Activation of terminal B cell differentiation by inhibition of histone deacetylation

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Abstract

A role for histone acetylation, which can alter the accessibility of DNA to transcriptional regulatory proteins and contribute to gene expression, in regulating terminal B cell differentiation was investigated in the mature B lymphoma L10A and mouse splenic B cells. Incubation of the L10A cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and butyrate increased expression of Blimp-1, J chain, and mad genes, decreased expression of c-myc and BSAP/Pax-5 genes, increased the expression of surface CD43 and Syndecan-1, and decreased surface IgM. Incubation of splenic B cells with TSA and dextran conjugated anti-IgD Ab increased Blimp-1 gene and Syndecan-1 surface expression. The alteration in gene expression and cell surface markers was consistent with induction of the onset of terminal B cell differentiation. Co-incubation of L10A cells with TSA and cycloheximide (CHX) abrogated the up-regulation of Blimp-1 expression, indicating that TSA-activated Blimp-1 expression required synthesis of a transcriptional activator. In contrast, mad expression was increased in L10A cells cultured with TSA and cycloheximide or cycloheximide alone, suggesting mad expression may occur independent of Blimp-1 expression and is regulated by a labile, HDAC associated transcriptional repressor. The results demonstrate that histone acetylation regulates transcription of genes controlling terminal B cell differentiation.

Introduction

Eukaryotic DNA is packaged into repeating nucleosomal units of chromatin around an octamer of the core histones H2A, H2B, H3, and H4 (Wolffe, 1996, Bartl et al., 1997). The core histone amino terminal tail domain, which can interact with regulatory proteins or DNA (Wade et al., 1997), is subject to multiple post-translational modifications such as phosphorylation, methylation, polyADP-ribosylation, and acetylation (Van et al., 1996). Histone acetylation, mediated by a histone acetyltransferase (HAT) complex, has been shown to play an important role in regulation of gene expression, DNA replication, and DNA repair (Bartl et al., 1997, Armstrong and Emerson, 1998). Acetylation destabilizes the nucleosomal structure by neutralizing the positively charged lysine residues of the N-terminal tail domain of core histones (DePinho, 1998, Strahl and Allis, 2000), and increases the accessibility of DNA to transcriptional regulatory proteins (Struhl, 1998). Conversely, histone deacetylation, mediated by a multicomponent histone deacetylase (HDAC) complex, removes the acetyl group of terminal lysine residues to silence transcription (DePinho, 1998, Wolffe, 1997).

Alteration of chromatin structure has been shown to play a critical role in the regulation of early lymphocyte development and differentiation (Agarwal and Rao, 1998, Schlissel, 2000). V(D)J recombination has been hypothesized to be controlled through modulation of DNA accessibility to the recombination machinery (Schlissel and Stanhope-Baker, 1997). Indeed, both V(D)J recombination and transcription of episomal substrates are inhibited in cells transfected with a deacetylated chromatin substrate. Conversely, disruption of nucleosomal organization of the substrate with the HDAC inhibitor sodium butyrate activates both V(D)J recombination and transcription (Cherry and Baltimore, 1999). V(D)J rearrangement of the T cell receptor (TCR) loci requires TCR δ and α enhancer function and is associated with histone H3 hyperacetylation. Thus, cis-regulatory elements appear to developmentally regulate histone acetylation and result in chromatin accessibility for V(D)J recombination (McMurry and Krangel, 2000, Krangel, 2001, Agata et al., 2001). Helper T (Th) cell commitment and preferential transcription of cytokine genes have also been shown to correlate with DNA accessibility, as evaluated by analysis of DNase I hypersensitivity. With differentiation of naı̈ve T cells to Th1 cells, the chromatin at the IL-2 and IFNγ gene loci becomes more accessible. In contrast, differentiation of naı̈ve T cells to Th2 cells increases accessibility at the IL-4 and IL-13 loci (Agarwal and Rao, 1998).

A specific role for histone acetylation or DNA accessibility in terminal B cell differentiation has not been determined. During immune responses to foreign antigens, B lymphocytes terminally differentiate to antibody-secreting plasma cells. This terminal B cell differentiation is associated with alteration in gene expression, cell surface markers, and cell morphology. Terminal differentiation of B cells is associated with increased production of Ig secretion and decrease in surface Ig, de novo expression of J chain protein, increased cell surface expression of CD138 (Syndecan-1) and CD43, and down-regulation of MHC class II antigens and CD20 (Turner et al., 1994, Henderson and Calame, 1998, Burrows et al., 1997). It has been estimated that the expression of approximately 100 genes is altered with terminal B cell differentiation (Turner et al., 1994).

To explore whether chromatin accessibility may regulate terminal B cell differentiation, the effect of the specific HDAC inhibitor Trichostatin A (TSA) on the terminal differentiation of a mature murine B cell line, L10A and primary splenic B cells was studied. We report here that TSA induces gene expression characteristic of late B cell differentiation, suggesting that modulation of chromatin may participate directly in regulation of the pathway of terminal B cell differentiation.

Section snippets

Mice

C57BL/6 mice were obtained from the Jackson laboratories (Bar Harbor, ME) and were used at 6–8 weeks of age. These animals were maintained in specific pathogen-free conditions in the animal facility at the University of Rochester.

Culture and treatment of the L10A cell line and splenic cells

L10A, a sIgM+, sIgD+ mouse lymphoma line (Laskov et al., 1981), was grown in RPMI 1640 containing 10% FBS, 5×10−5 M 2-ME, 2 mM l-glutamine and penicillin–streptomycin. A total of 2×106 L10A cells in 4 ml of media was cultured in 100×15 mm polystyrene petri dishes (VWR,

TSA induces expression of genes associated with terminal B cell differentiation in the L10A cell line

We explored whether incubation with TSA, a known hydroxamic acid-based specific HDAC inhibitor (Marks et al., 2000), altered gene expression of the L10A murine cell line, a mature murine cell line that expresses surface IgM and IgD and has been used as a model to analyze terminal B cell differentiation (Turner et al., 1994, Laskov et al., 1981, Messika et al., 1998). To confirm that TSA is able to induce a hyperacetylated state of core histones in L10A cells, the nuclei of TSA-treated L10A

Discussion

The terminal differentiation of B cells has been subdivided into distinct stages based on analysis of cell surface markers and gene expression of B cell malignancies and in vitro studies of B cells. A “preplasma” cell stage of terminal B cell differentiation is associated with expression of surface Syndecan-1 and CD43 and up-regulation of Blimp-1 gene expression but without Ig secretion. This stage is followed by a plasma cell stage associated with Ig secretion and high levels of Blimp-1 and

Acknowledgements

We thank Dr. M. Davis from Stanford University for providing the Blimp-1 expression vector, Dr. C.D. Allis from the University of Virginia Health Science Center for providing anti-acetylated histone H4 Ab, Dr. C.M. Snapper from Uniformed Services, University of the Health Sciences for providing the dextran conjugated anti-IgD Abs, Drs. J.-I. Abe, T. Bushnell, H. Federoff, P. Kingsley, I. Kuzin, E. Lord, J. Olschowka, and R. Phipps for mouse splenic cells and reagents, and Drs. J. Hayes and P.

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