V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh V H region in pro-B cells, remodels chromatin for V H -to-DJ H recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh D H J H region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer E and is abrogated in mice lacking this enhancer, indicating that E regulates D H antisense transcription. E was recently demonstrated to regulate D H -to-J H recombination of the Igh locus. Together, these data suggest that E controls D H -to-J H recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which E can regulate chromatin accessibility throughout the D H region. In contrast, E deletion has no effect on V H antisense intergenic transcription, which is rarely associated with D H antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.In order to generate the primary repertoire of immunoglobulin (Ig) and T-cell receptor (TCR) molecules, antigen receptor loci undergo variable, diversity, and joining [V(D)J] recombination in B and T lymphocytes. Recombination is catalyzed by a recombinase complex containing the protein products of the recombinase-activating genes Rag1 and Rag2 (28). Within precursor lymphocytes, this process is strictly lineage specific, with heavy (Igh) and light (Ig and Ig) immunoglobulin loci fully recombining only in B lymphocytes and T-cell receptor loci (Tcra, Tcrb, Tcrg, and Tcrd) recombining only in T cells. Further, within lineages, loci are recombined in a precise order. Recombination of the Igh locus is the earliest step in the generation of the mature antibody repertoire in B lymphocytes. The Igh locus of the C57BL/6 mouse spans 3 Mb and comprises 195 V H genes spanning 2.5 Mb, 10 D H genes (ϳ60 kb), 4 J H genes (2 kb), and 8 constant (C H ) genes (200 kb) (31, 68). D H -to-J H recombination occurs on both Igh alleles before V H -to-DJ H recombination takes place (16).Lineage and stage specificity of V(D)J recombination are regulated by differential chromatin accessibility to the RAG proteins. Several mechanisms may contribute, but their relative importance is still unclear. The first process discovered was germ line transcription, which occurs in all antigen receptor loci across gene segments competent for recombination (34).This transcription was termed "sterile" or "germ line" to distinguish it from coding V(D)J transcription. In the Igh locus, the earliest germ line transc...

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour.DOI: http://dx.doi.org/10.7554/eLife.08494.001

SummaryGene expression in living cells is highly dynamic, but temporal patterns of gene expression in intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used to quantify patterns of reporter gene expression in dispersed lactotrophic cells or intact pituitary tissue from bacterial artificial chromosome (BAC) transgenic rats in which a large prolactin genomic fragment directed expression of luciferase or destabilised enhanced green fluorescent protein (d2EGFP). Prolactin promoter activity in transgenic pituitaries varied with time across different regions of the gland. Although amplitude of transcriptional responses differed, all regions of the gland displayed similar overall patterns of reporter gene expression over a 50-hour period, implying overall coordination of cellular behaviour. By contrast, enzymatically dispersed pituitary cell cultures showed unsynchronised fluctuations of promoter activity amongst different cells, suggesting that transcriptional patterns were constrained by tissue architecture. Short-term, high resolution, single cell analyses in prolactin-d2EGFP transgenic pituitary slice preparations showed varying transcriptional patterns with little correlation between adjacent cells. Together, these data suggest that pituitary tissue comprises a series of cell ensembles, which individually display a variety of patterns of short-term stochastic behaviour, but together yield long-range and long-term coordinated behaviour.

V(D)J recombination of the multigene antigen receptor loci is essential for the generation of a diverse antigen receptor repertoire. Recombination is strictly regulated, occurring only in lymphocytes due to restricted expression of the recombination activating gene enzymes, RAG1 and RAG2, therein. Further, T cell receptors only recombine in T cells, B cell receptors only recombine in B cells, and the loci only recombine at specific stages in lymphocyte differentiation. In B cells, the Igh recombines before the Ig light chains. Finally some antigen receptor loci (e.g. the Igh) have two ordered recombination events. A D gene first recombines with a J gene on both alleles, followed by recombination of a V gene to the DJ recombined segment. Once a productive VDJ rearrangement has been generated, further V to DJ recombination is prevented on the second allele, a process termed allelic exclusion, which in B cells ensures that each B cell expresses a monoclonal IgH (1).Ordered recombination is crucial for antigen receptor integrity, but key questions remain: how is recombination order achieved, and how is it regulated? Numerous studies have suggested that order is achieved through alterations in the chromatin conformation of individual gene domains at sequential stages of lymphocyte development (2). In the mouse Igh locus, the D-J-C region acquires histone post-translational modifications characteristic of open chromatin before the V region (3, 4). Non-coding RNA transcripts, including I, generated from E, located 3Ј of the J genes (5), and o, transcribed from the promoter of the most 3Ј D gene, DQ52, occur on germ line alleles (6). Following D to J recombination, non-coding transcripts are generated from the V genes (7,8). Furthermore, extensive antisense intergenic transcription occurs throughout the D and J domains before D to J, and then throughout the V domain before V to DJ recombination (9, 10). Nuclear positioning may also play a role in ordered V(D)J recombination. The Igh locus is tethered at the nuclear periphery via the V region in non-B cells (11,12). Relocation toward euchromatic regions occurs preferentially from the DJC end, favoring D to J recombination. Furthermore, locus compaction through DNA looping is required for distal V gene recombination (13,14). Several transcription factors, including Pax5 (13), YY1 (15), and Ikaros (16), play a role in looping, and in their absence, only the D-proximal V genes recombine. Following productive V(D)J recombination and cell surface expression of an IgH polypeptide, several of the above processes are reversed to silence V to DJ recombination of the second allele by allelic exclusion. Both Igh V regions decontract, V region germ line transcription is lost, and the second Igh allele is recruited to pericentric heterochromatin via the D-distal V genes (1). In contrast, both DJC regions remain transcriptionally active (9, 17). Thus, there is differential chromatin regulation of both activation and inactivation of the DJC versus V regions of the Igh locus.

