Caroline Bartman

Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping

Mammalian genes transcribe RNA not continuously, but in bursts. Transcriptional output can be modulated by altering burst fraction or burst size, but how regulatory elements control bursting parameters remains unclear. Single-molecule RNA FISH experiments revealed that the β-globin enhancer (LCR) predominantly augments transcriptional burst fraction of the β-globin gene with modest stimulation of burst size. To specifically measure the impact of long-range chromatin contacts on transcriptional bursting, we forced an LCR-β-globin promoter chromatin loop. We observed that raising contact frequencies increases burst fraction but not burst size. In cells in which two developmentally distinct LCR-regulated globin genes are cotranscribed in cis, burst sizes of both genes are comparable. However, allelic co-transcription of both genes is statistically disfavored, suggesting mutually exclusive LCR-gene contacts. These results are consistent with competition between the β-type globin genes for LCR contacts and suggest that LCR-promoter loops are formed and released with rapid kinetics.

A hyperactive transcriptional state marks genome reactivation at the mitosis–G1 transition

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

The influence of the microbiota on the immune response to transplantation

Purpose of reviewIn the past decade, appreciation of the important effects of commensal microbes on immunity has grown exponentially. The effect of the microbiota on transplantation has only recently begun to be explored; however, our understanding of the mechanistic details of host–microbe interactions is still lacking. Recent findingsIt has become clear that transplantation is associated with changes in the microbiota in many different settings, although what clinical events and therapeutic interventions contribute to these changes remains to be parsed out. Research groups have begun to identify associations between specific communities of organisms and transplant outcomes, but it remains to be established whether microbial changes precede or follow transplant rejection episodes. Finally, results from continuing exploration of basic mechanisms by which microbial communities affect innate and adaptive immunity in various animal models of disease continue to inform research on the microbiotas effects on immune responses against transplanted organs. SummaryCommensal microbes may alter immune responses to organ transplantation, but direct experiments are only beginning in the field to identify species and immune pathways responsible for these putative effects.

The composition of the microbiota modulates allograft rejection

Transplantation is the only cure for end-stage organ failure, but without immunosuppression, T cells rapidly reject allografts. While genetic disparities between donor and recipient are major determinants of the kinetics of transplant rejection, little is known about the contribution of environmental factors. Because colonized organs have worse transplant outcome than sterile organs, we tested the influence of host and donor microbiota on skin transplant rejection. Compared with untreated conventional mice, pretreatment of donors and recipients with broad-spectrum antibiotics (Abx) or use of germ-free (GF) donors and recipients resulted in prolonged survival of minor antigen-mismatched skin grafts. Increased graft survival correlated with reduced type I IFN signaling in antigen-presenting cells (APCs) and decreased priming of alloreactive T cells. Colonization of GF mice with fecal material from untreated conventional mice, but not from Abx-pretreated mice, enhanced the ability of APCs to prime alloreactive T cells and accelerated graft rejection, suggesting that alloimmunity is modulated by the composition of microbiota rather than the quantity of bacteria. Abx pretreatment of conventional mice also delayed rejection of major antigen-mismatched skin and MHC class II-mismatched cardiac allografts. This study demonstrates that Abx pretreatment prolongs graft survival, suggesting that targeting microbial constituents is a potential therapeutic strategy for enhancing graft acceptance.

Microbes and allogeneic transplantation.

Microbial products can be recognized by pattern recognition receptors expressed by immune and parenchymal cells and drive innate immunity that can in turn shape adaptive immune responses to microbial and transplant antigens. In transplanted patients, the signals and their downstream inflammatory cytokines elicited in response to infections can modulate ongoing alloimmune responses and modify the fate of transplanted organs. In recent years, it has become apparent that microbial signals can be generated not only by active pathogenic infections but also by commensal microbiota, thus opening a new field of research into the interplay between the microbiota and the immune system in homeostasis and disease. The wide use of antibiotics and immunosuppressive drugs in transplanted patients can have dramatic consequences on the microbiota that can in turn shape immune responses and perhaps alloresponses, whereas the ongoing immune responses can in turn affect the commensal or pathogenic microorganisms in a feed-forward circle. Here, we discuss known and hypothesized mechanisms for how infections or microbiota-derived signals may affect local or systemic alloimmunity and briefly review data on downstream effects of antibiotics and vaccinations.

