Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis

Abstract

To understand the relationships between nuclear organization and gene expression in a model system, we employed three-dimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques to investigate the topographies of the immunoglobulin (Ig) genes and transcripts during B-cell development. Remarkably, in plasma cells, when antibody synthesis peaks, active Ig genes residing on three different chromosomes exhibit pronounced colocalizations in transcription factories, often near the nuclear periphery, and display trans-chromosomal enhancer interactions, and their transcripts frequently share interchromatin trafficking channels. Conceptually, these features of nuclear organization maximize coordinated transcriptional and transcript trafficking control for potentiating the optimal cytoplasmic assembly of the resulting translation products into protein multimers.

Contribution of 3D Chromatin Architecture to the Maintenance of Pluripotency

“…This is consistent with numerous studies linking transcriptional co-regulation to spatial clustering of the relevant genes and their cis-regulatory elements [26][27][28][29][30][31][32] around the nucleoplasmic supra-molecular entities that harbor most nuclear transcription-around transcription factories [19].…”

A Major Deletion in the Vκ–Jκ Intervening Region Results in Hyperelevated Transcription of Proximal Vκ Genes and a Severely Restricted Repertoire

“…Using our published techniques (15, 16, 30), pre-B cells were processed for 3D DNA FISH. Briefly, probes for 3D FISH were prepared from bacterial artificial chromosomes (BACs).…”

“…To make probes for each slide, 1 μg BAC DNA samples were labeled by nick translation with ChromaTide Alexa Fluor 488–5-deoxyuridine triphosphate (dUTP), ChromaTide Alexa Fluor 594–5-dUTP (Invitrogen), or Cy5-dUTP (GE Healthcare). Hybridization conditions were as described previously (15, 16, 30). FISH signals were analyzed by Leica TCS SP5 confocal microscopy with Z slice-sections separated by 0.3 μm, and the center-to-center distances between different hybridizing signals were measured using a plug-in of ImageJ software.…”

Gene Positioning Effects on Expression in Eukaryotes

“…Immunostaining coupled with DNA/RNA FISH revealed that actively transcribing immunoglobulin genes (IgK, IgH, and IgJ) from three different chromosomes colocalize at TNs, as marked by RNAPII (74). Further ChIP-3C-seq experiments with antibodies against RNAPII demonstrated that enhancer elements act in trans (interchromosomally) at these TNs to promote both the positioning of the different genes and their activation.…”

“…These immunoglobulin TNs were also found positioned in the ICD. It was speculated that the existence of the TNs in this space allowed for rapid and efficient export of immunoglobulin transcripts out of the nucleus for translation, demonstrating additional functionality of TNs in the ICD (74).…”