Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.

The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single "master regulator" of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.

Loss of functional peroxisomes leads to increased mitochondrial biogenesis and reduced autophagy that preserve mitochondrial function.

Peroxisomes are essential for mitochondrial health, as the absence of peroxisomes leads to altered mitochondria. However, it is unclear whether the changes in mitochondria are a function of preserving cellular function or a response to cellular damage caused by the absence of peroxisomes. To address this, we developed conditional hepatocyte-specific Pex16 deficient (Pex16 KO) mice that develop peroxisome loss and subjected them to a low-protein diet to induce metabolic stress. Loss of PEX16 in hepatocytes led to increased biogenesis of small mitochondria and reduced autophagy flux but with preserved capacity for respiration and ATP capacity. Metabolic stress induced by low protein feeding led to mitochondrial dysfunction in Pex16 KO mice and impaired biogenesis. Activation of PPARα partially corrected these mitochondrial disturbances, despite the absence of peroxisomes. The findings of this study demonstrate that the absence of peroxisomes in hepatocytes results in a concerted effort to preserve mitochondrial function, including increased mitochondrial biogenesis, altered morphology, and modified autophagy activity. Our study underscores the relationship between peroxisomes and mitochondria in regulating the hepatic metabolic responses to nutritional stressors.

Integrated small RNA, mRNA and protein omics reveal a miRNA network orchestrating metabolic maturation of the developing human heart.

BACKGROUND: As the fetal heart develops, cardiomyocyte proliferation potential decreases while fatty acid oxidative capacity increases in a highly regulated transition known as cardiac maturation. Small noncoding RNAs, such as microRNAs (miRNAs), contribute to the establishment and control of tissue-specific transcriptional programs. However, small RNA expression dynamics and genome-wide miRNA regulatory networks controlling maturation of the human fetal heart remain poorly understood. RESULTS: Transcriptome profiling of small RNAs revealed the temporal expression patterns of miRNA, piRNA, circRNA, snoRNA, snRNA and tRNA in the developing human heart between 8 and 19 weeks of gestation. Our analysis demonstrated that miRNAs were the most dynamically expressed small RNA species throughout mid-gestation. Cross-referencing differentially expressed miRNAs and mRNAs predicted 6200 mRNA targets, 2134 of which were upregulated and 4066 downregulated as gestation progressed. Moreover, we found that downregulated targets of upregulated miRNAs, including hsa-let-7b, miR-1-3p, miR-133a-3p, miR-143-3p, miR-499a-5p, and miR-30a-5p predominantly control cell cycle progression. In contrast, upregulated targets of downregulated miRNAs, including hsa-miR-1276, miR-183-5p, miR-1229-3p, miR-615-3p, miR-421, miR-200b-3p and miR-18a-3p, are linked to energy sensing and oxidative metabolism. Furthermore, integrating miRNA and mRNA profiles with proteomes and reporter metabolites revealed that proteins encoded in mRNA targets and their associated metabolites mediate fatty acid oxidation and are enriched as the heart develops. CONCLUSIONS: This study presents the first comprehensive analysis of the small RNAome of the maturing human fetal heart. Our findings suggest that coordinated activation and repression of miRNA expression throughout mid-gestation is essential to establish a dynamic miRNA-mRNA-protein network that decreases cardiomyocyte proliferation potential while increasing the oxidative capacity of the maturing human fetal heart. Our results provide novel insights into the molecular control of metabolic maturation of the human fetal heart.

MiR-30 promotes fatty acid beta-oxidation and endothelial cell dysfunction and is a circulating biomarker of coronary microvascular dysfunction in pre-clinical models of diabetes.

BACKGROUND: Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. METHODS: The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). RESULTS: We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid ß-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid ß-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. CONCLUSIONS: MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart.

Persistent myopathy despite release of partial obstruction: in vivo reversal of dysfunction and transcriptional responses using rapamycin.

