Deletion Of Enhancer Research Articles

CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription.

Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identified a total of 39 functional enhancers for four dosage-sensitive genes, APP , FMR1 , MECP2 , and SIN3A , using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. We found that enhancer annotation provides potential pathological insights into disease-associated copy number variants. More importantly, we discovered that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription is stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A , and potentially other dosage-sensitive genes.

LRP1 Repression by SNAIL Results in ECM Remodeling in Genetic Risk for Vascular Diseases.

Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown. Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry. Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and pulmonary function trait. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix, connective tissue development, and lung development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3. Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61, a known LRP1 ligand involved in vascular integrity and TIMP3, implicated in extracellular matrix maintenance and also known to interact with LRP1. Our findings support allele-specific LRP1 gene repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control.

PD-1 is a key negative regulator of CD8+ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8+ T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8+ T cell dysfunction while avoiding excess immunopathology.

CRISPR-Cas9-mediated deletion enhancer of MECOM play a tumor suppressor role in ovarian cancer.

MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion.

CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant.

Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ∼664 kb from the ONECUT1 promoter, since coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

A transcriptional enhancer regulates cardiac maturation.

Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. In this study, we investigated a clinically relevant, conserved ACTN2 enhancer's effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling, promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer's role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology.

Diverse Gene Regulatory Mechanisms Alter Rattlesnake Venom Gene Expression at Fine Evolutionary Scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.

P124 Elucidating the role of risk variants in the BIM gene enhancer in T-Cell dysregulation and rheumatoid arthritis pathogenesis

Abstract Background/Aims The balance governing T-cell responses is pivotal for immune homeostasis. Disruptions to this balance may lead to aberrant behaviours, potentially resulting in autoimmunity. Rheumatoid Arthritis (RA), an autoimmune disorder primarily manifesting in the joints, is characterised by a defective immune response, resulting in chronic inflammation. Enhanced T-cell apoptosis resistance in this context can contribute to sustained activation and abnormal proliferation in a pro-inflammatory environment. Understanding these disease mechanisms could lead to improvements in clinical care. Genome-wide association studies (GWAS) have identified hundreds of single nucleotide polymorphisms (SNPs) associated with RA, preferentially enriched in non-coding, T-cell-specific putative enhancers. Enhancers are DNA elements that can regulate the transcription of specific genes, often from many kilobases away. Our previous work identified BCL2L11, or BIM, as the probable causal gene within the RA locus rs13396472, despite the GWAS variants' intronic positioning relative to ACOXL. The RA risk variant disrupts a distant enhancer regulating the BIM gene, pivotal for apoptosis. Given the role of apoptosis in immune tolerance, disruption of this pathway driven by RA risk variants could pave the way for autoimmune responses. Our aim was to investigate the effect of this enhancer on T-cell proliferation and function in an in vivo model. Methods We mapped the human RA-locus to the mouse genome and utilised CRISPR-Cas9 to delete this putative RA enhancer in mice. Two sgRNAs, combined with Cas9, were microinjected into C57BL6/J zygote pronuclei, followed by embryo transfer to pseudo-pregnant mice. PCR and Sanger sequencing confirmed enhancer deletion in pups, establishing an RA-enhancer deletion colony. To gauge cell proportion differences, murine splenocytes or thymocytes were stained with specific fluorochrome markers and analysed via a flow cytometer. For the antigen-induced arthritis (AIA) model, wildtype and RA-enhancer deletion mice were subjected to arthritis induction via intra-articular injection of methylated bovine serum albumin, following prior immunisation, and monitored for arthritis signs. Results We observed an increased CD4+CD8+ double positive (DP) T-cell population in aged mice homozygous and heterozygous for the RA-enhancer deletion, but not in younger and wild-type controls. Knockout mice exhibited visibly enlarged spleens in response to the immunisation step prior to AIA induction. Further work will establish the mechanisms underlying this and disease progression after arthritis induction. These observations underscore a potential impact this enhancer might have on immune responses and survival upon immune system alteration. Conclusion Our research indicates that perturbing mouse non-coding sequences syntenic to human GWAS loci can provide a physiological model to elucidate novel mechanisms underlying RA, potentially suggesting therapeutic targets. Future research will explore the mechanism by which the enhancer deletion leads to an increase in DP T-cells and the altered phenotype, and the implications of such a change for T- and B-cell biology and RA. Disclosure S. Rossi: None. E. Richards: None. J. Ding: None. C. Shi: None. M. Menon: None. J. Gibbs: None. R. Grencis: None. A. Adamson: None. G. Orozco: None.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Two redundant transcription factor binding sites in a single enhancer are essential for mammalian sex determination.

