Locus architecture and RAG scanning determine antibody diversity.

Diverse mammalian antibody repertoires are produced via distant genomic contacts involving immunoglobulin Igh variable (V), diversity (D), and joining (J) gene segments and result in V(D)J recombination. How such interactions determine V gene usage remains unclear. The recombination-activating gene (RAG) chromatin scanning model posits that RAG recombinase bound to the recombination center (RC) linearly tracks along chromatin by means of cohesin-mediated loop extrusion; a proposition supported by cohesin depletion studies. A mechanistic role for chromatin loop extrusion has also been implicated for Igh locus contraction. In this opinion, we provide perspective on how loop extrusion interfaces with the 3D conformation of the Igh locus and newly identified enhancers that regionally regulate VH gene usage during V(D)J recombination, shaping the preselected repertoire.

Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells.