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1.
Methods Enzymol ; 573: 97-117, 2016.
Article in English | MEDLINE | ID: mdl-27372750

ABSTRACT

Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specificities and mode of action. Here, we describe an in vivo method using SNAP-tag-based imaging to assess the de novo deposition of histones and the role of histone chaperones. This method exploits cells expressing SNAP-tagged histones combined with individual cell imaging to visualize directly de novo histone deposition in vivo. We show how, by combining this method with siRNA-based depletion, we could assess the function of two distinct histone chaperones. For this, we provide the details of the method as applied in two examples to characterize the function of the histone chaperones CAF-1 and HIRA. In both cases, we document the impact of their depletion on the de novo deposition of the histone variants H3.1 and H3.3, first in a normal context and second in response to DNA damage. We discuss how this cellular assay offers means to define in a systematic manner the function of any chosen chaperone with respect to the deposition of a given histone variant.


Subject(s)
Histone Chaperones/metabolism , Histones/metabolism , O(6)-Methylguanine-DNA Methyltransferase/genetics , Animals , Cell Culture Techniques/methods , Chromatin/metabolism , DNA Damage , Fluorescent Dyes/analysis , Fluorescent Dyes/metabolism , Guanine/analogs & derivatives , Guanine/metabolism , Histone Chaperones/analysis , Histone Chaperones/genetics , Histones/analysis , Histones/genetics , Humans , Microscopy, Fluorescence/methods , Mutation , O(6)-Methylguanine-DNA Methyltransferase/analysis , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Optical Imaging/methods , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
FEBS Lett ; 464(3): 164-8, 1999 Dec 31.
Article in English | MEDLINE | ID: mdl-10618498

ABSTRACT

The Spi-B transcription factor is an Ets protein expressed in B lymphoid cells and closely related to the Spi-1/PU.1 oncoprotein. By mutational analysis, we showed that Spi-B is phosphorylated by casein kinase II in vitro on four serine residues. Mutation of these four serines to alanines prevented the phosphorylation of Spi-B in vivo, increased the ability of Spi-B to transactivate expression of a reporter gene and led to a decrease of Spi-B stability. We propose that the phosphorylation of Spi-B may participate in the modulation of Spi-B functional activity by controlling its intracellular protein level.


Subject(s)
DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Casein Kinase II , HeLa Cells , Humans , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Transcriptional Activation
4.
Biochem Biophys Res Commun ; 223(2): 257-63, 1996 Jun 14.
Article in English | MEDLINE | ID: mdl-8670269

ABSTRACT

Spi-B is an Ets transcription factor related to the oncoprotein Spi-1/PU.1 and highly expressed in B lymphoid cells. The Ets proteins share a conserved Ets domain that mediates specific DNA binding. Spi-B binds DNA sequences containing a core 5'-GGAA-3' and activates transcription through this motif. Up to date, the biological function of Spi-B remains unknown. Here, we describe the characterization of an alternatively spliced variant of Spi-B, named deltaSpi-B, which has lost the Ets domain. In B lymphoid cells, deltaspi-B and spi-B mRNAs were present simultaneously in a ratio of around 10%. DeltaSpi-B product was not able to bind DNA and was recovered in cytoplasmic cellular extracts. We raise the hypothesis that delta Spi-B might affect Spi-B function by recruiting factors involved in Spi-B activity.


Subject(s)
Alternative Splicing , B-Lymphocytes/metabolism , Burkitt Lymphoma/metabolism , DNA-Binding Proteins/biosynthesis , RNA, Messenger/metabolism , Transcription Factors/biosynthesis , Amino Acid Sequence , Base Sequence , Cell Line , DNA Primers , DNA, Complementary , DNA-Binding Proteins/genetics , Exons , Gene Expression , Genomic Library , Humans , Introns , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Transcription Factors/genetics , Transfection , Tumor Cells, Cultured
5.
Oncogene ; 12(4): 863-73, 1996 Feb 15.
Article in English | MEDLINE | ID: mdl-8632909

ABSTRACT

Spi-1/PU-1 and Spi-B are hematopoietic transcription factors, which, in vitro, display similar affinities for DNA target sequences containing the consensus binding site 5'-GGAA-3'. While the role of Spi-1 in the transcriptional regulation of B cell and myeloid specific genes has been largely demonstrated, the biological function of Spi-B still remains to be elucidated. Since Spi-B and Spi-1 are very divergent in their transactivator domain, these domains might acquire functional specificity in vivo by interacting with different co-factors and/or by undergoing different phosphorylations. First, we observed that casein kinase II phosphorylates Spi-B as well as Spi-1, in vitro. Then, by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione-S-transferase and the transactivator domain of Spi-B, two kinases were identified on their ability to interact and phosphorylate this domain; the MAP kinase ERK1 and the stress activated protein kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation site by using phosphoamino-acid analyses and a Spi-B mutant version with the substitution T56 to A56. Strikingly, ERK1 failed to phosphorylate Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and Spi-1. In addition, other purified Spi-B-kinase activities, unidentified as yet, display similar specificity than ERK1 for Spi-B versus Spi-1. Furthermore, the evident interaction of pRb protein with the transactivator domain of Spi-B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK1 or by the purified Spi-B-kinase activities. Our data revealed multiple phosphorylation sites within Spi-B whose some of them appeared specific for Spi-B versus Spi-1 and which may account for differential regulation of their activities.


