Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci

Abstract

CCCTC-binding factor (CTCF) is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR) loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B vs. pro-B) in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated convergent loops throughout the loci. CTCF/cohesin loops, along with transcription factors, drives contraction of AgR loci to facilitate the creation of a diverse repertoire of antibodies and T cell receptors.

Keywords: 3D chromatin topology; CCCTC-binding factor; T cell receptor loci; cohesin; immunoglobulin loci; long-range looping.

Figure 1

Plots of the number of significant peaks within the V gene-containing portion of each AgR locus called by MACS from ChIP-seq of pre-pro-B cells, pro-B cells, pre-B cells, double-negative (DN) and double-positive (DP) thymocytes, and murine embryonic fibroblast (MEF) and ES cells. The Rad21 and Smc1 peaks for ES and MEF were obtained from GEO, and the number of called peaks were obtained from GEO files. The cell type which is rearranging the locus being analyzed is plotted in red. The TCRα/δ locus rearranges at two stages since the TCRδ genes rearrange in DN thymocytes, and the TCRα genes rearrange in DP thymocytes. Statistics are shown in Tables S4 and S5 in Supplementary Material.

Figure 2

Genome Browser views of CCCTC-binding factor (CTCF) and cohesin subunit (Rad21 or Smc1) ChIP-seqs for the Igh locus. IGCR1 (the set of two CTCF sites upstream of DFL16.1) is highlighted in blue and the set of nine CTCF sites in the 3′ regulatory region are highlighted in yellow.

Figure 3

Genome Browser views of CCCTC-binding factor (CTCF) and cohesin subunit (Rad21 or Smc1) ChIP-seqs are plotted for the Igκ locus. The two CTCF sites in Cer are highlighted in yellow and the two CTCF sites in the adjacent Ser region are highlighted in blue.

Figure 4

(A) H3K4me3 and H3ac ChIP/qPCR on RAG1^−/−CD19⁺ pro-B cells, RAG1^−/−IgH Tg⁺ CD19⁺ pro-B cells, and RAG1^−/− pro-B cells cultured with IL7. (B) Relative levels of transcription of ncRNA (germline transcription) in the Igh (PAIR and J558 sense) and Igκ (κ° and Vκ38-93) loci in RAG1^−/− CD19⁺ pro-B cells, RAG1^−/−IgH Tg⁺ CD19⁺ pre-B cells, and RAG1^−/− pro-B cells cultured with IL7. (C) H3K4me1 ChIP-seq on RAG1^−/− CD19⁺ pro-B cells (38), RAG1^−/− pro-B cells cultured with IL7 (58), and RAG1^−/−IgHTg⁺ CD19⁺ pre-B cells throughout the Igκ locus. Significance determined with Mann–Whitney test. * is <0.05, ** is <0.01, *** is <0.001, and **** is <0.0001.

Figure 5

Genome Browser views of CCCTC-binding factor (CTCF) and cohesin subunit (Rad21 or Smc1) ChIP-seqs are plotted for the TCRα/δ locus. The TEA element is highlighted in blue and INT1/INT2 is highlighted in yellow.

Figure 6

Genome Browser views of CCCTC-binding factor (CTCF) and cohesin subunit (Rad21 or Smc1) ChIP-seqs are plotted for the TCRβ locus. 5′PC is highlighted in blue and the low CTCF site near Dβ1 is highlighted in yellow.

Figure 7

Orientation of CCCTC-binding factor (CTCF) sites in the immunoglobulin heavy chain (Igh) locus, with close-up of the D-J-C-enhancer region in the middle panel, and close-up of the 3′ regulatory region in the bottom panel.

Figure 8

(A) Orientation of CCCTC-binding factor (CTCF) sites in the TCRβ locus. (B) Orientation of CTCF sites in the Tcrα/δ locus.

Figure 9

Orientation of CCCTC-binding factor (CTCF) sites in the Igκ locus, with close-up of the Cer, Sis, Jκ-Cκ-enhancer region.

Figure 10

CCCTC-binding factor (CTCF) and cohesin subunit (Rad21 or Smc1) ChIP-seq are plotted for the TCRγ locus, along with the orientation of CTCF sites. Sites of the probable long-range CTCF loop is indicated.

Figure 11

CCCTC-binding factor (CTCF) and cohesin subunit (Rad21 or Smc1) ChIP-seq are plotted for the Igλ locus, along with the orientation of CTCF sites. Sites of probable long-range CTCF loops are indicated.