Chicken Histone Deacetylase-2 Controls the Amount of the IgM H-chain at the Steps of Both Transcription of Its Gene and Alternative Processing of Its Pre-mRNA in the DT40 Cell Line

Yasuda, Takami; Kikuchi, Hidehiko; Nakayama, Tatsuo

doi:10.1074/jbc.274.34.23977

Cited by 47 publications

(78 citation statements)

References 56 publications

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“…2A). Although HDAC1 and -2 have 85% amino acid sequence similarity (34) and are found in the same repressor complexes, it was recently shown that in the DT40 chicken B-cell line, disruption of either HDAC1 or -2 resulted in different protein expression patterns (35). This observation indicates that HDAC1 and -2 may have both specific and common interaction partners, which is consistent with the data on the differential interaction with Smad7 described in the present study.…”

Section: Discussionsupporting

confidence: 82%

“…The amount of protein on the beads was estimated by Coomassie staining of SDS-PAGE gels, and equal amounts of protein were used in the binding assays. Proteins were in vitro translated using the T7 TNT kit (Promega) and 35 S-labeled methionine and cysteine (Promix, Amersham Biosciences) and incubated with GST fusion proteins prebound to glutathione beads at 4°C for 3 h in the presence of bovine serum albumin (1 mg/ml) to avoid unspecific binding. The samples were treated as described for immunoprecipitates and resolved by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

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The Balance between Acetylation and Deacetylation Controls Smad7 Stability

Simonsson

Heldin

Ericsson

et al. 2005

Journal of Biological Chemistry

152

129

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Transforming growth factor beta (TGF␤) regulates multiple cellular processes via activation of Smad signaling pathways. We have recently demonstrated that the inhibitory Smad7 interacts with the acetyl transferase p300 and that p300 acetylates Smad7 on two lysine residues. These lysine residues are critical for Smurfmediated ubiquitination of Smad7, and acetylation protects Smad7 from TGF␤-induced degradation. In this study we demonstrate that Smad7 interacts with specific histone deacetylases (HDACs) and that the same HDACs are able to deacetylate Smad7. The interaction with HDACs is dependent on the C-terminal MH2 domain of Smad7. In addition, HDAC1-mediated deacetylation of Smad7 decreases the stability of Smad7 by enhancing its ubiquitination. Thus, our results demonstrate that the degradation of Smad7 is regulated by the balance between acetylation, deacetylation and ubiquitination, indicating that this could be a general mechanism to regulate the stability of cellular proteins.Transforming growth factor beta (TGF␤) 1 is a member of the TGF␤ superfamily of cytokines that regulate multiple cellular processes including extracellular matrix production, cell growth, apoptosis, and differentiation. Dysfunction of TGF␤ signaling has been implicated in various human disorders ranging from vascular diseases to cancer progression (for a recent review see Ref. 1). TGF␤ exerts its cellular effects via formation of a heteromeric complex of type I and type II serine/ threonine kinase receptors. The type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates and activates the receptor-activated Smads (Smad2 and Smad3) in their C-terminal SSXS motif. The activated Smads then interact with Smad4 and translocate into the nucleus where they act as transcription factors together with co-activators and co-repressors (2).The subfamily of inhibitory Smads consists of Smad6 and Smad7, which are immediate early target genes of TGF␤ (3-6). Smad7 is a nuclear protein in resting cells (7); after TGF␤ stimulation, Smad7 translocates out of the nucleus to the plasma membrane in a manner that is dependent on the Smurf E3-ubiquitin ligases (8 -10). At the cell membrane, the Smad7-Smurf complex interacts with the activated receptors and inhibits TGF␤ signaling by blocking the interaction between the receptor-activated Smads and the activated receptors (3, 6). In addition, Smurf ubiquitinates both the receptors and Smad7, thereby inducing the degradation of both the receptors and Smad7 (8, 9).We have recently shown that Smad7 interacts with the transcriptional co-activator p300, a protein acetyl transferase (11). p300-mediated acetylation transfers the acetyl moiety from acetyl coenzyme A to the amino group of a lysine residue of the acceptor protein. Acetylation is a dynamic process and the balance between acetylated and non-acetylated histones has major effects on chromatin structure and transcription (for a recent review see Ref. 12). Histones H3 and H4 are acetylated on specific lysine residues in ...

