rev-DR2 specificity did not require the C terminus in vivo, since fusion of C-terminally truncated Rev-Erb to a heterologous transactivation domain created a transcriptional activator specific for Rev-DR2. In addition to idealized Rev-DR2 sites, Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily members by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element.

Base Sequence↗
PubMed1996

The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between transcriptional repression by FVIII cDNA elements and the stimulatory effect of butyrate on FVIII cDNA expression.

Base Sequence↗
PubMed1997

Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes.

Nuclear RNA synthesis is repressed during the mitotic phase of each cell cycle. Although total RNA synthesis remains low throughout mitosis, the degree of RNA polymerase II transcription repression on specific genes has not been examined. In addition, it is not known whether mitotic repression of RNA polymerase II transcription is due to polymerase pausing or ejection of transcription elongation complexes from mitotic chromosomes. In this study, we show that RNA polymerase II transcription is repressed in mammalian cells on a number of specific gene regions during mitosis. We also show that the majority of RNA polymerase II transcription elongation complexes are physically excluded from mitotic chromosomes between late prophase and late telophase. Despite generalized transcription repression and stripping of RNA polymerase II complexes from DNA, arrested RNA polymerase II ternary complexes appear to remain on some gene regions during mitosis. The cyclic repression of transcription and ejection of RNA polymerase II transcription elongation complexes may help regulate the transcriptional events that control cell cycle progression and differentiation.

Chromatin↗
PubMed1998

Regulation of cell size by glucose is exerted via repression of the CLN1 promoter.

Yeast cells are keenly sensitive to the availability and quality of nutrients. Addition of glucose to cells growing on a poorer carbon source elicits a cell cycle delay during G1 phase and a concomitant increase in the cell size. The signal is transduced through the RAS-cyclic AMP pathway. Using synchronized populations of G1 cells, we show that the increase in cell size required for budding depends upon CLN1 but not other G1 cyclins. This delay in cell cycle initiation is associated specifically with transcriptional repression of CLN1. CLN2 is not repressed. Repression of CLN1 is not limited to the first cycle following glucose addition but occurs in each cell cycle during growth on glucose. A 106-bp fragment of the CLN1 promoter containing the three MluI cell cycle box (MCB) core elements responsible for the majority of CLN1-associated upstream activation sequence activity is sufficient to confer glucose-induced repression on a heterologous reporter. A mutant CLN2 promoter that is rendered dependent upon its three MCB core elements due to inactivation of its Swi4-dependent cell cycle box (SCB) elements is also repressed by glucose. The response to glucose is partially suppressed by inactivation of SWI4, but not MBP1, which is consistent with the dependence of MCB core elements upon the SCB-binding transcription factor (SBF). We suggest that differential regulation of CLN1 and CLN2 by glucose results from differences in the capacity of SBF to activate transcription driven by SCB and MCB core elements. Finally, we show that transcriptional repression is sufficient to explain the cell cycle delay that occurs in response to glucose.

Cyclins↗
PubMed1998

Brachyury-related transcription factor Tbx2 and repression of the melanocyte-specific TRP-1 promoter.

Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.

Animals↗
PubMed1999

p53-mediated repression of alpha-fetoprotein gene expression by specific DNA binding.

Aberrant expression of the alpha-fetoprotein (AFP) gene is characteristic of a majority of hepatocellular carcinoma cases and serves as a diagnostic tumor-specific marker. By dissecting regulatory mechanisms through electromobility gel shift, transient-transfection, Western blot, and in vitro transcription analyses, we find that AFP gene expression is controlled in part by mutually exclusive binding of two trans-acting factors, p53 and hepatic nuclear factor 3 (HNF-3). HNF-3 protein activates while p53 represses AFP transcription through sequence-specific binding within the previously identified AFP developmental repressor domain. A single mutation within the DNA binding domain of p53 protein or a mutation of the p53 DNA binding element within the AFP developmental repressor eliminates p53-repressive effects in both transient-transfection and cell-free expression systems. Coexpression of p300 histone acetyltransferase, which has been shown to acetylate p53 and increase specific DNA binding, amplifies the p53-mediated repression. Western blot analysis of proteins present in developmentally staged, liver nuclear extracts reveal a one-to-one correlation between activation of p53 protein and repression of AFP during hepatic development. Induction of p53 in response to actinomycin D or hypoxic stress decreases AFP expression. Studies in fibroblast cells lacking HNF-3 further support a model for p53-mediated repression that is both passive through displacement of a tissue-specific activating factor and active in the presence of tissue-specific corepressors. This mechanism for p53-mediated repression of AFP gene expression may be active during hepatic differentiation and lost in the process of tumorigenesis.

