scholarly journals Differentiation-Induced Cleavage of Cutl1/CDP Generates a Novel Dominant-Negative Isoform That Regulates Mammary Gene Expression

2006 ◽  
Vol 26 (20) ◽  
pp. 7466-7478 ◽  
Author(s):  
Urmila Maitra ◽  
Jin Seo ◽  
Mary M. Lozano ◽  
Jaquelin P. Dudley
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Late Pregnancy ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Dominant Negative ◽  
Full Length ◽  
Cysteine Protease Inhibitor ◽  
Specific Gene ◽  
C Terminus

ABSTRACT Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland.

Upregulation of eIF4E in Nucleophosmin 1 (NPM1) Haploinsufficient Cells Alters CCAAT Enhancer Binding Protein Alpha (C/EBPα) Activity: Implications for MDS and AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2432-2432
Author(s):  
Nirmalee Abayasekara ◽  
Michelle Levine ◽  
Niccolo Bolli ◽  
Hong Sun ◽  
Matthew Silver ◽  
...  
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Translational Control ◽  
Conflicts Of Interest ◽  
Mrna Translation ◽  
Dominant Negative ◽  
Full Length ◽  
Initiation Factor ◽  
Specific Gene ◽  
Specific Gene Expression

Abstract Abstract 2432 NPM1, is a highly conserved, ubiquitous nucleolar phosphoprotein that belongs to the nucleoplasmin family of nuclear chaperones. NPM1−/− mice die at mid-gestation (E11.5) from anemia, underscoring the gene's role in embryonic development. NPM1 is one of the most frequently mutated genes in AML. Mutations in NPM1 are found in 50% of normal karyotype AML patients, and mutant NPM1 (NPMc+) is aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Furthermore, NPM1 maps to a region on chromosome 5q that is the target of deletions in both de novo and therapy-associated human MDS. NPM1 thus acts as a haploinsufficient tumor suppressor in the hematological compartment, although the mechanism of its contribution to dysmyelopoiesis remains unknown. NPM-1+/− mice develop a hematological syndrome similar to that observed in human MDS, and develop AML over time. The NPM1 deficient model therefore provides a platform to interrogate the molecular basis of MDS. We identified nucleophosmin (NPM1) in a screen for protein binding partners of C/EBPα. C/EBPα is a single exon gene, but is expressed as two isoforms that arise by alternate translation start sites to yield a full length C/EBPα p42 and a truncated dominant negative C/EBPα p30 isoform. Translational control of isoform expression is orchestrated by a conserved upstream open reading frame (uORF) in the 5' untranslated region (5'UTR) and modulated by the translation initiation factors eIF4E and eIF2. We generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. These lines are IL-3-dependent and inducible toward neutrophil maturation with GM-CSF and/ or all- trans retinoic acid (ATRA). Neutrophils derived from MNPM1+/− cells display defective neutrophil-specific gene expression, including a cassette of C/EBPα-dependent genes. These observations led us to postulate that myeloid abnormalities in NPM1 deficiency reflect an aberrant NPM1-C/EBPα axis. We show that NPM1 haploinsufficiency upregulates eIF4E (eukaryotic initiation factor 4E) (but not eIF2), which binds the mRNA-Cap (m7-GTP) as part of the mRNA translation initiation complex, eIF4F. Increased eIF4E is observed in about 30% of all malignancies. Initial increased eIF4E levels in MNPM+/− cells likely reflect transcriptional activation by the oncoprotein c-Myc, protein levels of which are also elevated in MNPM1+/− cells. We propose that increased eIF4E then induces increased C/EBPαp30 translation. C/EBPαp30 is a dominant negative inhibitor of full length C/EBPαp42 activity and disrupts normal neutrophil development. Furthermore, we demonstrate that C/EBPαp30 but not C/EBPαp42, activates the eIF4E promoter. We propose a positive feedback loop, wherein increased C/EBPαp30 induced by eIF4E further increases the expression of eIF4E. Our data suggest that NPM1 deficiency modulates neutrophil-specific gene expression by altering C/EBPα. We propose an aberrant feed-forward mechanism that increases levels of both eIF4E and C/EBPαp30 and likely contributes to MDS associated with NPM1 deficiency. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Human SWI-SNF Complex Protein p270 Is an ARID Family Member with Non-Sequence-Specific DNA Binding Activity