Gene expression is made up of inherently stochastic processes within single cells and can be modeled through stochastic reaction networks (SRNs). In particular, SRNs capture the features of intrinsic variability arising from intracellular biochemical processes. We extend current models for gene expression to allow the transcriptional process within an SRN to follow a random step or switch function which may be estimated using reversible jump Markov chain Monte Carlo (MCMC). This stochastic switch model provides a generic framework to capture many different dynamic features observed in single cell gene expression. Inference for such SRNs is challenging due to the intractability of the transition densities. We derive a model-specific birth–death approximation and study its use for inference in comparison with the linear noise approximation where both approximations are considered within the unifying framework of state-space models. The methodology is applied to synthetic as well as experimental single cell imaging data measuring expression of the human prolactin gene in pituitary cells.

Our data support a role of BA in human energy metabolism and in thyroid hormone control. Even though no convincing response to BA was demonstrated in TSHoma and TαT1 cells, the TSH decrease after a nutritional challenge suggests an interaction of BA on the set point of the thyroid axis.

Transcription of numerous mammalian genes is highly pulsatile, with bursts of expression occurring with variable duration and frequency. The presence of this stochastic or ‘noisy’ expression pattern has been relatively unexplored in tissue systems. The prolactin gene provides a model of tissue‐specific gene regulation resulting in pulsatile transcription dynamics in both cell lines and endocrine tissues. In most cell culture models, prolactin transcription appears to be highly variable between cells, with differences in transcription pulse duration and frequency. This apparently stochastic transcription is constrained by a transcriptional refractory period, which may be related to cycles of chromatin remodelling. We propose that prolactin transcription dynamics result from the summation of oscillatory cellular inputs and by regulation through chromatin remodelling cycles. Observations of transcription dynamics in cells within pituitary tissue show reduced transcriptional heterogeneity and can be grouped into a small number of distinct patterns. Thus, it appears that the tissue environment is able to reduce transcriptional noise to enable coordinated tissue responses to environmental change. We review the current knowledge on the complex tissue‐specific regulation of the prolactin gene in pituitary and extra‐pituitary sites, highlighting differences between humans and rodent experimental animal models. Within this context, we describe the transcription dynamics of prolactin gene expression and how this may relate to specific processes occurring within the cell.

SummaryImportant questions in biology have emerged recently concerning the timing of transcription in living cells. Studies on clonal cell lines have shown that transcription is often pulsatile and stochastic, with implications for cellular differentiation. Currently, information regarding transcriptional activity at cellular resolution within a physiological context remains limited. To investigate single-cell transcriptional activity in real-time in living tissue we used bioluminescence imaging of pituitary tissue from transgenic rats in which luciferase gene expression is driven by a pituitary hormone gene promoter. We studied fetal and neonatal pituitary tissue to assess whether dynamic patterns of transcription change during tissue development. We show that gene expression in single cells is highly pulsatile at the time endocrine cells first appear but becomes stabilised as the tissue develops in early neonatal life. This stabilised transcription pattern might depend upon tissue architecture or paracrine signalling, as isolated cells, generated from enzymatic dispersion of the tissue, display pulsatile luminescence. Nascent cells in embryonic tissue also showed coordinated transcription activity over short distances further indicating that cellular context is important for transcription activity. Overall, our data show that cells alter their patterns of gene expression according to their context and developmental stage, with important implications for cellular differentiation.