TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

Tissue-specific Grb10/Ddc insulator drives allelic architecture for cardiac development.

Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architecture precipitated abnormal Grb10-Ddc expression with developmental consequences in the heart and muscle. Thus, we redefine the Grb10-Ddc imprinting domain by uncovering an unconventional intronic secondary DMR that functions as an insulator to instruct the tissue-specific, monoallelic expression of multiple genes-a feature previously ICR exclusive.

A new Elf5CreERT2-GFP BAC transgenic mouse model for tracing Elf5 cell lineages in adult tissues.

Elf5 is a transcription factor known to regulate critical developmental processes and has been shown to act as a tumour suppressor in multiple cancers. Elf5 knockout mice are embryonically lethal, limiting in vivo studies pertaining to its function. Moreover, haploinsufficiency of Elf5 limits the use of current mouse models to investigate adult tissue distribution of Elf5. Here, we successfully generated Elf5CreERT2-GFP bacterial artificial chromosome (BAC) transgenic mice and show that Elf5+ cells are present in several adult tissues, where its expression was previously not known. Our study demonstrates the unique distribution of Elf5+ cells in multiple adult organs, which will facilitate future studies investigating the function of Elf5 in these tissues during homeostasis, repair and cancer.

Rapid and dynamic arginylation of the leading edge ß-actin is required for cell migration.

ß-actin plays key roles in cell migration. Our previous work demonstrated that ß-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of ß-actin arginylation in cell migration. We found that arginylated ß-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated ß-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of ß-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.

Enhancing a Wnt-Telomere Feedback Loop Restores Intestinal Stem Cell Function in a Human Organotypic Model of Dyskeratosis Congenita.

Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues, including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation. We show that DC tissue has suboptimal Wnt pathway activity causing intestinal stem cell failure and that enhanced expression of the telomere-capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with the clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. Thus, the isogenic DC cell model provides a platform for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients.

Reduced passive force in skeletal muscles lacking protein arginylation.

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.

Arginylation of myosin heavy chain regulates skeletal muscle strength.

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

Contractility of myofibrils from the heart and diaphragm muscles measured with atomic force cantilevers: effects of heart-specific deletion of arginyl-tRNA-protein transferase.

BACKGROUND: Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. OBJECTIVE: The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. METHODS: We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. RESULTS: In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89±9 nN/µm(2)) when compared to WT (132±11 nN/µm(2)), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K(act)), redevelopment (K(tr)), and relaxation (K(rel)). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209±31 nN/µm(2)) when compared to WT (123±20 nN/µm(2)). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K(act), but α-MHCAte1 myofibrils presented a faster K(rel) (6.11±0.41s(-1) vs 4.63±0.41 s(-1)). CONCLUSION: Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.