Centromeric transcription maintains centromeric cohesion in human cells

Centromeric transcription has been shown to play an important role in centromere functions. However, lack of approaches to specifically manipulate centromeric transcription calls into question that the proposed functions are a direct consequence of centromeric transcription. By monitoring nascent RNAs, we found that several transcriptional inhibitors exhibited distinct, even opposing, efficacies on the suppression of ongoing gene and centromeric transcription in human cells, whereas under the same conditions, total centromeric RNAs were changed to a lesser extent. The inhibitor suppressing ongoing centromeric transcription weakened centromeric cohesion, whereas the inhibitor increasing ongoing centromeric transcription strengthened centromeric cohesion. Furthermore, expression of CENP-B DNA-binding domain or CENP-B knockdown moderately increased centromeric transcription without altering gene transcription; as a result, centromeric cohesion was accordingly strengthened. Targeting of the Kox1-KRAB domain with CENP-B DB to centromeres specifically decreased centromeric transcription and weakened centromeric cohesion. Thus, based on these findings, we propose that a major function of centromeric transcription is to maintain centromeric cohesion in human cells.

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The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2297-2308.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2297-2308 ◽

Cited By ~ 60

Author(s):

Daniel Menendez ◽

Alberto Inga ◽

Michael A. Resnick

Keyword(s):

Dna Binding ◽

Target Genes ◽

Tumor Development ◽

Human Tumor ◽

Human Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

The Relationship ◽

Biological Outcomes

ABSTRACT Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for “master genes of diversity,” p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

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BZLF1 Activation of the Methylated Form of the BRLF1 Immediate-Early Promoter Is Regulated by BZLF1 Residue 186

Journal of Virology ◽

10.1128/jvi.79.12.7338-7348.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7338-7348 ◽

Cited By ~ 55

Author(s):

Prasanna M. Bhende ◽

William T. Seaman ◽

Henri-Jacques Delecluse ◽

Shannon C. Kenney

Keyword(s):

Dna Binding ◽

Gene Transcription ◽

Viral Genome ◽

Gene Promoter ◽

Binding Domain ◽

Immediate Early ◽

Dna Binding Domain ◽

Barr Virus ◽

Z Dna ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the BRLF1 IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.

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Dynamic Cell Type Specificity of SRC-1 Coactivator in Modulating Uterine Progesterone Receptor Function in Mice

Molecular and Cellular Biology ◽

10.1128/mcb.25.18.8150-8165.2005 ◽

2005 ◽

Vol 25 (18) ◽

pp. 8150-8165 ◽

Cited By ~ 35

Author(s):

Sang Jun Han ◽

Jaewook Jeong ◽

Francesco J. DeMayo ◽

Jianming Xu ◽

Sophia Y. Tsai ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Progesterone Receptor ◽

Gene Transcription ◽

Fluorescent Protein ◽

Binding Domain ◽

Dna Binding Domain ◽

Female Reproduction ◽

Functional Relationships ◽

A Cell

ABSTRACT Regulation of gene transcription by the progesterone receptor (PR) in cooperation with coactivator/corepressor complexes coordinates crucial processes in female reproduction. To investigate functional relationships between PR and steroid receptor coactivators (SRCs) in distinct cell types of uterine tissue during gene transcription, we generated a new transgenic mouse model utilizing a Progesterone Receptor Activity Indicator (PRAI) system that could monitor PR activity in vivo. The PRAI system consists of a modified PR bacterial artificial chromosome (BAC) clone in which the DNA binding domain of the PR was replaced with the yeast Gal4 DNA binding domain. A humanized green fluorescent protein (hrGFP) reporter controlled by the Upstream Activating Sequences for the Gal4 gene (UASG) was inserted in tandem with the modified PR gene. Expression of hrGFP in the uterus demonstrated that the PRAI animal model faithfully replicated PR signaling under various endocrine states. Bigenic PRAI-SRC-1−/− mice revealed that SRC-1 modulates PR activity in the uterus in a cell-specific fashion and is involved in PR gene activation in stroma and myometrium of the uterus in response to estrogen and progesterone. In contrast, SRC-1 was involved in the down-regulation of PR target gene expression in the luminal and glandular epithelial compartments of the uterus after chronic progesterone treatment. Finally, we dissected the means by which SRC-1 dynamically regulates PR activity in each uterine cell compartment and demonstrated that it involves the differential ability of SRC-1 to modulate expression levels of distinct coactivators, corepressors, and PR in a cell-specific fashion.

