Mechanism for Nucleocytoplasmic Shuttling of Histone Deacetylase 7

Here we show that HDAC7, a member of the class II histone deacetylases, specifically targets several members of myocyte enhancer factors, MEF2A, -2C, and -2D, and inhibits their transcriptional activity. Furthermore, we demonstrate that DNA-bound MEF2C is capable of recruiting HDAC7, demonstrating that the HDAC7-dependent repression of transcription is not due to the inhibition of the MEF2 DNA binding activity. The data also suggest that the promoter bound MEF2 is potentially capable of remodeling adjacent nucleosomes via the recruitment of HDAC7. We have also observed a nucleocytoplasmic shuttling of HDAC7 and dissected the mechanism involved. In NIH3T3 cells, HDAC7 was primarily localized in the cytoplasm, essentially due to an active CRM1-dependent export of the protein from the nucleus. Interestingly, in HeLa cells, HDAC7 was predominantly nuclear. In these cells we could restore the cytoplasmic localization of HDAC7 by expressing CaMK I. This CaMK I-induced nuclear export of HDAC7 was abolished when three critical serines, Ser-178, Ser-344, and Ser-479, of HDAC7 were mutated. We show that these serines are involved in the direct interaction of HDAC7 with 14-3-3. Mutations of these serine residues weakened the association with 14-3-3 and dramatically enhanced the repression activity of HDAC7 in NIH3T3 cells, but not in HeLa cells. Data presented in this work clearly show that the signal dependent subcellular localization of HDAC7 is essential in controlling its activities. The data also show that the cellular concentration of factors such as 14-3-3, CaMK I, and other yet unknown molecules may determine the subcellular localization of an individual HDAC member in a cell type and HDAC-specific manner.

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p65 controls NF-κB activity by regulating cellular localization of IκBβ

Biochemical Journal ◽

10.1042/bj20101220 ◽

2011 ◽

Vol 434 (2) ◽

pp. 253-263 ◽

Cited By ~ 11

Author(s):

Taras Valovka ◽

Michael O. Hottiger

Keyword(s):

Dna Binding ◽

Nuclear Export ◽

Cellular Localization ◽

Nuclear Export Signal ◽

Nuclear Factor Κb ◽

Binding Activity ◽

Cellular Processes ◽

Dna Binding Activity ◽

Localization Signal ◽

Export Signal

NF-κB (nuclear factor κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by IκB (inhibitory κB) proteins which control nuclear–cytoplasmic shuttling and DNA binding of NF-κB. In the present paper, we describe a novel role for p65 as a critical regulator of the cellular localization and functions of NF-κB and its inhibitor IκBβ. In genetically modified p65−/− cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the NLS (nuclear localization signal) and the NES (nuclear export signal) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to the nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65-specific NLS and NES. The results of our present study also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA-binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB–IκBβ complexes. Taken together, the findings of the present study indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway.

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Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2464-2476.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2464-2476

Author(s):

M Cockell ◽

B J Stevenson ◽

M Strubin ◽

O Hagenbüchle ◽

P K Wellauer

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Dna Interaction ◽

Binding Activity ◽

Alpha Amylase ◽

Dna Binding Activity ◽

A Cell ◽

Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

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E2A-PBX1 functions as a coactivator for RUNX1 in acute lymphoblastic leukemia

Blood ◽

10.1182/blood.2019003312 ◽

2020 ◽

Vol 136 (1) ◽

pp. 11-23 ◽

Cited By ~ 1

Author(s):

Wen-Chieh Pi ◽

Jun Wang ◽

Miho Shimada ◽

Jia-Wei Lin ◽

Huimin Geng ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Fate ◽

Cell Transformation ◽

Lymphoblastic Leukemia ◽

Gene Activation ◽

Chromosomal Translocation ◽

Direct Interaction ◽

Binding Activity ◽

Specific Cell ◽

Dna Binding Activity

Abstract E2A, a basic helix-loop-helix transcription factor, plays a crucial role in determining tissue-specific cell fate, including differentiation of B-cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. Although previous studies have shown the oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1–enforced cistrome, the E2A-PBX1 interactome, and related mechanisms underlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)–positive pre-B ALL cells and show that, compared with normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci cobound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetylation). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Transcriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1–mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

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Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2464 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2464-2476 ◽

Cited By ~ 83

Author(s):

M Cockell ◽

B J Stevenson ◽

M Strubin ◽

O Hagenbüchle ◽

P K Wellauer

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Dna Interaction ◽

Binding Activity ◽

Alpha Amylase ◽

Dna Binding Activity ◽

A Cell ◽

Flanking Regions

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Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(90)90196-9 ◽

1990 ◽

Vol 1087 (2) ◽

pp. 127-136 ◽

Cited By ~ 29

Author(s):

Nili Feuerstein ◽

James J. Mond ◽

Paul R. Kinchington ◽

Robert Hickey ◽

Marja-Liisa Karjalainen Lindsberg ◽

...