Current and potential medical therapy for obstruction-induced myopathic bladder dysfunction (from benign prostatic hyperplasia or posterior urethral valves) focuses on symptoms. The persistent tissue pathology and dysfunction after release of obstruction is often deemed irreversible without any systematic therapeutic approaches. As rapamycin can attenuate bladder smooth muscle hypertrophy and dysfunction during the genesis of partial obstruction in vivo, we tested whether rapamycin could improve persistent function after release of obstruction (de-obstruction or REL). Female Sprague-Dawley rat bladders were partially obstructed (PBO) by suturing around both the urethra and a para-urethral steel rod, then removing the rod. One day prior to release of obstruction (preREL), voiding parameters and residual urine volume of preREL+future rapa, preREL+future veh groups were recorded. Release of obstruction (REL) was performed by suture removal following 6 weeks of PBO. For 4 more weeks after the de-obstruction, REL animals were randomized to rapamycin (REL+rapa) or vehicle (REL+veh). PBO for 6 weeks were used as positive controls. In shams, the urethra was exposed, but no suture tied. Voiding parameters and residual urine volume were measured prior to sacrifice of sham and REL+veh or REL+rapa, and PBO. Rapamycin efficacy was tested by pair-wise comparison of changes in individual voiding data from preREL+future veh or preREL+future rapa versus REL+veh or REL+rapa, respectively, as well as by comparisons of REL+veh to REL+rapa groups. Bladders were weighed and processed for a high-throughput QPCR array, and histopathology. Bladder/body mass ratios with PBO increased significantly and remained higher in the release phase in REL+veh animals. REL+rapa versus REL+veh improved residual volumes and micturition fractions toward sham levels. Three genes encoding extracellular proteins, BMP2, SOD3, and IGFBP7, correlated with functional improvement by Pearson's correlations. The promoters of these genes showed enrichment for several motifs including circadian E-boxes. While obstruction and REL augmented CLOCK and NPAS2 expression above sham levels, rapamycin treatment during release significantly blocked their expression. This experimental design of pharmaco-intervention during the de-obstruction phase revealed a novel pathway dysregulated during the clinically relevant treatment phase of obstructive bladder myopathy.

G9a controls placental vascular maturation by activating the Notch Pathway.

Defective fetoplacental vascular maturation causes intrauterine growth restriction (IUGR). A transcriptional switch initiates placental maturation, during which blood vessels elongate. However, the cellular mechanisms and regulatory pathways involved are unknown. We show that the histone methyltransferase G9a, also known as Ehmt2, activates the Notch pathway to promote placental vascular maturation. Placental vasculature from embryos with G9a-deficient endothelial progenitor cells failed to expand owing to decreased endothelial cell proliferation and increased trophoblast proliferation. Moreover, G9a deficiency altered the transcriptional switch initiating placental maturation and caused downregulation of Notch pathway effectors including Rbpj Importantly, Notch pathway activation in G9a-deficient endothelial progenitors extended embryonic life and rescued placental vascular expansion. Thus, G9a activates the Notch pathway to balance endothelial cell and trophoblast proliferation and coordinates the transcriptional switch controlling placental vascular maturation. Accordingly, G9A and RBPJ were downregulated in human placentae from IUGR-affected pregnancies, suggesting that G9a is an important regulator in placental diseases caused by defective vascular maturation.

Cell-extrinsic hematopoietic impact of Ezh2 inactivation in fetal liver endothelial cells.

Despite the well-established cell-intrinsic role of epigenetic factors in normal and malignant hematopoiesis, their cell-extrinsic role remains largely unexplored. Herein we investigated the hematopoietic impact of inactivating Ezh2, a key component of polycomb repressive complex 2 (PRC2), in the fetal liver (FL) vascular niche. Hematopoietic specific (Vav-iCre) Ezh2 inactivation enhanced FL hematopoietic stem cell (HSC) expansion with normal FL erythropoiesis. In contrast, endothelium (Tie2-Cre) targeted Ezh2 inactivation resulted in embryonic lethality with severe anemia at embryonic day 13.5 despite normal emergence of functional HSCs. Ezh2-deficient FL endothelium overexpressed Mmp9, which cell-extrinsically depleted the membrane-bound form of Kit ligand (mKitL), an essential hematopoietic cytokine, in FL. Furthermore, Mmp9 inhibition in vitro restored mKitL expression along with the erythropoiesis supporting capacity of FL endothelial cells. These data establish that Ezh2 is intrinsically dispensable for FL HSCs and provides proof of principle that modulation of epigenetic regulators in niche components can exert a marked cell-extrinsic impact.

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

The transcriptional regulator CCCTC-binding factor limits oxidative stress in endothelial cells.

The CCCTC-binding factor (CTCF) is a versatile transcriptional regulator required for embryogenesis, but its function in vascular development or in diseases with a vascular component is poorly understood. Here, we found that endothelial Ctcf is essential for mouse vascular development and limits accumulation of reactive oxygen species (ROS). Conditional knockout of Ctcf in endothelial progenitors and their descendants affected embryonic growth, and caused lethality at embryonic day 10.5 because of defective yolk sac and placental vascular development. Analysis of global gene expression revealed Frataxin (Fxn), the gene mutated in Friedreich's ataxia (FRDA), as the most strongly down-regulated gene in Ctcf-deficient placental endothelial cells. Moreover, in vitro reporter assays showed that Ctcf activates the Fxn promoter in endothelial cells. ROS are known to accumulate in the endothelium of FRDA patients. Importantly, Ctcf deficiency induced ROS-mediated DNA damage in endothelial cells in vitro, and in placental endothelium in vivo Taken together, our findings indicate that Ctcf promotes vascular development and limits oxidative stress in endothelial cells. These results reveal a function for Ctcf in vascular development, and suggest a potential mechanism for endothelial dysfunction in FRDA.

DNA Methylation Reduces the Yes-Associated Protein 1/WW Domain Containing Transcription Regulator 1 Pathway and Prevents Pathologic Remodeling during Bladder Obstruction by Limiting Expression of BDNF.

Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction in vivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P < 0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change in vitro.

Ezh2-mediated repression of a transcriptional pathway upstream of Mmp9 maintains integrity of the developing vasculature.

Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity.

Ezh2 regulates anteroposterior axis specification and proximodistal axis elongation in the developing limb.

Specification and determination (commitment) of positional identities precedes overt pattern formation during development. In the limb bud, it is clear that the anteroposterior axis is specified at a very early stage and is prepatterned by the mutually antagonistic interaction between Gli3 and Hand2. There is also evidence that the proximodistal axis is specified early and determined progressively. Little is known about upstream regulators of these processes or how epigenetic modifiers influence axis formation. Using conditional mutagenesis at different time points, we show that the histone methyltransferase Ezh2 is an upstream regulator of anteroposterior prepattern at an early stage. Mutants exhibit posteriorised limb bud identity. During later limb bud stages, Ezh2 is essential for cell survival and proximodistal segment elongation. Ezh2 maintains the late phase of Hox gene expression and cell transposition experiments suggest that it regulates the plasticity with which cells respond to instructive positional cues.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His-Purkinje network.

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Generation and characterization of a novel mouse model of Becker Muscular Dystrophy with a deletion of exons 52 to 55.

Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder frequently caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. The consequences of BMD-causing in-frame deletions on the organism are difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks by performing histology and echocardiography analyses and assessing motor functions. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart histology and functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of this exon52-55 in-frame deletion, we performed RNA-Seq pre- and post-exercise and identified several differentially expressed pathways that reflect the abnormal muscle phenotype observed at 52 weeks in the BMD model.