Male development in mammals depends on the activity of the two SOX gene: Sry and Sox9, in the embryonic testis. As deletion of Enhancer 13 (Enh13) of the Sox9 gene results in XY male-to-female sex reversal, we explored the critical elements necessary for its function and hence, for testis and male development. Here, we demonstrate that while microdeletions of individual transcription factor binding sites (TFBS) in Enh13 lead to normal testicular development, combined microdeletions of just two SRY/SOX binding motifs can alone fully abolish Enh13 activity leading to XY male-to-female sex reversal. This suggests that for proper male development to occur, these few nucleotides of non-coding DNA must be intact. Interestingly, we show that depending on the nature of these TFBS mutations, dramatically different phenotypic outcomes can occur, providing a molecular explanation for the distinct clinical outcomes observed in patients harboring different variants in the same enhancer.

Anatomy of a superenhancer.

Interferon regulatory factor-8 (IRF8) is the lineage determining transcription factor for the type one classical dendritic cell (cDC1) subset, a terminal selector for plasmacytoid dendritic cellsand important for the function of monocytes. Studies of Irf8 gene regulation have identified several enhancers controlling its activity during development of progenitors in the bone marrowthat precisely regulate expression at distinct developmental stages. Each enhancer responds to distinct transcription factors that are expressed at each stage. IRF8 is first expressed in early progenitors that form the monocyte dendritic cell progenitor (MDP) in response to induction of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) acting at the Irf8 +56kb enhancer. IRF8 levels increase further as the MDP transits into the common dendritic cell progenitor (CDP) in response to E protein activity at the Irf8 +41kb enhancer. Upon Nfil3-induction in CDPs leading to specification of the cDC1 progenitor, abrupt induction of BATF3 forms the JUN/BATF3/IRF8 heterotrimer that activates the Irf8 +32kb enhancer that sustains Irf8 autoactivation throughout the cDC1 lifetime. Deletions of each of these enhancers has revealed their stage dependent activation. Surprisingly, studies of compound heterozygotes for each combination of enhancer deletions revealed that activation of each subsequent enhancer requires the successful activation of the previous enhancer in strictly cis-dependent mechanism. Successful progression of enhancer activation is finely tuned to alter the functional accessibility of subsequent enhancers to factors active in the next stage of development. The molecular basis for these phenomenon is still obscure but could have implications for genomic regulation in a broader developmental context.

Study of the role of evolutionary new enhancers in the development of the <i>corpus callosum</i>

One important aspect of the mammalian brain is the exchange of information between neurons located in different hemispheres. This process evolved during the development of mammals. In marsupial (Marsupialia) and monotreme (Monotremata) mammals, the communication between hemispheres is facilitated through an enlarged anterior commissure. In placental (Eutheria) mammals, a new brain structure, the corpus callosum, emerged during the evolutionary process. The corpus callosum, comprising 80% of the brain’s commissural axons, is the largest commissure in the human body. The corpus callosum is a major contributor to the efficiency of higher neural activities, including memory, decision making, social interaction, and language. A possible explanation for the emergence of a novel structure for interhemispheric interaction is a change in the growth direction of neocortical axons during development. Changes in gene expression levels can regulate this process, which involves controlling axon growth and cell migration in the neocortex. A full comprehension of this process will enable the creation of new animal models for studying cortical malformations and the navigation of axons in the cerebral cortex leading to the formation of interhemispheric connections. Several enhancers were identified for protein-coding genes with differing expression patterns in neocortical cells of placental and non-placental (marsupial) mammals. Throughout the genomes of the house opossum (Monodelphis domestica) and the house mouse (Mus musculus), the acetylation levels of histone H3 on lysine 27 (H3K27ac) were compared. H3K27ac is considered an epigenetic marker for active gene enhancers. Then, a screening of candidate genes was performed to evaluate their localization and expression levels in the cortex during embryonic development. Thus, the Tbr1 gene was identified. Incorrect expression of this gene may result in changes to cortical development. The CRISPR/Cas9 system was combined with in utero electroporation to completely delete the active Tbr1 gene enhancer in developing neocortical cells of mouse embryos at day 14 of embryonic development. The impact of this enhancer deletion was then analyzed on the expression of Tbr1 in the upper layers of the cortex, as well as the direction of axon growth and neuronal migration on the 18th day of embryonic development. A significant reduction in Tbr1 expression was observed in the upper layers of the cortex after deletion of the active enhancer. Only 30% of the electroporated neurons retained Tbr1 expression. Moreover, a considerable delay in neuronal migration was observed in the subventricular zone (41% versus 17% in the control group) and in the upper layers of the cortex (20% versus 35% in the control group). However, the direction of axonal growth remained unchanged: callosal axons effectively crossed the midline and created the corpus callosum. Thus, expression of the evolutionary novel Tbr1 enhancer is important for neuronal migration during corticogenesis. However, its contribution to the development of the corpus callosum is not fully understood. A detailed analysis of the corpus callosum morphology post-enhancer deletion will be the subsequent step.