Animals↗
PubMed1999

Corepressor required for adenovirus E1B 55,000-molecular-weight protein repression of basal transcription.

Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.

Adenovirus E1B Proteins↗
PubMed1999

A novel role for helix 12 of retinoid X receptor in regulating repression.

Nutrients, drugs, and hormones influence transcription during differentiation and metabolism by binding to high-affinity nuclear receptors. In the absence of ligand, some but not all nuclear receptors repress transcription as a heterodimer with retinoid X receptor (RXR). Here we define a novel role for helix 12 (H12) in sterically masking the corepressor (CoR) binding site in apo-RXR. Removing H12 converts RXR to a potent transcriptional repressor. The length but not the specific sequence of H12 is critical for masking RXR's intrinsic repression function. This contrasts with the amphipathic character required for mediating ligand-dependent activation and coactivator recruitment. Physiologically, we show that heterodimerization of RXR with apo-thyroid hormone receptor (TR) unmasks the CoR binding site in RXR and allows the TR-RXR heterodimer to repress. A molecular mechanism that involves sequence-specific interaction between RXR H12 and the coactivator-binding surface of the nuclear receptor is proposed for this heterodimerization-mediated unmasking. Peroxisome proliferator-activated receptor gamma does not interact as well with RXR H12, thus explaining its inability to repress transcription as an RXR heterodimer. The requirement to unmask RXR's latent repression function explains why only certain RXR partners repress transcription.

Amino Acid Sequence↗
PubMed2000

Rapid deadenylation and Poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos.

The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.

3' Untranslated Regions↗
PubMed2000

Transcriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase.

B-lymphocyte-induced maturation protein (Blimp-1) is a transcriptional repressor that is considered to be a master regulator of terminal B-cell development because it is sufficient to trigger differentiation in the BCL(1)-cell model. Transcription of the c-myc gene is repressed by Blimp-1 during B-cell differentiation. In this study, we have explored the mechanism by which Blimp-1 represses transcription by using Gal4-fusion protein assays and assays in which Blimp-1 represses the natural c-myc promoter. The results show that Blimp-1 represses the c-myc promoter by an active mechanism that is independent of the adjacently bound activator YY1. Blimp-1 contains two regions that independently associate with histone deacetylase (HDAC) and endogenous Blimp-1 in nuclear extracts binds in vitro to the c-myc Blimp-1 site in a complex containing HDAC. The functional importance of recruiting HDAC for Blimp-1-dependent repression of c-myc transcription is supported by two experiments. First, the HDAC inhibitor tricostatin A inhibits Blimp-1-dependent repression in cotransfection assays. Second, a chromatin immunoprecipitation assay shows that expression of Blimp-1 causes deacetylation of histone H3 associated with the c-myc promoter, and this deacetylation depends on the Blimp-1 binding site in the c-myc promoter.

Acetylation↗
PubMed2001

The Hsp70-Ydj1 molecular chaperone represses the activity of the heme activator protein Hap1 in the absence of heme.

In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

Blotting, Western↗
PubMed2002

The E2 ubiquitin conjugase Rad6 is required for the ArgR/Mcm1 repression of ARG1 transcription.

Transcription of the Saccharomyces cerevisiae ARG1 gene is under the control of both positive and negative elements. Activation of the gene in minimal medium is induced by Gcn4. Repression occurs in the presence of arginine and requires the ArgR/Mcm1 complex that binds to two upstream arginine control (ARC) elements. With the recent finding that the E2 ubiquitin conjugase Rad6 modifies histone H2B, we examined the role of Rad6 in the regulation of ARG1 transcription. We find that Rad6 is required for repression of ARG1 in rich medium, with expression increased approximately 10-fold in a rad6 null background. Chromatin immunoprecipitation analysis indicates increased binding of TATA-binding protein in the absence of Rad6. The active-site cysteine of Rad6 is required for repression, implicating ubiquitination in the process. The effects of Rad6 at ARG1 involve two components. In one of these, histone H2B is the likely target for ubiquitination by Rad6, since a strain expressing histone H2B with the principal ubiquitination site converted from lysine to arginine shows a fivefold relief of repression. The second component requires Ubr1 and thus likely the pathway of N-end rule degradation. Through the analysis of promoter constructs with ARC deleted and an arg80 rad6 double mutant, we show that Rad6 repression is mediated through the ArgR/Mcm1 complex. In addition, analysis of an ada2 rad6 deletion strain indicated that the SAGA acetyltransferase complex and Rad6 act in the same pathway to repress ARG1 in rich medium.

Arginine↗