A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation - PubMed (original) (raw)

. 2011 Aug 10;476(7361):467-71.

doi: 10.1038/nature10312.

Bingtao Hao, Kikuë Tachibana-Konwalski, Thais Lavagnolli, Hegias Mira-Bontenbal, Karen E Brown, Grace Teng, Tom Carroll, Anna Terry, Katie Horan, Hendrik Marks, David J Adams, David G Schatz, Luis Aragon, Amanda G Fisher, Michael S Krangel, Kim Nasmyth, Matthias Merkenschlager

Affiliations

A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation

Vlad C Seitan et al. Nature. 2011.

Abstract

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.

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Figures

Figure 1

Figure 1. Genetic cohesin depleted in non-dividing thymocytes

a) Thymocyte differentiation from left to right: CD4− CD8− double negative (DN) stages 1 to 4; CD4+ CD8+ double positive (DP), CD4+8lo; CD4 or CD8 single positive (SP) cells. Proliferation is in green, cell cycle arrest in red. Histograms show DNA content. b) Conditional Rad21 allele (see supplementary fig. 1a). c) Real time genomic PCR of Rad21 locus deletion, RT-PCR of Rad21 RNA and western blotting of Rad21 protein. d) Cell numbers and flow cytometric analysis of thymocyte subsets in 6 week-old CD4Cre _Rad21_lox/lox and CD4Cre _Rad21_lox/lwt mice (mean ± SD, n = 12). e) Pulse chase analysis of CD4Cre _Rad21_lox/wt and CD4Cre _Rad21_lox/lox thymocytes. Dot blots are gated on BrdU+ cells (see supplementary fig. 2b). f) Continuous BrdU labelling for DP thymocyte turnover. See supplementary Fig. 2c for CD4+8lo and CD4 SP subsets (mean ± SD, n = 3–5 per data point). g) Top: metaphase spreads of 2 day activated thymocytes stained for alpha-tubulin (green) and DNA (DAPI, blue, see supplementary fig. 3). Bottom: Cells recovered after 5 days.

Figure 2

Figure 2. Cohesin affects Tcra transcription Rag recombinase recruitment

a) Rad21 ChIP-seq of the 3' part of the Tcra locus in DP thymocytes. Arrowheads highlight cohesin sites at the Eα enhancer (black), the Tcra locus control region (grey), Jα promoters (turquoise), the TEA promoter (green) and between Tcrd elements and V gene segments (blue). b) RNA-seq of Tcra in CD4Cre _Rad21_lox/lox (red) and control _Rad21_lox/wt (black) DP thymocytes. Rad21 ChIP-seq is in blue. c) ChIP of H3K4me3 relative to total H3 in CD4Cre _Rad21_lox/lox and control DP thymocytes. Hbb is a negative and Actg and Elp4 are positive control loci (mean ± SE of 2 independent experiments). p=0.016 for all Jα elements; p=0.38 (NS) for proximal (Jα61–48) and p=0.004 for distal (Jα37–16) Jα elements. d) ChIP of Rag2 relative to total H3 as in c) (mean ± SE of 3 independent experiments). p=0.0001 for all Jα elements; p=0.054 (NS) for proximal (Jα61–48) and p=0.0001 for distal (Jα37–16) Jα elements. e) ChIP of Rag1 relative to total H3 as in c) (mean ± SE of 2 independent experiments). p=0.005 for all Jα elements; p=0.17 (NS) for proximal (Jα61–48) and p=0.003 for distal (Jα37–16) Jα elements.

Figure 3

Figure 3. Cohesin affects Tcra rearrangement

a) Three-fold dilutions of genomic Vα8-Jα PCR products from CD71+ or CD71− DP thymocytes visualised with ethidium bromide. Cd14 is a genomic control. b) Genomic PCR products from small DP thymocytes visualised by Southern blotting with Jα-specific probes (left). Usage of the distal Jα22 element was reduced by 86% (middle, mean ± SE, n=3). Southern blotting of Vα8-Jα RT-PCR products from CD4Cre _Rad21_lox/lox normalised to control DP thymocytes (right, mean ± SE, n=3). c) Double strand breaks in three-fold serially diluted genomic DNA from CD4Cre Rad21lox/lox and control DP thymocytes detected by ligation-mediated PCR.

Figure 4

Figure 4. Cohesin mediates long-range interactions between regulatory elements that control Tcra transcription

a) Transcript copy number of the unrearranged Tcra J region in _Rag1_-deficient CD4Cre _Rad21_lox/lox and _Rad21_lox/wt DP thymocytes (mean ± SE, n=3). b) 3C analysis of long-range interactions between Eα and Tcra restriction fragments (shaded) in _Rag1_-deficient CD4Cre _Rad21_lox/wt (black, mean ± SD, n=3), CD4Cre _Rad21_lox/lox (red, mean ± SD, n=3) DP thymocytes and pre-B cells (grey, mean ± SD, n=3). Intervening HindIII fragment numbers and genomic distances are indicated. Stars: p < 0.05 (grey: control thymocytes versus pre-B cells; red: control versus cohesin-depleted thymocytes; NS = not significant). c) TCR transgenes rescue the differentiation of cohesin-depleted thymocytes. Top: Percentages BrdU+ cells in CD4+8lo, CD4 SP and CD8 SP thymocytes. The differentiation of cohesin-deficient thymocytes is rescued by MHC class II-restricted (middle) and MHC class I-restricted TCR transgenes (bottom), n=3–5 per data point ± SD.

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