G9a inactivation in progenitor cells with Isl1-Cre with reduced recombinase activity models aspects of Dandy-Walker complex.

G9a, also known as EHMT2, is essential for embryogenesis and has specific functions in multiple developmental processes. G9a inactivation affects development of the nervous system, which is formed with contribution of descendants of progenitor cells expressing the transcription factor Isl1. However, the function of G9a in Isl1-expressing progenitors is unknown. Here, we show that G9a is required for proper development of multiple structures formed with contribution of Isl1-expressing progenitors. A Cre-dependent GFP reporter revealed that the recombinase activity of the Isl1-Cre used in this study to inactivate G9a was reduced to a subset of Isl1-expressing progenitor cells. G9a mutants reached endpoint by 7 weeks of age with cardiac hypertrophy, hydrocephalus, underdeveloped cerebellum and hind limb paralysis, modeling aspects of Dandy-Walker complex. Moreover, neuroepithelium of the lateral ventricle derived from Isl1-expressing progenitors was thinner and disorganized, potentially compromising cerebrospinal fluid dynamics in G9a mutants. Micro-computed tomography after iodine staining revealed increased volume of the heart, eye lens and brain structures in G9a mutant fetuses. Thus, altered development of descendants of the second heart field and the neural crest could contribute to multicomponent malformation like Dandy-Walker.

Prevention of early-onset cardiomyopathy in Dmd exon 52-54 deletion mice by CRISPR-Cas9-mediated exon skipping.

Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal muscle and fibrosis of cardiac muscle. The overwhelming majority of mutations are multiexonic deletions. We previously established a dystrophic mouse model with deletion of exons 52-54 in Dmd that develops an early-onset cardiac phenotype similar to DMD patients. Here we employed CRISPR-Cas9 delivered intravenously by adeno-associated virus (AAV) vectors to restore functional dystrophin expression via excision or skipping of exon 55. Exon skipping with a solitary guide significantly improved editing outcomes and dystrophin recovery over dual guide excision. Some improvements to genomic and transcript editing levels were observed when the guide dose was enhanced, but dystrophin restoration did not improve considerably. Editing and dystrophin recovery were restricted primarily to cardiac tissue. Remarkably, our exon skipping approach completely prevented onset of the cardiac phenotype in treated mice up to 12 weeks. Thus, our results demonstrate that intravenous delivery of a single-cut CRISPR-Cas9-mediated exon skipping therapy can prevent heart dysfunction in DMD in vivo.

Spontaneous urinary bladder regeneration after subtotal cystectomy increases YAP/WWTR1 signaling and downstream BDNF expression: Implications for smooth muscle injury responses.

Rodents have the capacity for spontaneous bladder regeneration and bladder smooth muscle cell (BSMC) migration following a subtotal cystectomy (STC). YAP/WWTR1 and BDNF (Brain-derived neurotrophic factor) play crucial roles in development and regeneration. During partial bladder outlet obstruction (PBO), excessive YAP/WWTR1 signaling and BDNF expression increases BSMC hypertrophy and dysfunction. YAP/WWTR1 and expression of BDNF and CYR61 were examined in models of regeneration and wound repair. Live cell microscopy was utilized in an ex vivo model of STC to visualize cell movement and division. In Sprague-Dawley female rats, STC was performed by resection of the bladder dome sparing the trigone, followed by closure of the bladder. Smooth muscle migration and downstream effects on signaling and expression were also examined after scratch wound of BSMC with inhibitors of YAP and BDNF signaling. Sham, PBO and incision (cystotomy) were comparators for the STC model. Scratch wound in vitro increased SMC migration and expression of BDNF, CTGF and CYR61 in a YAP/WWTR1-dependent manner. Inhibition of YAP/WWTR1 and BDNF signaling reduced scratch-induced migration. BDNF and CYR61 expression was elevated during STC and PBO. STC induces discrete genes associated with endogenous de novo cell regeneration downstream of YAP/WWTR1 activation.