Structural variants in Lewy body dementia and frontotemporal dementia spectrum

AbstractBackgroundStructural variants range from simple loss or gain of genetic material to complex events that restructure entire chromosomes. This heterogeneity coupled with variant size greater than sequencing read‐length makes structural variant mapping from short‐read sequencing data difficult and error‐prone. Consequently, the role of structural variants is poorly explored in many phenotypes, including dementia.MethodWe used the GATK‐SV pipeline that combines four external structural variant mapping algorithms with machine‐learning to build high‐quality consensus structural variant calls. We applied GATK‐SV to short‐read whole‐genome sequencing data of 2,601 frontotemporal dementia‐amyotrophic lateral sclerosis spectrum (FTD/ALS) patients, 2,612 Lewy body dementia (LBD) patients, and 4,132 neurologically unaffected participants.ResultAfter stringent quality‐control, we performed genome‐wide association studies on 2,307 ALS/FTD patients versus 3,677 controls with 4,699 common structural variants and 2,355 LBD patients versus 3,700 controls with 4,889 common structural variants. In the FTD/ALS cohort, we identified well‐known risk variants at the C9orf72 (p‐value = 4.99×10‐18, OR = 14.47, 95% CI = 7.90–26.49) and MAPT loci (p‐value = 3.48×10‐6, OR = 0.77, 95% CI = 0.68–0.86) corroborating the ability of our pipeline to identify disease‐associated structural variants. Moreover, we discovered, replicated, and validated TPCN1 as a novel risk locus for LBD (p‐value = 9.18×10‐6, OR = 1.43, 95% CI = 1.22–1.67). Further, we identified rare exonic variants with known pathogenic or likely pathogenic effects, such as an SNCA gene duplication in LBD and a deletion of exons 1 and 2 in CHCHD10 in FTD/ALS. We additionally observed structural variants in regulatory regions, e.g. a deletion of a key PSEN2 enhancer, which resulted in an approximate 0.70 fold‐change in PSEN2 expression in LBD. Finally, we built an interactive web app where structural variants can be visualized and explored (https://ndru-ndrs-lng-nih.shinyapps.io/non_ad_dementias_sv_app/).ConclusionCommon and rare structural variants play a role in LBD and FTD/ALS. Our structural variant resource is publicly available and can be used to further explore the role of structural variants in LBD and FTD/ALS.

Mef2c regulates bone mass through Sost-dependent and -independent mechanisms

Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost−/−; Mef2cfl/fl; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost−/− alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2cfl/fl; Dmp1-Cre and Mef2cfl/fl; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2cfl/fl; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2cfl/fl; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp−/− mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost−/−; Mef2cfl/fl; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.

Deletion of the Artery Specific Enhancer Promotes B Cell and Impairs T Cell Development from the Hemogenic Endothelium

HSCs and HSC-independent lymphoid progenitors in the embryo arise from hemogenic endothelium (HE) at multiple anatomical locations. Although activation of arterial program in HE has been shown to play an important role in HSC development, the impact of the arterial program on specification of HSC-independent lymphoid cell remains unclear. During vascular development, arterial fate is specified following induction of DLL4 expression by VEGF by signaling through a highly conserved VEGF-responsive artery-specific enhancer located within the third intron of DLL4 ( DLL4-i3) which is controlled by ETS, SOXF and RBPJ factors. To investigate how the arterial program affects T and B cell lymphoid development, we generated DLL4-i3:Venus reporter and knockout ( DLL4 Di3/Di3) human pluripotent stem cells (hPSCs). Using a 3-dimensional, serum- and feeder-free spheroid differentiation system, we demonstrated that DLL4: i3 enhancer activity recapitulates endogenous DLL4 expression and arterial endothelial cell specification in human hPSC cultures. Moreover, DLL4-i3: Venus + HE and hematopoietic progenitors produced more myeloid CFCs and T cells as compared DLL4-i3: Venus - hemogenic cells. Deletion of DLL4i3 enhancer impaired arterial specification from hPSCs including formation of arterial DLL4 +CXCR4 +/- HE. In contrast to wild type (WT), hematopoietic progenitors (HPs) generated from DLL4 Di3/Di3hPSCs showed impaired CFC potential with significantly reduced G-CFCs and markedly diminished T cell potential. However, DLL4 Di3/Di3hPSCs showed efficient and highly reproducible B cell potential as compared to WT. To define the impact of artery-specific enhancer deletion on transcriptional program during endothelial-to-hematopoietic transition (EHT) and HP formation, we performed scRNAseq of all endothelial and hematopoietic cells from D5 and D8 differentiation cultures of DLL4-i3:Venus and DLL4 Di3/Di3 hPSCs. Through unsupervised clustering, 10 cell clusters (C0-C9) were distinguished and visualized using UMAP. Based on the expression panel of known pan-, arterial and hemogenic endothelial and hematopoietic markers we defined three clusters of HE with arterial features (AHE; high EFNB2, CDH5 and RUNX1 positive and SPN negative), two non-arterial HE clusters (low EFNB2, CDH5 and RUNX1 positive and SPN negative), two EHT clusters (CDH5 and SPN positive, high RUNX1, and GFI1b), and three HP clusters (SPN positive, high RUNX1 and CDH5 negative). GSEA of DLL4i3: Venus+ cells with active enhancer vs DLL4 Di3/Di3 cells revealed enrichment in ribosomes and oxidative phosphorylation pathways across all clusters and focal adhesion and tight junction pathways in HE and EHT clusters. DEG analysis demonstrated upregulation of EFNB2, MECOM HES4 in DLL4-i3: Venus+ cells within HE and EHT clusters, while DLL4 Di3/Di3 cells demonstrated upregulation of NRP2 and CDH13 genes. Interestingly, in EHT and HP clusters, we noticed that DLL4-i3 knockout significantly upregulates the expression of GAS5 lncRNAs known to be involved in growth arrest of human peripheral blood T cells. Using siRNA, we demonstrated that knockdown of GAS5 in D5 cultures of DLL4 Di3/Di3 cells rescued the T cell potential of the hematopoietic progenitors suggesting that NOTCH-mediated GAS5 suppression is essential for the T cell specification and expansion following EHT. In contrast to T cells, GAS5 downregulation had no effect on B cell specification of DLL4 Di3/Di3 HE. Overall, we established that DLL4-i3 enhancer is required for arterial programming of HE with T cell potential while deletion of this enhancers promotes B cell development. In addition, we demonstrated an important role of GAS5 lncRNA in the regulation of T cell development from HE.

Genomic Determinants of Hematopoietic Progenitor Sensitivity to Innate Immune Signaling

