The cis-regulatory architecture of a gene desert controlling pleiotropic Shox2 expression and cardiac pacemaker development

Trabajo presentado en Weinstein Meeting, celebrado en Marsella (Francia) del 12 al 14 de mayo de 2022

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development

Single cell evaluation of endocardial HAND2 gene regulatory networks reveals critical HAND2 dependent pathways impacting cardiac morphogenesis.

The transcription factor HAND2 plays critical roles during cardiogenesis. Hand2 endocardial deletion (H2CKO) results in tricuspid atresia or double inlet left ventricle with accompanying intraventricular septum defects, hypo-trabeculated ventricles, and an increased density of coronary lumens. To understand the regulatory mechanisms of these phenotypes, single cell transcriptome analysis of E11.5 H2CKO hearts was performed revealing a number of disrupted endocardial regulatory pathways. Utilizing HAND2 DNA occupancy data, we identify several HAND2-dependent enhancers, including two endothelial enhancers for the shear-stress master regulator, KLF2. A 1.8kb enhancer located 50kb upstream of the Klf2 TSS imparts specific endothelial/endocardial expression within the vasculature and endocardium. This enhancer is HAND2-dependent for ventricular endocardium expression but HAND2-independent for Klf2 vascular and valve expression. Deletion of this Klf2 enhancer results in reduced Klf2 expression within ventricular endocardium. These data reveal that HAND2 functions within endocardial gene regulatory networks including shear-stress response

Increased enhancer–promoter interactions during developmental enhancer activation in mammals

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals

ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and Function.

RationaleCardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified.ObjectiveTo define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function.Methods and resultsWe used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations.Conclusions(1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function

DNA Repair and Cell Cycle Biomarkers of Radiation Exposure and Inflammation Stress in Human Blood

<div><p>DNA damage and repair are hallmarks of cellular responses to ionizing radiation. We hypothesized that monitoring the expression of DNA repair-associated genes would enhance the detection of individuals exposed to radiation versus other forms of physiological stress. We employed the human blood <em>ex vivo</em> radiation model to investigate the expression responses of DNA repair genes in repeated blood samples from healthy, non-smoking men and women exposed to 2 Gy of X-rays in the context of inflammation stress mimicked by the bacterial endotoxin lipopolysaccharide (LPS). Radiation exposure significantly modulated the transcript expression of 12 genes of 40 tested (2.2E-06CDKN1A, FDXR, BBC3, PCNA, GADD45a, XPC, POLH and <em>DDB2</em>). This panel demonstrated excellent dose response discrimination (0.5 to 8 Gy) in an independent human blood <em>ex vivo</em> dataset, and 100% accuracy for discriminating patients who received total body radiation. Three genes of this panel (<em>CDKN1A, FDXR</em> and <em>BBC3)</em> were also highly sensitive to LPS treatment in the absence of radiation exposure, and LPS co-treatment significantly affected their radiation responses. At the protein level, BAX and pCHK2-thr68 were elevated after radiation exposure, but the pCHK2-thr68 response was significantly decreased in the presence of LPS. Our combined panel yields an estimated 4-group accuracy of ∼90% to discriminate between radiation alone, inflammation alone, or combined exposures. Our findings suggest that DNA repair gene expression may be helpful to identify biodosimeters of exposure to radiation, especially within high-complexity exposure scenarios.</p> </div

Conserved enhancer logic controls the notochord expression of vertebrate Brachyury

The cell type-specific expression of key transcription factors is central to development. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three Brachyury -controlling notochord enhancers T3, C , and I in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, deletion of all three enhancers in mouse abolishes Brachyury/T expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. Sequence and functional conservation of Brachyury -driving notochord enhancers with the brachyury/tbxtb loci from diverse lineages of fishes dates their origin to the last common ancestor of jawed vertebrates. Our data define the enhancers for Brachyury/T/TBXTB notochord expression as ancient mechanism in axis development

Nanosensor dosimetry of mouse blood proteins after exposure to ionizing radiation.

Giant magnetoresistive (GMR) nanosensors provide a novel approach for measuring protein concentrations in blood for medical diagnosis. Using an in vivo mouse radiation model, we developed protocols for measuring Flt3 ligand (Flt3lg) and serum amyloid A1 (Saa1) in small amounts of blood collected during the first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6 Gy. Flt3lg concentrations showed excellent dose discrimination at ≥ 1 Gy in the time window of 1 to 7 days after exposure except 1 Gy at day 7. Saa1 dose response was limited to the first two days after exposure. A multiplex assay with both proteins showed improved dose classification accuracy. Our magneto-nanosensor assay demonstrates the dose and time responses, low-dose sensitivity, small volume requirements, and rapid speed that have important advantages in radiation triage biodosimetry

Radiation-induced transcript responses of <i>CDKN1A</i>, <i>BBC3</i> and <i>FDXR</i> are confounded by LPS treatment.

<p>Transcript level responses measured by quantitative RT-PCR analysis 24 hrs after exposure to 2 Gy, LPS treatment, and combined LPS and 2 Gy of whole blood of <i>CDKN1A</i> (A), <i>BBC3</i> (B), and <i>FDXR</i> (C) genes with respect to transcript levels in un-treated blood cultures. A. A radiation exposure of 2 Gy in the absence of LPS (left panel) or LPS treatment alone (middle panel) induced <i>CDKN1A</i> to approximately the same level at 24 hrs: 7.3 vs 8.2-fold, respectively (T-Test p = 0.47). LPS treatment in the presence of a 2 Gy radiation exposure induced <i>CDKN1A</i> expression ∼10.2-fold (right panel), which is a 1.4-fold increase compared to 2 Gy alone (T-test p = 0.03). B. In the absence of LPS, radiation induced <i>BBC3</i> ∼2.7-fold (left panel). LPS treatment alone (middle panel) suppresses <i>BBC3</i> ∼2.9-fold. LPS treatment in the presence of a 2 Gy radiation exposure induced <i>BBC3</i> expression ∼1.7-fold (right panel), a ∼1.6-fold decrease in <i>BBC3</i> expression when compared to 2 Gy alone (T-test p = 0.03). C. In the absence of LPS, radiation induced <i>FDXR</i> ∼17-fold (left panel). LPS treatment alone (middle panel) suppressed <i>FDXR</i> ∼1.5-fold. LPS treatment in the presence of a 2 Gy radiation exposure induced <i>FDXR</i> expression ∼10-fold (right panel), a ∼1.7-fold decrease in <i>FDXR</i> expression when compared to 2 Gy alone (T-test p = 1.2E-04).</p

Independent human <i>ex vivo</i> and <i>in vivo</i> confirmation of the radiation response of the 8-gene panel.

<p>The robustness of our panel of 8 non-overlapping radiation biomarkers was confirmed using two published expression array data sets: (A) <i>ex vivo</i> irradiated (0, 0.5, 2, 5, 8 Gy) human blood samples obtained from five independent donors 6 and 24 hrs after radiation exposure (GSE8917; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048619#pone.0048619-Paul1" target="_blank">[10]</a>) and (B) human <i>in vivo</i> irradiated blood samples obtained from patients undergoing total body irradiation (GSE20162; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048619#pone.0048619-Paul2" target="_blank">[12]</a>). A. Shown is the average of the summed expression for the samples in each exposure group (+/− standard error) normalized to the average expression of the 0 Gy samples for each time-point. B. Shown is the plot of the summed expression of the 8-gene panel of each blood sample in the <i>in vivo</i> study, normalized to the average of the healthy donor samples.</p