Subject(s)
DNA-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Burkitt Lymphoma , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cell Nucleus/enzymology , Chlorocebus aethiops , Chromatography, Affinity , Genes, Retinoblastoma , Glutathione Transferase/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase 3 , Molecular Sequence Data , Phosphorylation , Protein Biosynthesis , Recombinant Fusion Proteins/metabolism , Retinoblastoma Protein/biosynthesis , Retroviridae Proteins, Oncogenic , Substrate Specificity , Transcription, Genetic , Transfection , Tumor Cells, Cultured
6.
Oncogene ; 11(8): 1549-60, 1995 Oct 19.
Article in English | MEDLINE | ID: mdl-7478579

ABSTRACT

PU.1 (Spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid (granulocytes, monocytes and macrophages) and B cells. PU.1 is upregulated early during commitment of multipotential progenitors to the myeloid lineages and inhibition of PU.1 function in human CD34+ progenitors prior to this upregulation blocks myeloid colony formation. Since PU.1 expression appears to play a role in hematopoietic development, we characterized the PU.1 promoter. Here we report that the murine PU.1 promoter, as well as the human promoter, demonstrate tissue-specific reporter gene expression in myeloid cell lines but not in T cells and HeLa (non-hematopoietic cells) cells. Deletion analysis of the PU.1 promoter indicates that tissue-specific functional elements are encoded in the -61 to -39 bp and -7 to +34 bp regions. The first region contains a functional octamer (Oct) site at -54 bp and an Sp1 site at -39 bp. The second contains a binding site at +20 bp for both PU.1 itself and the related ets family member Spi-B. In vivo footprinting assays demonstrate that a hypersensitive band was detected at the PU.1 site in myeloid cells but not in HeLa. A mutation of the PU.1 site which abolished PU.1 binding caused a significant decrease in promoter activity. Mutation of the Oct and/or Sp1 site results in a lesser decrease of promoter activity in myeloid cells. Co-transfection of PU.1 or Spi-B in cells lacking PU.1 and Spi-B specifically transactivated a minimal promoter containing the PU.1 binding site, indicating that PU.1 can activate its own promoter elements in an autoregulatory loop. Positive autoregulation of the PU.1 promoter may play an important role in the function of PU.1 in myeloid cells.


Subject(s)
DNA-Binding Proteins/genetics , Hematopoiesis , Hematopoietic Stem Cells/physiology , Promoter Regions, Genetic , Animals , Base Sequence , Binding Sites , DNA Footprinting , DNA-Binding Proteins/physiology , Gene Expression Regulation , Humans , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Retroviridae Proteins, Oncogenic , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription Factors/physiology
7.
Oncogene ; 11(2): 303-13, 1995 Jul 20.
Article in English | MEDLINE | ID: mdl-7624145

ABSTRACT

Spi-1/PU.1 and Spi-B encode hematopoietic-specific transcription factors that are the most distantly related members of the Ets family. The Ets proteins share a conserved 85 amino acids DNA binding domain, the Ets domain and recognize various DNA target sites around a common core 5'-GGAA/T-3'. The DNA binding specificities of Spi-1 and Spi-B were investigated by using the method of polymerase chain reaction (PCR)-mediated random site selection. The deduced Spi-1 and Spi-B consensus binding sites are very similar suggesting that the functional activities of Spi-1 and Spi-B cannot be distinguished on the basis of their DNA binding specificities. We identified a putative Spi-1/Spi-B binding site in the promoter region of the c-fes/c-fps protooncogene which encodes a tyrosine kinase expressed predominantly in myeloid cells. In vitro translated Spi-1 and Spi-B proteins were capable to bind this site similarly and to activate the c-fes promoter in HeLa transfected cells. We showed that Spi-1 binds the Spi-1/Spi-B binding site of c-fes in HL-60 cells suggesting that Spi-1 may be involved in the regulation of c-fes transcription in myeloid cells. Intriguingly, we detected only Spi-1 binding to this site in the Raji cell line which express both Spi-1 and Spi-B proteins. This suggests that Spi-1 and Spi-B exhibit different DNA binding activities in vivo although they share similar DNA binding specificities in vitro.


Subject(s)
DNA-Binding Proteins/metabolism , Oncogenes/physiology , Promoter Regions, Genetic , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , DNA/metabolism , HeLa Cells/metabolism , Humans , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Mice , Molecular Sequence Data , Oncogenes/genetics , Polymerase Chain Reaction , Protein Binding , Rabbits , Transcription, Genetic , Transfection , Tumor Cells, Cultured/metabolism
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