Alterations in chromatin at antigen receptor loci define lineage progression during B lymphopoiesis.

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis.

The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus.

Low-Input MNase Accessibility of Chromatin (Low-Input MACC).

An understanding of the dynamic structural properties of chromatin requires techniques that allow the profiling of regions of both open and closed chromatin as well as the assessment of nucleosome occupancy. The recently developed MNase accessibility (MACC) technique allows for the simultaneous measurement of chromatin opening and compaction, as well as nucleosome occupancy, on a genome-wide scale in a single assay. This article presents a low-input MACC procedure that considerably extends the utility of the original MACC assay.

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice.

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination.

We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineage-specific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase.