scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814 ◽  
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

scholarly journals Components of the ESCRT Pathway, DFG16, and YGR122w Are Required for Rim101 To Act as a Corepressor with Nrg1 at the Negative Regulatory Element of the DIT1 Gene of Saccharomyces cerevisiae

2005 ◽  
Vol 25 (15) ◽  
pp. 6772-6788 ◽  
Author(s):  
Karen Rothfels ◽  
Jason C. Tanny ◽  
Enikö Molnar ◽  
Helena Friesen ◽  
Cosimo Commisso ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Proteins ◽  
Target Genes ◽  
Regulatory Element ◽  
Active Form ◽  
Dna Binding Proteins ◽  
Negative Regulatory Element

ABSTRACT The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.

scholarly journals RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins

1994 ◽  
Vol 14 (2) ◽  
pp. 1230-1244
Author(s):  
W Reith ◽  
C Ucla ◽  
E Barras ◽  
A Gaud ◽  
B Durand ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Dna Binding ◽  
Hepatitis B ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Mrna Levels ◽  
B Virus ◽  
Factor C

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

scholarly journals Differential Contributions of DNA-Binding Proteins to Polycomb Response Element Activity at the Drosophila giant Gene

Genetics ◽  
2020 ◽  
Vol 214 (3) ◽  
pp. 623-634
Author(s):  
Elnaz Ghotbi ◽  
Kristina Lackey ◽  
Vicki Wong ◽  
Katie T. Thompson ◽  
Evan G. Caston ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Target Genes ◽  
Dna Binding Proteins ◽  
Polycomb Group ◽  
Link Type ◽  
Element Activity

Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic regulators whose primary function is to maintain the transcriptional repression of target genes. Recruitment of Drosophila melanogaster PcG proteins to target genes requires the presence of one or more Polycomb Response Elements (PREs). The functions or necessity for more than one PRE at a gene are not clear and individual PREs at some loci may have distinct regulatory roles. Various combinations of sequence-specific DNA-binding proteins are present at a given PRE, but only Pleiohomeotic (Pho) is present at all strong PREs. The giant (gt) locus has two PREs, a proximal PRE1 and a distal PRE2. During early embryonic development, Pho binds to PRE1 ∼30-min prior to stable binding to PRE2. This observation indicated a possible dependence of PRE2 on PRE1 for PcG recruitment; however, we find here that PRE2 recruits PcG proteins and maintains transcriptional repression independently of Pho binding to PRE1. Pho-like (Phol) is partially redundant with Pho during larval development and binds to the same DNA sequences in vitro. Although binding of Pho to PRE1 is dependent on the presence of consensus Pho-Phol-binding sites, Phol binding is less so and appears to play a minimal role in recruiting other PcG proteins to gt. Another PRE-binding protein, Sp1/Kruppel-like factor, is dependent on the presence of Pho for PRE1 binding. Further, we show that, in addition to silencing gene expression, PcG proteins dampen transcription of an active gene.

scholarly journals Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

scholarly journals RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins.

1994 ◽  
Vol 14 (2) ◽  
pp. 1230-1244 ◽  
Author(s):  
W Reith ◽  
C Ucla ◽  
E Barras ◽  
A Gaud ◽  
B Durand ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Dna Binding ◽  
Hepatitis B ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Mrna Levels ◽  
B Virus ◽  
Factor C

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

scholarly journals Fluorescent Single-Stranded DNA-Binding Proteins Enable In Vitro and In Vivo Studies

2012 ◽  
pp. 235-244
Author(s):  
Piero R. Bianco ◽  
Adam J. Stanenas ◽  
Juan Liu ◽  
Christopher S. Cohan
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
In Vivo Studies ◽  
Single Stranded Dna

scholarly journals Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jenny Kaur Singh ◽  
Rebecca Smith ◽  
Magdalena B. Rother ◽  
Anton J. L. de Groot ◽  
Wouter W. Wiegant ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Binding Proteins ◽  
Zinc Finger Protein ◽  
Dna Binding Proteins ◽  
End Joining ◽  
Dsb Repair ◽  
Non Homologous End Joining

AbstractDNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.

scholarly journals Complementary roles of Pif1 helicase and single stranded DNA binding proteins in stimulating DNA replication through G-quadruplexes

2019 ◽  
Author(s):  
Melanie A Sparks ◽  
Saurabh P Singh ◽  
Peter M Burgers ◽  
Roberto Galletto
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Proteins ◽  
Protective Role ◽  
Dna Binding Proteins ◽  
Single Stranded Dna ◽  
Fork Stalling ◽  
Pif1 Helicase

Abstract G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5′ to 3′ helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5′-3′-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3′-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.

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