Innate immune processes impact diverse hematopoietic stem and progenitor cell (HSPC) functions, yet how innate immune signaling networks are sensed by HSPC genomes and how HSPC genomic activity alters cell responsiveness to innate immune regulators are not fully understood. We demonstrated that E14.5 primary (-77 -/-) and immortalized (hi-77 -/-) GATA2-deficient fetal progenitors from Gata2 -77 enhancer deletion mice upregulate Interferon-gamma (IFNγ) and Toll-like receptor (TLR) signaling components (Johnson et al. JEM 2020) and are hypersensitive to IFNγ and TLR signaling (Tran et al. iScience 2023). Combinatorial signaling involving both pathways regulates genes that are not regulated by individual pathways and amplifies responses beyond that induced by single pathways. Innate immune-activated genes harbor motifs for signal-dependent transcription factors, including the ETS factor PU.1, and GATA2 deficiency elevates PU.1 activity to upregulate B-lineage and myeloid genes (Jung et al. JCI 2023). We hypothesize that PU.1 links innate immune signaling networks and HSPC genome control. In new multi-omic studies, we asked if PU.1 is required to sense exclusively IFNγ or TLR signaling, both pathways independently or combinatorially, or is not essential. We used hi-77 -/- progenitors lacking a PU.1 enhancer ( Spi1URE-/-) with ~2-fold lower PU.1 vs. control hi-77 -/- progenitors. RNA-seq was conducted with hi-77 -/- and hi-77 -/-; Spi1URE-/- progenitors, with or without IFNγ, TLR1/2 agonist Pam 3CSK 4, or both agents for 4 h. hi-77 -/-; Spi1URE-/- progenitors lost the responsiveness of 27 TLR-activated genes and retained the responsiveness of 6 TLR-activated genes. 80% of IFNγ-activated genes (115 of 144) retained IFNγ-responsiveness when PU.1 was reduced. In hi-77 -/-; Spi1URE-/- progenitors, 68.2% (148 of 217 genes) retained their responsiveness to combinatorial signaling. These studies revealed the disproportionate importance of PU.1 for TLR- vs. IFNγ-mediated transcriptional control and a PU.1 requirement for combinatorial signaling at the majority of loci. Elevated IFNγ and TLR1/2 signaling in GATA2-deficient fetal progenitors is associated with increased monocytic and reduced granulocytic progenitors (Johnson et al. JEM 2020; Blood Adv 2022). Ablation of Irf8, encoding an IFNγ pathway component, partially normalizes the granulocytic progenitor deficit (Johnson et al. Blood Adv. 2022). Since GATA2 deficiency creates crosstalk between IFNγ and TLR1/2 pathways to elevate cytokine/chemokine production (Tran et al . iScience 2023), we asked if attenuating TLR signaling reverses the differentiation defect. As MYD88 Innate Immune Signal Transduction Adaptor (MYD88) is crucial for TLR1/2 signaling, we generated -77 +/-; Myd88-/- mice. Timed matings were conducted to obtain -77 +/+, -77 -/-, Myd88-/-, and -77 -/-; Myd88-/- E14.5 embryos. Myd88 ablation in -77 -/- embryos did not reverse the skewed granulocytic vs. monocytic progenitor ratio. To determine if MYD88 loss impacts -77 -/- progenitor function, we analyzed CMP and GMP populations from E14.5 embryos with CFU assays. In comparison with -77 +/+, -77 -/- CMP and Myd88-/- CMP produced fewer CFU-G colonies (1.9-fold decrease, P = 0.0097 and 2.0-fold decrease, P = 0.007, respectively). Surprisingly, -77 -/-; Myd88-/- CMP exhibited a further reduction in CFU-G colonies (5.2-fold decrease, P < 0.0001). The CFU-G colonies produced by -77 +/-; Myd88-/- CMP resembled that of -77 +/+ CMP. Thus, GATA2 and MYD88 synergistically promote CFU-G production from CMP, and a single -77 allele maintains CMP activity to produce CFU-G without Myd88. Myd88 deletion did not affect the reduced CFU-G nor the increased CFU-M with -77 -/- GMP. We are testing if TLR1/2 signaling is not required to maintain balanced fetal liver progenitor populations or if another signaling adapter nullifies the impact of MYD88 loss. Our studies revealed a mechanism underlying the hypersensitivity of GATA2-deficient fetal progenitors to innate immune signaling, and MYD88 loss further decreases the reduced CFU-G generated from -77 -/- CMP. We are testing the hypothesis that GATA2 restricts assembly of PU.1-containing chromatin complexes and analyzing models to unveil functional ramifications of elevated innate immune signaling in single HSPCs.

De novo enhancer deletion of LMX1B produces a mild nail-patella clinical phenotype.

Critical genes involved in embryonic development are often transcription factors, regulating many downstream genes. LMX1B is a homeobox gene that is involved in formation of the limbs, eyes and kidneys, heterozygous loss-of-function sequence variants and deletions cause Nail-Patella syndrome. Most of the reported variants are localised within the gene's coding sequence, however, approximately 5%-10% of affected individuals do not have a pathogenic variant identified within this region. In this study, we present a family with four affected individuals across two generations with a deletion spanning a conserved upstream LMX1B-binding sequence. This deletion is de novo in the mother of three affected children. Furthermore, in this family, the manifestations appear limited to the nails and limbs, and therefore may reflect an attenuated phenotype of the classic Nail-Patella phenotype that includes ophthalmological and renal manifestations.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Interlocking of co-opted developmental gene networks in Drosophila and the evolution of pre-adaptive novelty

The re-use of genes in new organs forms the base of many evolutionary novelties. A well-characterised case is the recruitment of the posterior spiracle gene network to the Drosophila male genitalia. Here we find that this network has also been co-opted to the testis mesoderm where is required for sperm liberation, providing an example of sequentially repeated developmental co-options. Associated to this co-option event, an evolutionary expression novelty appeared, the activation of the posterior segment determinant Engrailed to the anterior A8 segment controlled by common testis and spiracle regulatory elements. Enhancer deletion shows that A8 anterior Engrailed activation is not required for spiracle development but only necessary in the testis. Our study presents an example of pre-adaptive developmental novelty: the activation of the Engrailed transcription factor in the anterior compartment of the A8 segment where, despite having no specific function, opens the possibility of this developmental factor acquiring one. We propose that recently co-opted networks become interlocked, so that any change to the network because of its function in one organ, will be mirrored by other organs even if it provides no selective advantage to them.