Perturbing Chromatin Structure to Understand Mechanisms of Gene Expression

The study of nuclear structure falls between the fields of cell biology and molecular biology and draws on techniques from both fields. In recent years, many exciting advances have been made in these areas, including single-molecule and superresolution imaging and the development of chromosome conformation capture (3C)-based technologies, which have brought the advent of genome-wide analysis of chromatin structure and contacts. However, many questions remain as to the function of nuclear structures, in particular their influence on transcription. Here we describe studies that have directly manipulated nuclear architecture at various levels and thus have clarified the causal influence of structure on transcription. We will also highlight open questions in the field, most notably regarding our understanding of the dynamics and variability in nuclear structure and its influence on gene expression.

Impact of Staphylococcus aureus USA300 Colonization and Skin Infections on Systemic Immune Responses in Humans

Staphylococcus aureus is both a commensal and a pathogen, and USA300, a strain that is usually methicillin-resistant but can sometimes be methicillin-susceptible, has been causing skin and soft tissue infections (SSTIs) in epidemic proportions among otherwise healthy individuals. Although many people are colonized with S. aureus strains, including some with USA300, few of these colonized individuals develop SSTIs. This prompts the hypothesis that infections may develop in individuals with somewhat reduced innate and/or adaptive immune responses to S. aureus, either because prior S. aureus colonization has dampened such responses selectively, or because of more globally reduced immune reactivity. In this study, we analyzed the S. aureus colonization status and PBMC responses to innate and adaptive stimuli in 72 patients with SSTIs and 143 uninfected demographically matched controls. Contrary to the hypothesis formulated, PBMCs from infected patients obtained at the time of infection displayed enhanced innate cytokine production upon restimulation compared with PBMCs from controls, a difference that disappeared after infection resolution. Notably, PBMCs from patients infected with a documented USA300 SSTI displayed greater innate cytokine production than did those from patients infected with documented non-USA300 genotypes. Moreover, colonization with USA300 in infected patients, regardless of their infecting strain, correlated with increased production of IL-10, IL-17A, and IL-22 compared with patients colonized with non-USA300 subtypes. Thus, our results demonstrate that infected patients associated with USA300 either as an infecting strain, or as a colonizing strain, have systemic immune responses of greater magnitude than do those associated with other S. aureus subtypes.

Long-term Maintenance of Sterility Following Skin Transplantation in Germ-free Mice.

Background There is considerable interest in investigating the role of the microbiota in various diseases, including transplant rejection. Germ-free (GF) and gnotobiotic mice are powerful models for this line of investigation, but performing surgery within the confines of a sterile housing isolator is exceptionally challenging. Development of rigorous protocols to be able to remove axenic mice from their sterile isolator for surgical intervention in a class II biological safety cabinet (BSC) without compromising sterility would give many investigators access to this model and broaden possible studies. However, it is assumed that GF animals will most often become colonized with environmental microbiota on leaving the isolator. In this study, we tested whether applying sterile techniques for animal transport out of the isolator and skin transplantation in a class II BSC could maintain animal sterility. Methods Quantitative polymerase chain reaction of the bacterial 16S ribosomal RNA gene, and cultures in various aerobic and anaerobic conditions were used to probe for bacterial contamination before and after transplantation. Results Of 28 surgeries performed, only 3 mice acquired bacterial contamination coincident with a transient shutdown of the ventilation system in the BSC. Conclusions Our results indicate that skin transplantation can be successfully performed in GF mice using sterile conditions for transport and surgery in a class II BSC, but requires continuous positive airflow. Our approach paves the way to investigating the role of the microbiota in modulating immune responses to skin allografts as a first model of solid organ transplantation in GF mice.

Transcriptional burst initiation and polymerase pause release are key control points of transcriptional regulation

Transcriptional regulation occurs via changes to the rates of various biochemical processes. Sequencing-based approaches that average together many cells have suggested that polymerase binding and polymerase release from promoter-proximal pausing are two key regulated steps in the transcriptional process. However, single cell studies have revealed that transcription occurs in short, discontinuous bursts, suggesting that transcriptional burst initiation and termination might also be regulated steps. Here, we develop and apply a quantitative framework to connect changes in both Pol II ChIP-seq and single cell transcriptional measurements to changes in the rates of specific steps of transcription. Using a number of global and targeted transcriptional regulatory perturbations, we show that burst initiation rate is indeed a key regulated step, demonstrating that transcriptional activity can be frequency modulated. Polymerase pause release is a second key regulated step, but the rate of polymerase binding is not changed by any of the biological perturbations we examined. Our results establish an important role for transcriptional burst regulation in the control of gene expression.