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Nuclear Matrix ◽

Matrix Protein ◽

Binding Activity ◽

Nuclear Matrix Protein ◽

Dna Binding Activity ◽

A Cell

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Global H-NS counter-silencing by LuxR activates quorum sensing gene expression

Nucleic Acids Research ◽

10.1093/nar/gkz1089 ◽

2019 ◽

Cited By ~ 6

Author(s):

Ryan R Chaparian ◽

Minh L N Tran ◽

Laura C Miller Conrad ◽

Douglas B Rusch ◽

Julia C van Kessel

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Cell Communication ◽

Binding Activity ◽

Primary Role ◽

Dna Binding Activity ◽

Cellular Behaviors ◽

A Cell ◽

Promoter Dna

Abstract Bacteria coordinate cellular behaviors using a cell–cell communication system termed quorum sensing. In Vibrio harveyi, the master quorum sensing transcription factor LuxR directly regulates >100 genes in response to changes in population density. Here, we show that LuxR derepresses quorum sensing loci by competing with H-NS, a global transcriptional repressor that oligomerizes on DNA to form filaments and bridges. We first identified H-NS as a repressor of bioluminescence gene expression, for which LuxR is a required activator. In an hns deletion strain, LuxR is no longer necessary for transcription activation of the bioluminescence genes, suggesting that the primary role of LuxR is to displace H-NS to derepress gene expression. Using RNA-seq and ChIP-seq, we determined that H-NS and LuxR co-regulate and co-occupy 28 promoters driving expression of 63 genes across the genome. ChIP-PCR assays show that as autoinducer concentration increases, LuxR protein accumulates at co-occupied promoters while H-NS protein disperses. LuxR is sufficient to evict H-NS from promoter DNA in vitro, which is dependent on LuxR DNA binding activity. From these findings, we propose a model in which LuxR serves as a counter-silencer at H-NS-repressed quorum sensing loci by disrupting H-NS nucleoprotein complexes that block transcription.

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M-phase-specific phosphorylation of the POU transcription factor GHF-1 by a cell cycle-regulated protein kinase inhibits DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6694 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6694-6701 ◽

Cited By ~ 44

Author(s):

C Caelles ◽

H Hennemann ◽

M Karin

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Protein Kinase ◽

Dna Binding ◽

Binding Activity ◽

Dependent Manner ◽

Mitotic Kinase ◽

Dna Binding Activity ◽

M Phase ◽

A Cell

GHF-1 is a member of the POU family of homeodomain proteins. It is a cell-type-specific transcription factor responsible for determination and expansion of growth hormone (GH)- and prolactin-expressing cells in the anterior pituitary. It was previously suggested that cyclic AMP (cAMP)-responsive protein kinase A (PKA) phosphorylates GHF-1 at a site within the N-terminal arm of its homeodomain, thereby inhibiting its binding to the GH promoter. These results, however, are inconsistent with the physiological stimulation of GH production by the cAMP pathway. As reported here, cAMP agonists and PKA do not inhibit GHF-1 activity in living cells and although they stimulate the phosphorylation of GHF-1, the inhibitory phosphoacceptor site within the homeodomain is not affected. Instead, this site, Thr-220, is subject to M-phase-specific phosphorylation. As a result, GHF-1 DNA binding activity is transiently inhibited during the M phase. This activity is regained once cells enter G1, a phase during which GHF-1 phosphorylation is minimal. Thr-220 of GHF-1 is the homolog of the mitotic phosphoacceptor site responsible for the M-phase-specific inhibition of Oct-1 DNA binding Ser-382. As this site is conserved in all POU proteins, it appears that all members of this group are similarly regulated. A specific kinase activity distinct in its substrate specificity and susceptibility to inhibitors from the Cdc2 mitotic kinase or PKA was identified in extracts of mitotic cells. This novel activity could be involved in regulating the DNA binding activity of all POU proteins in a cell cycle-dependent manner.

