Histone acetylation determines the developmentally regulated accessibility for T cell receptor gamma gene recombination

Abstract

Variable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner. Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control. Here, we investigate the role for histone acetylation in the developmentally regulated rearrangements of the mouse TCR-gamma gene, wherein predominant rearrangement is switched from Vgamma3 to Vgamma2 gene during the fetal to adult thymocyte development. Our results indicate that histone acetylation correlates with accessibility, as histone acetylation at the fetal-type Vgamma3 gene in accord with germline transcription is relatively high in fetal thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during the development of adult bone marrow-derived thymocytes by a specific histone deacetylase inhibitor, trichostatin A, leads to elevated histone acetylation, germline transcription, cleavage, and rearrangement of the Vgamma3 gene. These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.

Figure 1

Histone acetylation at Vγ genes correlates with accessibility during fetal to adult thymocyte development. (A) Schematic diagram of the TCR-γ locus and location of PCR primers used in RT-PCR and ChIP assays. The promoter (P) and RSS (S) regions of each Vγ2 and Vγ3 gene were analyzed in ChIP assays. GLTs of Vγ2, Vγ3, and Jγ1 genes were measured by RT-PCR. The Vγ RSSs with 23-bp spacer and Jγ1 RSS with 12-bp spacer are indicated by filled and open triangles. (B) RT-PCR analysis of E14 and E16 total, and adult CD3⁻CD4⁻CD8⁻ TN thymocyte RNA. Fivefold serial dilutions of cDNAs and reaction without reverse transcriptase (RT−) were amplified for indicated transcripts. For Jκ1 GLT and MyoD transcripts, cDNAs from adult bone marrow and C2C12 cells were used, respectively (Control). (C) Histone acetylation measured by ChIP assay in E14 and E16 total, and adult TN thymocytes. Threefold serial dilutions of input DNA and DNAs immunoprecipitated with antiacetylated histone H3 (αAcH3) and H4 (αAcH4) antibodies or with normal rabbit IgG (Control) were analyzed by PCR. Fivefold serial dilutions of input DNA from Ba/F3 cells (Standard) and no DNA control (DNA−) were also subjected to PCR. (D) Histone acetylation levels measured in C are shown as relative histone H3 (black bars) and H4 (white bars) acetylation levels to that of the CD3ε locus.

Figure 3

Accessibility at Vγ3 is increased by TSA in adult-derived thymocytes. (A) Ligation-mediated PCR analysis of organ-cultured thymocyte DNA used in Fig. 2 B to measure SBEs at Vγ2 and Vγ3. (B) RT-PCR analysis of RNA prepared from fetal liver- and adult bone marrow (BM)-derived thymocytes cultured for 7 d with (+) or without (−) TSA, and from E16 and adult TN (ATN) thymocytes (Thy). Fivefold serial dilutions of cDNAs and reaction without reverse transcriptase (RT−) were amplified for Vγ2, Vγ3 GLTs, and GAPDH transcripts. (C) Kinetic analysis of transcript levels in organ-cultured thymocytes. Transcript levels measured as in B are shown as the ratio to those in E16 thymocytes.

Figure 2

TSA induces Vγ3 rearrangements in adult-derived thymocytes. (A) Flow cytometric analysis of organ-cultured thymocytes in fetal thymic lobes repopulated with E14 fetal liver and adult bone marrow (BM) cells. Thymocytes cultured for 14 d with (+) or without (−) TSA were stained with FITC-Vγ2 and PE-CD3ε, or PE-Vγ3 and FITC-CD3ε antibodies. Average cell numbers per lobe from four lobes and percentages of cells for a given phenotype are shown above and inside each panel, respectively. (B) PCR analysis of organ-cultured thymocyte DNA to measure coding joints (CJ) and signal joints (SJ) of Vγ2-Jγ1 or Vγ3-Jγ1, and CD3ε gene for a control. Fivefold serial dilutions of E16 and adult TN (ATN) thymocyte (Thy) DNA and no DNA control (DNA−) were also subjected to PCR.

Figure 4

Histone acetylation at Vγ3 is increased by TSA in adult-derived thymocytes. (A) Histone acetylation measured by ChIP assay in adult bone marrow (BM)-derived thymocytes cultured for 15 days with (+) or without (−) TSA. ChIP and PCR analysis were performed as in the legend to Fig. 1 except that twofold serial dilutions of input DNA were amplified. (B) Histone acetylation levels in thymocytes cultured with (shaded bars) or without (black bars) TSA measured in A are shown as relative histone acetylation levels to that of the CD3ε locus.