2000 ◽  
Vol 20 (9) ◽  
pp. 3137-3146 ◽  
Author(s):  
Peter B. Dallas ◽  
Stephen Pacchione ◽  
Deborah Wilsker ◽  
Valerie Bowrin ◽  
Ryuji Kobayashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Structural Features ◽  
Binding Activity ◽  
Antigenic Specificity ◽  
Specific Gene ◽  
Binding Motif ◽  
Creb Binding Protein ◽  
Specific Gene Expression ◽  
Dna Binding Activity

ABSTRACT p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein. The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain). The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogastergene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.

scholarly journals Novel member of the zinc finger superfamily: A C2-HC finger that recognizes a glia-specific gene.

1992 ◽  
Vol 12 (12) ◽  
pp. 5632-5639 ◽  
Author(s):  
J G Kim ◽  
L D Hudson
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Chelating Agent ◽  
Proteolipid Protein ◽  
Polymerase Chain Reaction Analysis ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Order Structure ◽  
Specific Gene ◽  
Sequence Comparisons

A novel member of the zinc finger superfamily was cloned by virtue of its binding to cis-regulatory elements of a glia-specific gene, the myelin proteolipid protein (PLP) gene. Named MyTI (myelin transcription factor I), this gene is most highly transcribed in the developing nervous system, where expression precedes induction of its presumptive target, PLP. Low levels of MyTI transcripts can be detected in nonneural tissues only by polymerase chain reaction analysis. Zinc is a necessary cofactor for DNA binding of MyTI, as the zinc-chelating agent 1,10-orthophenanthroline eliminates binding activity. Zinc may stabilize the DNA-binding domain of MyTI by coordinating three cysteine and one histidine residue in a Cys-X5-Cys-X12-His-X4-Cys (C2-HC) arrangement. The MyTI protein has six fingers of the C2-HC class arranged in two widely separated clusters. These two domains of DNA binding can function independently and recognize the same DNA sequence, suggesting that MyTI may contribute to the higher-order structure of a target promoter by simultaneously binding both proximal and distal sites. The six fingers are highly conserved, suggesting that they arose from successive duplication events, while the linker regions diverge in size and sequence. Both amino acid sequence comparisons and secondary-structure predictions indicate that the C2-HC fingers of MyTI do not resemble the zinc-mediated loops of C2-H2 fingers, C2-C2 fingers, or Cx clusters. MyTI may therefore be the prototype of a new structural family of zinc-stabilized DNA binding proteins.

NF-κB Activation Is Required for C5a-Induced Interleukin-8 Gene Expression in Mononuclear Cells

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3241-3249 ◽  
Author(s):  
Matthew H. Hsu ◽  
Meiying Wang ◽  
Darren D. Browning ◽  
Naofumi Mukaida ◽  
Richard D. Ye
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Pertussis Toxin ◽  
Mononuclear Cells ◽  
Binding Activity ◽  
Dominant Negative ◽  
Interleukin 8 ◽  
Peripheral Blood Mononuclear ◽  
C5a Receptor ◽  
Dna Binding Activity

C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a κB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced κB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the κB sequence from IκB and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the κB sites and by coexpression of a dominant negative IκB construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-κB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway.

Novel member of the zinc finger superfamily: A C2-HC finger that recognizes a glia-specific gene

1992 ◽  
Vol 12 (12) ◽  
pp. 5632-5639
Author(s):  
J G Kim ◽  
L D Hudson
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Chelating Agent ◽  
Proteolipid Protein ◽  
Polymerase Chain Reaction Analysis ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Order Structure ◽  
Specific Gene ◽  
Sequence Comparisons

