Using genome-wide data to model signalling-responsive gene regulatory mechanisms in blood development

The control of gene expression driving developmental haematopoiesis crucially depends on distal cis-regulatory elements such as enhancers which directly interact with promoters in the nucleus. However, no global experiments have been conducted which identify the cell type and cell stage-specific activity of enhancers in a chromatin context. It is through these elements that lineage specific transcription factors orchestrate cell fate decisions and direct haematopoietic lineage development emerging from the mesoderm. The roles of transcriptional regulators are beginning to be understood, however, it is still unclear how the myriad of extracellular signals modulate their activity. In this work, we report a global method which enables the identification of thousands tissue-specifically active cisregulatory elements able to stimulate a minimal promoter in cells representing five stages of haematopoietic specification derived from embryonic stem cells. Using serum-free differentiation culture, we demonstrate that our method can identify signalling-responsive enhancer elements and we highlight that it can be adapted to any embryonic stem cell differentiation system generating different cell types. We demonstrate that thousands of cell stage-specific sets of cis-elements are responsive to cytokine signals terminating at signalling-responsive transcription factors. Integrating these data with chromatin accessibility and single cell RNA-Seq data provided important new insights into the regulatory dynamics of the gene regulatory network transitions driving haematopoiesis. Our work identified the cytokine signalling-responsive transcription factors mediating responsiveness of enhancers at each developmental stage. We validated enhancers for Sparc, Pxn, Hspg2, Cdh5, Dlk1 and Mrpl15 as being signalling responsive to VEGF. We found that the cytokine VEGF is a crucial factor that regulates the balance between endothelial and haematopoietic development and our scRNA-seq analysis revealed that in the presence of VEGF Sox17 fails to be downregulated and Runx1 fails to be upregulated in the haemogenic endothelium and progenitor cells. For two Runx1 enhancers (the +23kb and +3.7kb) we studied the transcription factors motifs mediating the responsiveness of the enhancers to VEGF by mutation of these sites. Taken together, our work generated an important novel resource for future studies of haematopoietic differentiation and provides insights into how and where in the genome extrinsic signals program the cell type-specific chromatin landscape driving this process

Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1

AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier

An integrative review of the impact of indirect trauma exposure in health professionals and potential issues of salience for midwives

Aims: To explore responses to indirect trauma reported by health professionals and to identify issues of potential salience for midwives. Background: Indirect exposure to a traumatic event can lead to the development of distressing and potentially enduring responses. Little is understood about the impact that perinatal trauma exposure could have on midwives. Design: An integrative review design was used. Data sources: PsychInfo, Medline, PsychArticles, Web of Knowledge, CINAHL, MIDIRS and Scopus databases were search for papers published between 1980–November 2012. Review methods: Studies providing quantitative or qualitative exploration of healthcare professionals' responses to indirectly experienced traumatic events were selected. Results: Forty‐two papers fulfilled the inclusion criteria. Four of these studies included professionals engaged in maternity care or exposed to traumatic perinatal events. Findings indicate evidence of intrusion, avoidance and arousal in healthcare professionals, with differing degrees of frequency. Empathy, work‐related stress and the extent of professional experience were identified as associated with traumatic stress responses. Conclusions: Evidence derived from healthcare professionals suggests that indirect exposure to the traumatic events of recipients of care can sometimes elicit traumatic stress responses. Factors increasing risk for traumatic stress were identified as empathy and organizational stress. These factors hold specific salience in midwifery. Responding to trauma in a midwifery context, as informed by findings from other healthcare professionals, could adversely affect midwives' well‐being, care provided to women and contribute to an adverse organizational climate. Large‐scale research considering the experiences of midwives is recommended

Thermal unfolding of smooth muscle and nonmuscle tropomyosin alpha-homodimers with alternatively spliced exons

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule

The impact of infant Bacille Calmette-Guérin vaccination on the immunogenicity of other vaccines: a randomized exploratory study

The effect of the Bacille Calmette-Guérin (BCG) vaccine on the immunogenicity of separately administered serogroup C meningococcal vaccine and other vaccinations was examined in 28 infants randomized to receive BCG at age ≤7 days, at 3 months or after study completion. Immunogenicity of the serogroup C meningococcal vaccine and other routine vaccines might be improved when BCG is administered in early infancy