KDM8 epigenetically controls cardiac metabolism to prevent initiation of dilated cardiomyopathy.

Cardiac metabolism is deranged in heart failure, but underlying mechanisms remain unclear. Here, we show that lysine demethylase 8 (Kdm8) maintains an active mitochondrial gene network by repressing Tbx15, thus preventing dilated cardiomyopathy leading to lethal heart failure. Deletion of Kdm8 in mouse cardiomyocytes increased H3K36me2 with activation of Tbx15 and repression of target genes in the NAD+ pathway before dilated cardiomyopathy initiated. NAD+ supplementation prevented dilated cardiomyopathy in Kdm8 mutant mice, and TBX15 overexpression blunted NAD+-activated cardiomyocyte respiration. Furthermore, KDM8 was downregulated in human hearts affected by dilated cardiomyopathy, and higher TBX15 expression defines a subgroup of affected hearts with the strongest downregulation of genes encoding mitochondrial proteins. Thus, KDM8 represses TBX15 to maintain cardiac metabolism. Our results suggest that epigenetic dysregulation of metabolic gene networks initiates myocardium deterioration toward heart failure and could underlie heterogeneity of dilated cardiomyopathy.

CTCF promotes muscle differentiation by modulating the activity of myogenic regulatory factors.

CTCF nuclear factor regulates many aspects of gene expression, largely as a transcriptional repressor or via insulator function. Its roles in cellular differentiation are not clear. Here we show an unexpected role for CTCF in myogenesis. Ctcf is expressed in myogenic structures during mouse and zebrafish development. Gain- and loss-of-function approaches in C2C12 cells revealed CTCF as a modulator of myogenesis by regulating muscle-specific gene expression. We addressed the functional connection between CTCF and myogenic regulatory factors (MRFs). CTCF enhances the myogenic potential of MyoD and myogenin and establishes direct interactions with MyoD, indicating that CTCF regulates MRF-mediated muscle differentiation. Indeed, CTCF modulates functional interactions between MyoD and myogenin in co-activation of muscle-specific gene expression and facilitates MyoD recruitment to a muscle-specific promoter. Finally, ctcf loss-of-function experiments in zebrafish embryos revealed a critical role of CTCF in myogenic development and linked CTCF to broader aspects of development via regulation of Wnt signaling. We conclude that CTCF modulates MRF functional interactions in the orchestration of myogenesis.

Structure-Function Relationships of Human Milk Oligosaccharides on the Intestinal Epithelial Transcriptome in Caco-2 Cells and a Murine Model of Necrotizing Enterocolitis.

SCOPE: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency affecting preterm infants. Breastmilk protects against NEC, partly due to human milk oligosaccharides (HMOs). HMO compositions are highly diverse, and it is unclear if anti-NEC properties are specific to carbohydrate motifs. Here, this study compares intestinal epithelial transcriptomes of five synthetic HMOs (sHMOs) and examines structure-function relationships of HMOs on intestinal signaling. METHODS AND RESULTS: This study interrogates the transcriptome of Caco-2Bbe1 cells in response to five synthetic HMOs (sHMOs) using RNA sequencing: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3FL), 6'-siallyllactose (6'-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT). Protection against intestinal barrier dysfunction and inflammation occurred in an HMO-dependent manner. Each sHMO exerts a unique set of host transcriptome changes and modulated unique signaling pathways. There is clustering between HMOs bearing similar side chains, with little overlap in gene regulation which is shared by all sHMOs. Interestingly, most sHMOs protect pups against NEC, exerting divergent mechanisms on intestinal cell morphology and inflammation. CONCLUSIONS: These results demonstrate that while structurally distinct HMOs impact intestinal physiology, their mechanisms of action differ. This finding establishes the first structure-function relationship of HMOs in the context of intestinal cell signaling responses and offers a functional framework by which to screen and design HMO-like compounds.