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Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA

Nucleic Acids Research ◽

10.1093/nar/gku1015 ◽

2014 ◽

Vol 42 (20) ◽

pp. 12523-12536 ◽

Cited By ~ 15

Author(s):

Sophie S. Nicod ◽

Robert O. J. Weinzierl ◽

Lynn Burchell ◽

Andres Escalera-Maurer ◽

Ellen H. James ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Transcription Factor ◽

Dna Binding ◽

Gene Transcription ◽

Mutational Analysis ◽

Virulence Gene ◽

Binding Domain ◽

Dna Binding Domain

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Cytosine base editing systems with minimized off-target effect and molecular size

10.21203/rs.3.rs-414230/v1 ◽

2021 ◽

Author(s):

Ang Li ◽

Hitoshi Mitsunobu ◽

Shin Yoshioka ◽

Suzuki Takahisa ◽

Akihiko Kondo ◽

...

Keyword(s):

Dna Binding ◽

Molecular Size ◽

Human Cells ◽

Binding Domain ◽

Enzyme Function ◽

Dna Binding Domain ◽

Aav Vector ◽

Base Editing ◽

Rational Engineering ◽

Target Effect

Abstract Structure-based rational engineering of the cytosine base editing system Target-AID was performed to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 was eliminated and additional mutations were introduced to restore enzyme function. The resulting tCDA1EQ was effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized gRNA-independent off-targets, as assessed in yeast and human cells. Combining with the smaller Cas9 ortholog system, the smallest cytosine base editing system was created that is within the size limit of AAV vector.

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Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

Epigenetics ◽

10.1080/15592294.2017.1294306 ◽

2017 ◽

Vol 12 (4) ◽

pp. 296-303 ◽

Cited By ~ 12

Author(s):

Jara Teresa Parrilla-Doblas ◽

Rafael R. Ariza ◽

Teresa Roldán-Arjona

Keyword(s):

Dna Binding ◽

Dna Demethylation ◽

Human Cells ◽

Binding Domain ◽

Dna Glycosylase ◽

Dna Binding Domain

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Structural and Functional Heterogeneity of Nuclear Bodies

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4423 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4423-4430 ◽

Cited By ~ 38

Author(s):

Donald B. Bloch ◽

Jean-Daniel Chiche ◽

Donald Orth ◽

Suzanne M. de la Monte ◽

Anthony Rosenzweig ◽

...

Keyword(s):

Dna Binding ◽

Reporter Gene ◽

Gene Transcription ◽

Nuclear Body ◽

Binding Domain ◽

Nuclear Bodies ◽

Biliary Cirrhosis ◽

Resting Cells ◽

Dna Binding Domain ◽

Ifn Γ

ABSTRACT The nuclear body is a cellular structure that appears to be involved in the pathogenesis of acute promyelocytic leukemia and viral infection. In addition, the nuclear body is a target of autoantibodies in patients with the autoimmune disease primary biliary cirrhosis. Although the precise function of the nuclear body in normal cellular biology is unknown, this structure may have a role in the regulation of gene transcription. In a previous investigation, we identified a leukocyte-specific, gamma interferon (IFN-γ)-inducible autoantigen designated Sp140. The objectives of the present study were to investigate the cellular location of Sp140 with respect to the nuclear-body components PML and Sp100 and to examine the potential role of Sp140 in the regulation of gene transcription. We used adenovirus-mediated gene transfer to express Sp140 in human cells and observed that the protein colocalized with PML and Sp100 in resting cells and associated with structures containing PML during mitosis. In cells infected with the adenovirus expressing Sp140 and incubated with IFN-γ, the number of PML-Sp100 nuclear bodies per cell increased but immunoreactive Sp140 was not evenly distributed among the nuclear bodies. Sp140 associated with a subset of IFN-γ-induced PML-Sp100 nuclear bodies. To examine the potential effect of Sp140 on gene transcription, a plasmid encoding Sp140 fused to the DNA-binding domain of GAL4 was cotransfected into COS cells with a chloramphenicol acetyltransferase (CAT) reporter gene containing five GAL4-binding sites and a simian virus 40 enhancer region. The GAL4-Sp140 fusion protein increased the expression of the reporter gene. In contrast, Sp100 fused to the GAL4 DNA-binding domain inhibited CAT activity in transfected mammalian cells. The results of this study demonstrate that Sp140 associates with a subset of PML-Sp100 nuclear bodies in IFN-γ-treated cells and that Sp140 may activate gene transcription. Taken together, these observations suggest that the nuclear bodies within a cell may be heterogeneous with respect to both composition and function.

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