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

Salvatore Loguercio,E Mauricio Barajas-Mora,Michael S Krangel,Han-Yu Shih,Ann J Feeney

doi:10.3389/fimmu.2018.00425

Salvatore Loguercio, E Mauricio Barajas-Mora + Show 3 more

Open Access

https://doi.org/10.3389/fimmu.2018.00425

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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.

Highlights

The evolutionarily conserved, ubiquitously expressed zinc finger protein CCCTC-binding factor (CTCF) plays many important roles in gene activation and/or gene repression [1]
We have found that the common method of analyzing ChIP-seq data by mapping only unique reads is not appropriate for antigen receptor (AgR) loci which have arisen by extensive gene duplication of individual V genes
Other AgR loci tend to display greater V segment sequence divergence than in the TCRα/δ locus, the v2m3 parameter is beneficial for all ChIP-seq analyses within multi-V gene AgR loci, and it was used for all of the analyses presented here

Summary

Introduction

The evolutionarily conserved, ubiquitously expressed zinc finger protein CCCTC-binding factor (CTCF) plays many important roles in gene activation and/or gene repression [1]. It was appreciated that CTCF exerts essentially all these activities by virtue of its ability to create long-range loops, creating domains within which promoters and enhancers can interact [4,5,6,7]. It has been appreciated that the entire genome is organized into “topologically associating domains” (TAD), which are megabase-sized chromatin domains that are largely conserved across different cell types [8,9,10]. Since TADs are generally conserved among various cell types and even conserved across species, it is not surprising that the majority of CTCF sites are invariant across different cell types [8, 13, 14]. There are CTCF sites that are cell type specific and which correlate with cell type-specific gene expression [14, 15]

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