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Section: Discussionsupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

The Balance between Acetylation and Deacetylation Controls Smad7 Stability

Simonsson

Heldin

Ericsson

et al. 2005

Journal of Biological Chemistry

152

129

View full text Add to dashboard Cite

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“…However, the analyses of regulation of polyadenylation during normal B-cell development argue against control influenced solely by changes in CstF-64 levels in human primary B cells (18), in mouse B-cell lines (7), and in chicken B cells, according to a recent study. In chicken B cells Ig secretory-form mRNA was induced by knocking out the histone deactylase 2 gene; CstF-64 levels remained unperturbed (37). In contrast, the RNA-binding activity of CstF-64 but not its amount increased in nuclear extracts prepared from plasma versus memory B-cell lines (7).…”

mentioning

confidence: 99%

hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells

Veraldi

Arhin

Martincic

et al. 2001

Molecular and Cellular Biology

127

146

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Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H, and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavychain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.The immunoglobulin (Ig) heavy-chain transcription unit and the two heavy-chain mRNAs it encodes are shown in Fig. 1A (reviewed in reference 8). In mature and memory B cells the promoter-distal membrane-specific poly(A) site (mb-pA) is selected and splicing to the downstream M1 exon occurs via a 5Ј splice site within the coding region of CH4. The secretoryspecies-specific poly(A) (sec-pA) and mb-pA sites are used with equal frequency in mature and memory B cells and their tumor analogs, lymphoma cells. Plasma cells are terminally differentiated B cells; myeloma cells are their tumor counterparts, which accurately reflect their pattern of Ig gene expression. In plasma and myeloma lines polyadenylation takes place preferentially at the weaker, promoter-proximal sec-pA site, precluding the splicing to membrane-specific exons; the sec-pA site is used up to 100-fold more often than the mb-pA site in plasma cells (23). Polyadenylation at the promoter-proximal secretory site and splicing of CH4 to M1 are mutually exclusive events; consequently, it is the balance between these two that determines the final ratio of secretory-form to membrane mRNA (sec-to-mb mRNA ratio) (26). Previous experiments demonstrated that regulation of Ig heavy-chain mRNA production occurs primarily at the level of polyadenylation, not message stability, transcription termination, or splicing efficiency (14-16, 19, 2...

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“…A possible system for such studies is the chicken B-cell line, DT40, which integrates transfected DNA constructs at high ratios by homologous recombination (Buerstedde and Takeda 1991). DT40 has been used successfully for the genetic analysis of a variety of cell biology processes (Wang et al 1996;Bezzubova et al 1997;Fukagawa et al 1999a;Kurosaki 1999;Takami et al 1999). Gene disruption in DT40 is also possible, if the gene of interest is essential for cell proliferation, as conditional loss-of-function mutations can be generated using either cre-recombinase-mediated excision (Fukagawa et al 1999b), tetracycline-regulated transcription (Wang et al 1996), or tamoxifen-regulated protein transport (Fukagawa and Brown 1997).…”

mentioning

confidence: 99%

A Large Database of Chicken Bursal ESTs as a Resource for the Analysis of Vertebrate Gene Function

Abdrakhmanov

Lodygin²,

Geroth³

et al. 2000

Genome Research

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Chicken B cells create their immunoglobulin repertoire within the Bursa of Fabricius by gene conversion. The high homologous recombination activity is shared by the bursal B-cell-derived DT40 cell line, which integrates transfected DNA constructs at high rates into its endogenous loci. Targeted integration in DT40 is used frequently to analyze the function of genes by gene disruption. In this paper, we describe a large database of >7000 expressed sequence tags (ESTs) from bursal lymphocytes that should be a valuable resource for the identification of gene disruption targets in DT40. ESTs of interest can be recognized easily by online BLAST or keyword searches. Because the database reflects the gene expression profile of bursal lymphocytes, it provides valuable hints as to which genes might be involved in B-cell-specific processes related to immunoglobulin repertoire formation, signal transduction, transcription, and apoptosis. This large collection of chicken ESTs will also be useful for gene expression studies and comparative gene mapping within the chicken genome project. Details of the bursal EST sequencing project and access to database search forms can be found on the DT40 web site

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Chicken Histone Deacetylase-2 Controls the Amount of the IgM H-chain at the Steps of Both Transcription of Its Gene and Alternative Processing of Its Pre-mRNA in the DT40 Cell Line

Cited by 47 publications

References 56 publications

The Balance between Acetylation and Deacetylation Controls Smad7 Stability

The Balance between Acetylation and Deacetylation Controls Smad7 Stability

hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells

A Large Database of Chicken Bursal ESTs as a Resource for the Analysis of Vertebrate Gene Function

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