Regulatory Architecture of the RCA Gene Cluster Captures an Intragenic TAD Boundary, CTCF-Mediated Chromatin Looping and a Long-Range Intergenic Enhancer.

The Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (CR2), are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of CR2 and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the CR1 gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (CR2 and CD55) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease.

Enhancing Early Tertiary Students' Education: a Novel Lecture Learning Objectives Strategy.

While university lectures enable large volumes of complex material to be taught efficiently, this format requires students to discriminate between core concepts and examples, applications and anecdotes. Here we present a lecture slide learning objectives method which builds this capability in Level 1 tertiary students in preclinical sciences. Our method applies the principles of constructive alignment to individual teaching activities. Students report the use of this lecturing methodology results in improved focus, decreased stress during lectures and greater preparedness for assessment (n = 93). This practicable addition to the lecture slides greatly improves the student experience both during and following teaching sessions.

To Be, or Notch to Be: Mediating Cell Fate from Embryogenesis to Lymphopoiesis.

Notch signaling forms an evolutionarily conserved juxtacrine pathway crucial for cellular development. Initially identified in Drosophila wing morphogenesis, Notch signaling has since been demonstrated to play pivotal roles in governing mammalian cellular development in a large variety of cell types. Indeed, abolishing Notch constituents in mouse models result in embryonic lethality, demonstrating that Notch signaling is critical for development and differentiation. In this review, we focus on the crucial role of Notch signaling in governing embryogenesis and differentiation of multiple progenitor cell types. Using hematopoiesis as a diverse cellular model, we highlight the role of Notch in regulating the cell fate of common lymphoid progenitors. Additionally, the influence of Notch through microenvironment interplay with lymphoid cells and how dysregulation influences disease processes is explored. Furthermore, bi-directional and lateral Notch signaling between ligand expressing source cells and target cells are investigated, indicating potentially novel therapeutic options for treatment of Notch-mediated diseases. Finally, we discuss the role of cis-inhibition in regulating Notch signaling in mammalian development.

Notch signaling induces a transcriptionally permissive state at the Complement C3d Receptor 2 (CR2) promoter in a pre-B cell model.

During mammalian lymphoid development, Notch signaling is necessary at multiple stages of T lymphopoiesis, including lineage commitment, and later stages of T cell effector differentiation. In contrast, outside of a defined role in the development of splenic marginal zone B cells, there is conflicting evidence regarding whether Notch signaling plays functional roles in other B cell sub-populations. Complement receptor 2 (CR2) modulates BCR-signaling and is tightly regulated throughout differentiation. During B lymphopoiesis, CR2 is detected on immature and mature B cells with high surface expression on marginal zone B cells. Here, we have explored the possibility that Notch regulates human CR2 transcriptional activity using in vitro models including a co-culture system, co-transfection gene reporters and chromatin accessibility assays. We provide evidence that Notch signaling regulates CR2 promoter activity in a mature B cell line, as well as the induction of endogenous CR2 mRNA in a non-expressing pre-B cell line. The dynamics of endogenous gene activation suggests additional unidentified factors are required to mediate surface CR2 expression on immature and mature B lineage cells.

Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells.

Complement receptor 2 (CR2/CD21) is predominantly expressed on the surface of mature B cells where it forms part of a coreceptor complex that functions, in part, to modulate B-cell receptor signal strength. CR2/CD21 expression is tightly regulated throughout B-cell development such that CR2/CD21 cannot be detected on pre-B or terminally differentiated plasma cells. CR2/CD21 expression is upregulated at B-cell maturation and can be induced by IL-4 and CD40 signaling pathways. We have previously characterized elements in the proximal promoter and first intron of CR2/CD21 that are involved in regulating basal and tissue-specific expression. We now extend these analyses to the CR2/CD21 core promoter. We show that in mature B cells, CR2/CD21 transcription proceeds from a focused TSS regulated by a non-consensus TATA box, an initiator element and a downstream promoter element. Furthermore, occupancy of the general transcriptional machinery in pre-B versus mature B-cell lines correlate with CR2/CD21 expression level and indicate that promoter accessibility must switch from inactive to active during the transitional B-cell window.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

TNF-inducible expression of lymphotoxin-ß in hepatic cells: an essential role for NF-κB and Ets1 transcription factors.

As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-ß by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-ß was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-ß expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-ß expression in hepatic cells at the transcriptional level by the binding of NF-κB p50/p65 heterodimers and Ets1 to their respective sites in the LT-ß promoter.

Hepatic expression of the tumor necrosis factor family member lymphotoxin-beta is regulated by interleukin (IL)-6 and IL-1beta: transcriptional control mechanisms in oval cells and hepatoma cell lines.

BACKGROUND: Lymphotoxin-beta (LT-beta) plays an important role in inflammation and its promoter contains a functional nuclear factor-kappaB (NF-kappaB) element, rendering it a likely target of pro-inflammatory cytokines. Inflammatory cytokines play a central role in liver regeneration resulting from acute or chronic liver injury, with interleukin (IL)-6 signaling essential for liver regeneration induced by partial hepatectomy. In hepatic oval cells observed following chronic liver injury, LT-beta levels are upregulated, suggesting a link between LT-beta and liver regeneration. RESULTS: The expression of LT-beta in hepatic oval cell and hepatocellular carcinoma cell lines was further investigated, along with its responsiveness to IL-6 and IL-1beta. Key regulatory cis-acting elements of the LT-beta promoter that mediate IL-6 responsiveness (Sp/BKLF, Ets, NF-kappaB and Egr-1/Sp1) and IL-1beta responsiveness (NF-kappaB and Ets) of hepatic LT-beta expression were identified. The novel binding of basic Kruppel-like factor (BKLF) proteins to an apparent composite Sp/BKLF site of the LT-beta promoter was shown to mediate IL-6 responsiveness. Binding of NF-kappaB p65/p50 heterodimers and Ets-related transcription factors to their respective sites mediates responsiveness to IL-1beta. CONCLUSION: The identification of IL-6 and IL-1beta as activators of LT-beta supports their involvement in LT-beta signaling in liver regeneration associated with chronic liver damage.

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.