scholarly journals Multiple Hemopoietic Defects and Lymphoid Hyperplasia in Mice Lacking the Transcriptional Activation Domain of the c-Rel Protein

1998 ◽  
Vol 187 (7) ◽  
pp. 973-984 ◽  
Author(s):  
Daniel Carrasco ◽  
Janet Cheng ◽  
Anne Lewin ◽  
Glenn Warr ◽  
Hyekyung Yang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Extramedullary Hematopoiesis ◽  
Lymphoid Hyperplasia ◽  
Homozygous Mutation ◽  
Immune Functions ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Bone Marrow Hypoplasia

The c-rel protooncogene encodes a member of the Rel/nuclear factor (NF)-κB family of transcriptional factors. To assess the role of the transcriptional activation domain of c-Rel in vivo, we generated mice expressing a truncated c-Rel (Δc-Rel) that lacks the COOH-terminal region, but retains a functional Rel homology domain. Mice with an homozygous mutation in the c-rel region encoding the COOH terminus of c-Rel (c-relΔCT/ΔCT) display marked defects in proliferative and immune functions. c-relΔCT/ΔCT animals present histopathological alterations of hemopoietic tissues, such as an enlarged spleen due to lymphoid hyperplasia, extramedullary hematopoiesis, and bone marrow hypoplasia. In older c-relΔCT/ΔCT mice, lymphoid hyperplasia was also detected in lymph nodes, liver, lung, and stomach. These animals present a more severe phenotype than mice lacking the entire c-Rel protein. Thus, in c-relΔCT/ΔCT mice, the lack of c-Rel activity is less efficiently compensated by other NF-κB proteins.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis

Development ◽  
2021 ◽  
Vol 148 (24) ◽  
Author(s):  
Samantha A. Russell ◽  
Kaitlin M. Laws ◽  
Greg J. Bashaw
Keyword(s):  
Cell Death ◽  
Axon Guidance ◽  
Transcriptional Activation ◽  
Nutrient Sensing ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Egg Chambers ◽  
Death Effector ◽  
Female Germline

ABSTRACT The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

scholarly journals Genetic evidence for transcriptional activation by the yeast IME1 gene product.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 775-784 ◽  
Author(s):  
H E Smith ◽  
S E Driscoll ◽  
R A Sia ◽  
H E Yuan ◽  
A P Mitchell
Keyword(s):  
Fusion Protein ◽  
Gene Product ◽  
Transcriptional Activation ◽  
Regulatory Region ◽  
Missense Mutations ◽  
Activation Domain ◽  
Rich Region ◽  
Transcriptional Activation Domain ◽  
Lexa Binding

Abstract IME1 is required in yeast for meiosis and for expression of IME2 and other early meiotic genes. IME1 is a 360-amino acid polypeptide with central and C-terminal tyrosine-rich regions. We report here that a fusion protein composed of the lexA DNA-binding domain and IME1 activates transcription in vivo of a reporter gene containing upstream lexA binding sites. Activation by the fusion protein shares several features with natural IME1 activity: both are dependent on the RIM11 gene product; both are impaired by the same ime1 missense mutations; both are restored by intragenic suppressors. The central tyrosine-rich region is sufficient to activate transcription when fused to lexA. Deletion of this putative activation domain results in a defective IME1 derivative. Function of the deletion derivative is restored by fusion to the acidic Herpesvirus VP16 activation domain. The C-terminal tyrosine-rich region is dispensable for transcriptional activation; rather it renders activation dependent upon starvation and RIM11. Immunofluorescence studies indicate that an IME1-lacZ fusion protein is concentrated in the nucleus. These observations are consistent with a model in which IME1 normally stimulates IME2 expression by providing a transcriptional activation domain at the IME2 5' regulatory region.

scholarly journals Characterization of Neurospora CPC1, a bZIP DNA-binding protein that does not require aligned heptad leucines for dimerization.

1991 ◽  
Vol 11 (2) ◽  
pp. 935-944 ◽  
Author(s):  
J L Paluh ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activator ◽  
Dimerization Domain ◽  
Activation Domain ◽  
Band Shift ◽  
Transcriptional Activation Domain ◽  
Chimeric Polypeptide

CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa. CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1. A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA. However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding. Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly. The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N. crassa. Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency. However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1. The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements. The basic region was required for DNA binding but not for dimerization. CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4. When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented. DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization. Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.

scholarly journals Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein

2002 ◽  
Vol 83 (3) ◽  
pp. 517-524 ◽  
Author(s):  
Nikita Avvakumov ◽  
Majdina Sahbegovic ◽  
Zhiying Zhang ◽  
Michael Shuen ◽  
Joe S. Mymryk
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Adenovirus Type ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Adenovirus Type 5

Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directly in vitro, it is not clear how this relates to its function in vivo. The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro. Deletion of E1A amino acids 201–220 inhibited binding and a minimal fragment encompassing amino acids 201–218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro. These data suggest that E1A does not bind DNA in vivo.

scholarly journals In Vivo Analysis of Functional Regions within Yeast Rap1p

1999 ◽  
Vol 19 (11) ◽  
pp. 7481-7490 ◽  
Author(s):  
Ian R. Graham ◽  
Robin A. Haw ◽  
Karen G. Spink ◽  
Kathryn A. Halden ◽  
Alistair Chambers
Keyword(s):  
Cell Growth ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Protein A ◽  
Point Mutations ◽  
Northern Analysis ◽  
Activation Domain ◽  
Linker Region ◽  
Transcriptional Activation Domain

ABSTRACT We have analyzed the in vivo importance of different regions of Rap1p, a yeast transcriptional regulator and telomere binding protein. A yeast strain (SCR101) containing a regulatable RAP1 gene was used to test functional complementation by a range of Rap1p derivatives. These experiments demonstrated that the C terminus of the protein, containing the putative transcriptional activation domain and the regions involved in silencing and telomere function, is not absolutely essential for cell growth, a result confirmed by sporulation of a diploid strain containing a C terminal deletion derivative ofRAP1. Northern analysis with cells that expressed Rap1p lacking the transcriptional activation domain revealed that this region is important for the expression of only a subset of Rap1p-activated genes. The one essential region within Rap1p is the DNA binding domain. We have investigated the possibility that this region has additional functions. It contains two Myb-like subdomains separated by a linker region. Individual point mutations in the linker region had no effect on Rap1p function, although deletion of the region abolished cell growth. The second Myb-like subdomain contains a large unstructured loop of unknown function. Domain swap experiments with combinations of elements from DNA binding domains of Rap1p homologues from different yeasts revealed that major changes can be made to the amino acid composition of this region without affecting Rap1p function.

scholarly journals Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067 ◽  
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

Characterization of Neurospora CPC1, a bZIP DNA-binding protein that does not require aligned heptad leucines for dimerization

1991 ◽  
Vol 11 (2) ◽  
pp. 935-944
Author(s):  
J L Paluh ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activator ◽  
Dimerization Domain ◽  
Activation Domain ◽  
Band Shift ◽  
Transcriptional Activation Domain ◽  
Chimeric Polypeptide

CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa. CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1. A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA. However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding. Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly. The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N. crassa. Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency. However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1. The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements. The basic region was required for DNA binding but not for dimerization. CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4. When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented. DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization. Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.

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