A novel member of the zinc finger superfamily was cloned by virtue of its binding to cis-regulatory elements of a glia-specific gene, the myelin proteolipid protein (PLP) gene. Named MyTI (myelin transcription factor I), this gene is most highly transcribed in the developing nervous system, where expression precedes induction of its presumptive target, PLP. Low levels of MyTI transcripts can be detected in nonneural tissues only by polymerase chain reaction analysis. Zinc is a necessary cofactor for DNA binding of MyTI, as the zinc-chelating agent 1,10-orthophenanthroline eliminates binding activity. Zinc may stabilize the DNA-binding domain of MyTI by coordinating three cysteine and one histidine residue in a Cys-X5-Cys-X12-His-X4-Cys (C2-HC) arrangement. The MyTI protein has six fingers of the C2-HC class arranged in two widely separated clusters. These two domains of DNA binding can function independently and recognize the same DNA sequence, suggesting that MyTI may contribute to the higher-order structure of a target promoter by simultaneously binding both proximal and distal sites. The six fingers are highly conserved, suggesting that they arose from successive duplication events, while the linker regions diverge in size and sequence. Both amino acid sequence comparisons and secondary-structure predictions indicate that the C2-HC fingers of MyTI do not resemble the zinc-mediated loops of C2-H2 fingers, C2-C2 fingers, or Cx clusters. MyTI may therefore be the prototype of a new structural family of zinc-stabilized DNA binding proteins.

scholarly journals The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
José L Ruiz ◽  
Lisa C Ranford-Cartwright ◽  
Elena Gómez-Díaz
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Anopheles Gambiae ◽  
Mosquito Control ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Malaria Vectors ◽  
Specific Gene ◽  
Mosquito Vector ◽  
Human Malaria

Abstract Anopheles gambiae mosquitoes are primary human malaria vectors, but we know very little about their mechanisms of transcriptional regulation. We profiled chromatin accessibility by the assay for transposase-accessible chromatin by sequencing (ATAC-seq) in laboratory-reared A. gambiae mosquitoes experimentally infected with the human malaria parasite Plasmodium falciparum. By integrating ATAC-seq, RNA-seq and ChIP-seq data, we showed a positive correlation between accessibility at promoters and introns, gene expression and active histone marks. By comparing expression and chromatin structure patterns in different tissues, we were able to infer cis-regulatory elements controlling tissue-specific gene expression and to predict the in vivo binding sites of relevant transcription factors. The ATAC-seq assay also allowed the precise mapping of active regulatory regions, including novel transcription start sites and enhancers that were annotated to mosquito immune-related genes. Not only is this study important for advancing our understanding of mechanisms of transcriptional regulation in the mosquito vector of human malaria, but the information we produced also has great potential for developing new mosquito-control and anti-malaria strategies.

A Global Vista of the Epigenomic State of the Mouse Submandibular Gland

2021 ◽  
pp. 002203452110120
Author(s):  
C. Gluck ◽  
S. Min ◽  
A. Oyelakin ◽  
M. Che ◽  
E. Horeth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Expression Patterns ◽  
Global Gene Expression ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Submandibular Salivary Gland ◽  
Data Sets ◽  
Control Mechanisms ◽  
Chromatin Immunoprecipitation Sequencing

The parotid, submandibular, and sublingual glands represent a trio of oral secretory glands whose primary function is to produce saliva, facilitate digestion of food, provide protection against microbes, and maintain oral health. While recent studies have begun to shed light on the global gene expression patterns and profiles of salivary glands, particularly those of mice, relatively little is known about the location and identity of transcriptional control elements. Here we have established the epigenomic landscape of the mouse submandibular salivary gland (SMG) by performing chromatin immunoprecipitation sequencing experiments for 4 key histone marks. Our analysis of the comprehensive SMG data sets and comparisons with those from other adult organs have identified critical enhancers and super-enhancers of the mouse SMG. By further integrating these findings with complementary RNA-sequencing based gene expression data, we have unearthed a number of molecular regulators such as members of the Fox family of transcription factors that are enriched and likely to be functionally relevant for SMG biology. Overall, our studies provide a powerful atlas of cis-regulatory elements that can be leveraged for better understanding the transcriptional control mechanisms of the mouse SMG, discovery of novel genetic switches, and modulating tissue-specific gene expression in a targeted fashion.

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

scholarly journals Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1166 ◽  
Author(s):  
Olivia R. Buonarati ◽  
Peter B. Henderson ◽  
Geoffrey G. Murphy ◽  
Mary C. Horne ◽  
Johannes W. Hell
Keyword(s):  
Gene Expression ◽  
Brain Tissue ◽  
Neuronal Excitability ◽  
Central Element ◽  
Proteolytic Processing ◽  
Full Length ◽  
Hek293 Cells ◽  
Specific Antibodies ◽  
C Terminus ◽  
Molecular Masses

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (MR)of the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

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