scholarly journals Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Pierre Khoueiry ◽  
Charles Girardot ◽  
Lucia Ciglar ◽  
Pei-Chen Peng ◽  
E Hilary Gustafson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Sequence Variation ◽  
Sequence Divergence ◽  
Protein Protein Interactions ◽  
Enhancer Activity ◽  
Neutral Site ◽  
Evolutionary Changes ◽  
Transcription Factor Occupancy

Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.

The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation

1994 ◽  
Vol 107 (8) ◽  
pp. 2055-2063 ◽  
Author(s):  
A.P. Wolffe
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Protein Interactions ◽  
Multigene Family ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Eukaryotic Genes ◽  
Rna Genes

Differential expression of the oocyte and somatic 5 S RNA genes during Xenopus development can be explained by changes in transcription factor and histone interactions with the two types of gene. Both factors and histones bind 5 S RNA genes with specificity. Protein-protein interactions determine the stability of potentially transcriptionally active or repressed nucleoprotein complexes. A decline in transcription factor abundance, differential binding of transcription factors to oocyte and somatic 5 S genes, and increased competition with the histones for association with DNA during early embryogenesis, can account for the developmental decision to selectively repress the oocyte genes, while retaining the somatic genes in the transcriptionally active state. The 5 S ribosomal genes of Xenopus are perhaps the simplest eukaryotic genes to show regulated expression during development. A large multigene family (oocyte 5 S DNA) is transcriptionally active in oocytes but is repressed in somatic cells, whereas a small multigene family (somatic 5 S DNA) is active in both cell types. A potential molecular mechanism to explain the developmental switch that turns off oocyte 5 S DNA transcription has been experimentally reconstructed in vitro and more recently tested in vivo. Central to this mechanism is the specific association of both transcription factors and histones with 5 S RNA genes. How the interplay of histones and transcription factors is thought to affect transcription, and how their respective contributions might change during development from an oocyte, to an embryo and eventually to a somatic cell is the focus of this review.

scholarly journals The transcription factor PU.1 is involved in macrophage proliferation.

1996 ◽  
Vol 184 (1) ◽  
pp. 61-69 ◽  
Author(s):  
A Celada ◽  
F E Borràs ◽  
C Soler ◽  
J Lloberas ◽  
M Klemsz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Protein Interactions ◽  
Surface Expression ◽  
Protein Protein Interactions ◽  
Expression Construct ◽  
Antisense Constructs ◽  
Ets Family ◽  
Macrophage Colony

PU.1 is a tissue-specific transcription factor that is expressed in cells of the hematopoietic lineage including macrophages, granulocytes, and B lymphocytes. Bone marrow-derived macrophages transfected with an antisense PU.1 expression construct or treated with antisense oligonucleotides showed a decrease in proliferation compared with controls. In contrast, bone marrow macrophages transfected with a sense PU.1 expression construct displayed enhanced macrophage colony-stimulating factor (M-CSF)-dependent proliferation. Interestingly, there was no effect of sense or antisense constructs of PU.1 on the proliferation of the M-CSF-independent cell line, suggesting that the response was M-CSF dependent. This was further supported by the finding that macrophages transfected with a sense or an antisense PU.1 construct showed, respectively, an increased or a reduced level of surface expression of receptors for M-CSF. The enhancement of proliferation seems to be selective for PU.1, since transfections with several other members of the ets family, including ets-2 and fli-1, had no effect. Various mutants of PU.1 were also tested for their ability to affect macrophage proliferation. A reduction in macrophage proliferation was found when cells were transfected with a construct in which the DNA-binding domain of PU.1 was expressed. The PEST (proline-, glutamic acid-, serine-, and threonine-rich region) sequence of the PU.1 protein, which is an important domain for protein-protein interactions in B cells, was found to have no influence on PU.1-enhanced macrophage proliferation when an expression construct containing PU.1 minus the PEST domain was transfected into bone marrow-derived macrophages. In vivo, PU.1 is phosphorylated on several serine residues. The transfection of plasmids containing PU.1 with mutations at each of five serines showed that only positions 41 and 45 are critical for enhanced macrophage proliferation. We conclude that PU.1 is necessary for the M-CSF-dependent proliferation of macrophages. One of the proliferation-relevant targets of this transcription factor could be the M-CSF receptor.

