scholarly journals The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

1998 ◽  
Vol 141 (2) ◽  
pp. 335-348 ◽  
Author(s):  
Ian de Belle ◽  
Shutao Cai ◽  
Terumi Kohwi-Shigematsu
Keyword(s):  
Nuclear Matrix ◽  
Cpg Island ◽  
Binding Protein ◽  
Jurkat Cells ◽  
Yeast Cells ◽  
Genomic Sequences ◽  
Specific Gene ◽  
Addition Line ◽  
Chromatin Loops

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation.

Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication

2001 ◽  
Vol 114 (10) ◽  
pp. 1861-1866 ◽  
Author(s):  
J. Postberg ◽  
S.A. Juranek ◽  
S. Feiler ◽  
H. Kortwig ◽  
F. Jonsson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Binding Protein ◽  
Specific Interaction ◽  
Eukaryotic Cells ◽  
Dna Molecules ◽  
Telomere Binding Protein ◽  
The Matrix

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8)individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

scholarly journals The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

1997 ◽  
Vol 17 (9) ◽  
pp. 5275-5287 ◽  
Author(s):  
J Liu ◽  
D Bramblett ◽  
Q Zhu ◽  
M Lozano ◽  
R Kobayashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Nuclear Matrix ◽  
Inverted Repeat ◽  
Binding Proteins ◽  
Binding Protein ◽  
Specific Gene ◽  
Lymphoid Tissues ◽  
Tissue Specific ◽  
Mmtv Ltr

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

scholarly journals Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts

1998 ◽  
Vol 111 (20) ◽  
pp. 3035-3043 ◽  
Author(s):  
J.Y. Choi ◽  
A.J. van Wijnen ◽  
F. Aslam ◽  
J.D. Leszyk ◽  
J.L. Stein ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Osteoblast Differentiation ◽  
Matrix Protein ◽  
Binding Protein ◽  
Protein Composition ◽  
Matrix Proteins ◽  
Specific Gene ◽  
Two Dimensional Gel Electrophoresis ◽  
Nuclear Matrix Proteins ◽  
Differential Retention

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.

scholarly journals Studies on the effect of an heterologous fatty acid-binding protein on acyl-CoA oxidase induction in Saccharomyces cerevisiae

1994 ◽  
Vol 301 (2) ◽  
pp. 615-620 ◽  
Author(s):  
I Smaczyńska ◽  
M Skoneczny ◽  
A Kurlandzka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Yeast Cells ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Acyl Coa Oxidase

The participation of fatty acid-binding protein (FABP) in the induction of peroxisomal beta-oxidation of fatty acids was investigated in vivo in an heterologous system. Bovine heart FABP was expressed in Saccharomyces cerevisiae under the control of two different promoters: a constitutive one and an oleic acid-inducible one. Constructs were introduced into yeast cells on multicopy and integrating plasmids. The heterologous FABP was present in yeast cells in two isoforms having pI values of about 5 and was able to bind oleic acid. The heterologous FABP had no significant effect on acyl-CoA oxidase activity at various concentrations of the inducing agent.

scholarly journals Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

1991 ◽  
Vol 11 (3) ◽  
pp. 1718-1723 ◽  
Author(s):  
Y Koltin ◽  
L Faucette ◽  
D J Bergsma ◽  
M A Levy ◽  
R Cafferkey ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Binding Protein ◽  
Interleukin 2 ◽  
Yeast Cells ◽  
Ppiase Activity ◽  
Resistant Mutants

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Function of Tubulin Binding Proteins in Vivo

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 69-80 ◽  
Author(s):  
James A Fleming ◽  
Leticia R Vega ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gene Products ◽  
Salvage Pathway ◽  
Tubulin Binding ◽  
Mutant Cells ◽  
Β Tubulin

Abstract Overexpression of the β-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant α-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-β-tubulin complex, as it does in the α-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant α-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free β-tubulin.

scholarly journals CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses

2020 ◽  
Author(s):  
Kristina Sakers ◽  
Yating Liu ◽  
Lorida Llaci ◽  
Michael J. Vasek ◽  
Michael A. Rieger ◽  
...  
Keyword(s):  
Rna Binding ◽  
Gene Knockout ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cell Types ◽  
Specific Gene ◽  
Specific Cell ◽  
Normal Brain ◽  
Oligodendrocyte Development

AbstractQuaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’ UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte’s territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

scholarly journals Discordant Influence of Blastomyces dermatitidis Yeast-Phase-Specific Gene BYS1 on Morphogenesis and Virulence

2010 ◽  
Vol 78 (6) ◽  
pp. 2522-2528 ◽  
Author(s):  
Theerapong Krajaejun ◽  
Marcel Wüthrich ◽  
Gregory M. Gauthier ◽  
Thomas F. Warner ◽  
Thomas D. Sullivan ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Yeast Cells ◽  
Blastomyces Dermatitidis ◽  
Mouse Lung ◽  
Specific Gene ◽  
Dimorphic Fungus ◽  
Yeast Phase ◽  
Different Strains

ABSTRACT Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic infections in immunocompetent animal hosts. With the publication of genomic sequences from three different strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become possible to easily ascertain the virulence and morphological effects of knocking down the expression of candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of BYS1 alters morphology at 37°C, it does not appear to impair the pathogenicity of B. dermatitidis.

scholarly journals Developmentally Programmed 3′ CpG Island Methylation Confers Tissue- and Cell-Type-Specific Transcriptional Activation

2013 ◽  
Vol 33 (9) ◽  
pp. 1845-1858 ◽  
Author(s):  
Da-Hai Yu ◽  
Carol Ware ◽  
Robert A. Waterland ◽  
Jiexin Zhang ◽  
Miao-Hsueh Chen ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activation ◽  
Functional Role ◽  
Cpg Island ◽  
Embryonic Stem ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Cell Type ◽  
Cell Type Specific

During development, a small but significant number of CpG islands (CGIs) become methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here, we used genome-wide DNA methylation microarrays to identify epigenetic changes during human embryonic stem cell (hESC) differentiation. We discovered a group of CGIs associated with developmental genes that gain methylation after hESCs differentiate. Conversely, erasure of methylation was observed at the identified CGIs during subsequent reprogramming to induced pluripotent stem cells (iPSCs), further supporting a functional role for the CGI methylation. Both global gene expression profiling and quantitative reverse transcription-PCR (RT-PCR) validation indicated opposing effects of CGI methylation in transcriptional regulation during differentiation, with promoter CGI methylation repressing and 3′ CGI methylation activating transcription. By studying diverse human tissues and mouse models, we further confirmed that developmentally programmed 3′ CGI methylation confers tissue- and cell-type-specific gene activationin vivo. Importantly, luciferase reporter assays provided evidence that 3′ CGI methylation regulates transcriptional activation via a CTCF-dependent enhancer-blocking mechanism. These findings expand the classic view of mammalian CGI methylation as a mechanism for transcriptional silencing and indicate a functional role for 3′ CGI methylation in developmental gene regulation.

scholarly journals The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

1989 ◽  
Vol 109 (3) ◽  
pp. 1015-1022 ◽  
Author(s):  
R A Bacon ◽  
A Salminen ◽  
H Ruohola ◽  
P Novick ◽  
S Ferro-Novick
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Transport Defect

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

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