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Nuclear export signal of IkappaBalpha interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I

Journal of Cell Science ◽

10.1242/jcs.110.22.2883 ◽

1997 ◽

Vol 110 (22) ◽

pp. 2883-2893

Author(s):

F. Bachelerie ◽

M.S. Rodriguez ◽

C. Dargemont ◽

D. Rousset ◽

D. Thomas ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Binding Activity ◽

Nuclear Accumulation ◽

Viral Rnas ◽

Dna Binding Activity ◽

Nf Kappab ◽

Export Signal ◽

Hiv 1

De novo synthesized IkappaBalpha accumulates transiently in the nucleus where it inhibits NF-kappaB-dependent transcription and reduces nuclear NF-kappaB content. A sequence present in the C-terminal domain of IkappaBalpha and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IkappaBalpha the ability to export IkappaBalpha/NF-kappaB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IkappaBalpha could interfere with Rev-dependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IkappaBalpha in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IkappaBalpha to inhibit NF-kappaB-DNA-binding activity and required a functional IkappaBalpha NES motif. Indeed, mutation of the NES abrogated the capacity of IkappaBalpha to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-kappaB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IkappaBalpha NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IkappaBalpha, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-kappaB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-kappaB is likely the consequence of interference in the IkappaBalpha-mediated export of NF-kappaB. These findings indicate that IkappaBalpha and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IkappaBalpha, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.

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Cell lines from xeroderma pigmentosum complementation group A lack a single-stranded-DNA-binding activity

Bioscience Reports ◽

10.1007/bf01172877 ◽

1983 ◽

Vol 3 (7) ◽

pp. 667-674 ◽

Cited By ~ 6

Author(s):

Urs Kuhnlein ◽

Siu Sing Tsang ◽

Opal Lokken ◽

Silvian Tong ◽

Daniel Twa

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Xeroderma Pigmentosum ◽

Human Fibroblasts ◽

Complementation Group ◽

Binding Activity ◽

Single Stranded Dna ◽

Dna Binding Activity ◽

A Cell ◽

D Cell

Human fibroblasts and HeLa cells contain two major DNA-binding activities for superhelical DNA, which can be separated by phosphocellulose chromatography. The DNA-binding activity which elutes first from the column coelutes with and is probably identical to a single-stranded-DNA-binding activity. The second activity has been characterized previously. It binds preferentially to super-helical DNA containing DNA damage, but does not bind to single-stranded DNA. Five cell lines derived from patients with the repairdeficiency syndrome xeroderma pigmentosum (XP) were analyzed for the presence of these binding activities. Four of the cell lines were from the A-complementation group and one was from the D-complementation group of XP. The binding activity with preference for damaged DNA was present in all cell lines. The single-stranded-DNA-binding activity was present in the XP-D cell line but was absent or reduced in all of the four XP-A cell lines tested.

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New Role for hPar-1 Kinases EMK and C-TAK1 in Regulating Localization and Activity of Class IIa Histone Deacetylases

Molecular and Cellular Biology ◽

10.1128/mcb.00231-06 ◽

2006 ◽

Vol 26 (19) ◽

pp. 7086-7102 ◽

Cited By ~ 45

Author(s):

Franck Dequiedt ◽

Maud Martin ◽

Julia Von Blume ◽

Didier Vertommen ◽

Emily Lecomte ◽

...

Keyword(s):

Subcellular Localization ◽

Binding Sites ◽

Nuclear Export ◽

Histone Deacetylases ◽

Serine Residue ◽

Developmental Programs ◽

Nuclear Exclusion ◽

Class Iia Hdacs ◽

Hierarchical Phosphorylation ◽

Constitutive Phosphorylation

ABSTRACT Class IIa histone deacetylases (HDACs) are found both in the cytoplasm and in the nucleus where they repress genes involved in several major developmental programs. In response to specific signals, the repressive activity of class IIa HDACs is neutralized through their phosphorylation on multiple N-terminal serine residues and 14-3-3-mediated nuclear exclusion. Here, we demonstrate that class IIa HDACs are subjected to signal-independent nuclear export that relies on their constitutive phosphorylation. We identify EMK and C-TAK1, two members of the microtubule affinity-regulating kinase (MARK)/Par-1 family, as regulators of this process. We further show that EMK and C-TAK1 phosphorylate class IIa HDACs on one of their multiple 14-3-3 binding sites and alter their subcellular localization and repressive function. Using HDAC7 as a paradigm, we extend these findings by demonstrating that signal-independent phosphorylation of the most N-terminal serine residue by the MARK/Par-1 kinases, i.e., Ser155, is a prerequisite for the phosphorylation of the nearby 14-3-3 site, Ser181. We propose that this multisite hierarchical phosphorylation by a variety of kinases allows for sophisticated regulation of class IIa HDACs function.

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