scholarly journals Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

2009 ◽  
Vol 29 (15) ◽  
pp. 4103-4115 ◽  
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Thomas E. Akie ◽  
Tyler B. Moran ◽  
Andrew J. Woo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fetal Liver ◽  
Gel Filtration ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

scholarly journals Author response: Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity

2017 ◽  
Author(s):  
Pierre Khoueiry ◽  
Charles Girardot ◽  
Lucia Ciglar ◽  
Pei-Chen Peng ◽  
E Hilary Gustafson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Sequence ◽  
Author Response ◽  
Enhancer Activity ◽  
Evolutionary Changes ◽  
Transcription Factor Occupancy

The MYST Domain Histone Acetyltransferase TIP60 Interacts with and Is as a Coactivator of the Myeloid Transcription Factor C/EBPα.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4338-4338
Author(s):  
Deepak Bararia ◽  
Arun K. Trivedi ◽  
Abdul Peer Zada ◽  
Hermann M. Behre ◽  
Gerhard Behre ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Myeloid Leukemia ◽  
Interacting Proteins ◽  
Protein Protein Interactions ◽  
Histone Acetyl Transferase ◽  
Nuclear Extracts ◽  
Important Player ◽  
Factor C

Abstract Recently, we and others have shown that protein-protein interactions play an important role in the pathogenesis of leukemia, and that the transcription factor C/EBPα is a key player in granulopoiesis and leukemogenesis. In the present study, we sought to identify C/EBPα interacting proteins. A glutathione-S-transferase-C/EBPα fusion protein was used to pull down interacting proteins from U937 nuclear extracts. These proteins were analyzed by 2-D gel electrophoresis, 1-D nano LC and identified by MALDI or MALDI-TOF/TOF. Several novel C/EBPα interactors were identified including known proteins like Rb, hnRNP and E2F4. Two novel interactions, one of cell cycle regulator protein MCM5 and the other with the MYST domain histone acetyl transferase TIP60 with C/EBPα were further confirmed by using pull-down and co-immunoprecipitation experiments. TP60 was able to markedly enhance C/EBPα mediated transcription in reporter gene assays, suggesting that TIP60 is a co-activator of C/EBPα. This co-activator function of TIP60 was dependent on an intact histone acetyl transferase domain and on the C/EBPα DNA binding domain. TIP60 was found to be associated with the human C/EBPα promoter in-vivo in a chromatin immunoprecipitation assay with a concomitant increase in histone H3 and H4 acetylation. Furthermore, we observed a lower expression of TIP60 mRNA in U937 CD11b− compared to retinoic acid induced U937 CD11b+ cells suggesting that higher TIP60 expression is associated with myeloid differentiation. There was also a marked correlation between the expression levels of TIP60 and C/EBPa in normal bone marrow, chronic myeloid leukemia and acute myeloid leukemia samples with t(8;21), inv(16) and t(15;17). This finding further confirms the functional synergism between C/EBPα and TIP60 and suggests that TIP60 might be an important player in leukemogenesis.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

scholarly journals Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021 ◽  
Vol 49 (7) ◽  
pp. 3856-3875
Author(s):  
Marina Kulik ◽  
Melissa Bothe ◽  
Gözde Kibar ◽  
Alisa Fuchs ◽  
Stefanie Schöne ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Dna Binding ◽  
Receptor Binding ◽  
Binding Sites ◽  
Specific Gene ◽  
Functional Diversification ◽  
Enhancer Activity ◽  
Sequence Composition

Abstract The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (&gt;10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

scholarly journals EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

2021 ◽  
Author(s):  
Liqing Jia ◽  
Xiaolu Ge ◽  
Chao Du ◽  
Linna Chen ◽  
Yanhong Zhou ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Protein Translation ◽  
Protein Protein Interactions ◽  
Promising Target ◽  
Tgfβ Receptor ◽  
Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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