DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

COVID-19 Infection May Drive EC-like Myofibroblasts towards Myofibroblasts to Contribute to Pulmonary Fibrosis.

COVID-19 has an extensive impact on Homo sapiens globally. Patients with COVID-19 are at an increased risk of developing pulmonary fibrosis. A previous study identified that myofibroblasts could be derived from pulmonary endothelial lineage cells as an important cell source that contributes to pulmonary fibrosis. Here, we analyzed publicly available data and showed that COVID-19 infection drove endothelial lineage cells towards myofibroblasts in pulmonary fibrosis of patients with COVID-19. We also discovered a similar differentiation trajectory in mouse lungs after viral infection. The results suggest that COVID-19 infection leads to the development of pulmonary fibrosis partly through the activation of endothelial cell (EC)-like myofibroblasts.

Developmental diversity and unique sensitivity to injury of lung endothelial subtypes during postnatal growth.

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

Regulating the cell shift of endothelial cell-like myofibroblasts in pulmonary fibrosis.

Pulmonary fibrosis is a common and severe fibrotic lung disease with high morbidity and mortality. Recent studies have reported a large number of unwanted myofibroblasts appearing in pulmonary fibrosis, and shown that the sustained activation of myofibroblasts is essential for unremitting interstitial fibrogenesis. However, the origin of these myofibroblasts remains poorly understood. Here, we create new mouse models of pulmonary fibrosis and identify a previously unknown population of endothelial cell (EC)-like myofibroblasts in normal lung tissue. We show that these EC-like myofibroblasts significantly contribute myofibroblasts to pulmonary fibrosis, which is confirmed by single-cell RNA sequencing of human pulmonary fibrosis. Using the transcriptional profiles, we identified a small molecule that redirects the differentiation of EC-like myofibroblasts and reduces pulmonary fibrosis in our mouse models. Our study reveals the mechanistic underpinnings of the differentiation of EC-like myofibroblasts in pulmonary fibrosis and may provide new strategies for therapeutic interventions.

Single-Cell RNA-Seq Identifies Dynamic Cardiac Transition Program from ADCs Induced by Leukemia Inhibitory Factor.

Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.

The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis.

Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes, including those for key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.

Shifting osteogenesis in vascular calcification.

Transitions between cell fates commonly occur in development and disease. However, reversing an unwanted cell transition in order to treat disease remains an unexplored area. Here, we report a successful process of guiding ill-fated transitions toward normalization in vascular calcification. Vascular calcification is a severe complication that increases the all-cause mortality of cardiovascular disease but lacks medical therapy. The vascular endothelium is a contributor of osteoprogenitor cells to vascular calcification through endothelial-mesenchymal transitions, in which endothelial cells (ECs) gain plasticity and the ability to differentiate into osteoblast-like cells. We created a high-throughput screening and identified SB216763, an inhibitor of glycogen synthase kinase 3 (GSK3), as an inducer of osteoblastic-endothelial transition. We demonstrated that SB216763 limited osteogenic differentiation in ECs at an early stage of vascular calcification. Lineage tracing showed that SB216763 redirected osteoblast-like cells to the endothelial lineage and reduced late-stage calcification. We also found that deletion of GSK3ß in osteoblasts recapitulated osteoblastic-endothelial transition and reduced vascular calcification. Overall, inhibition of GSK3ß promoted the transition of cells with osteoblastic characteristics to endothelial differentiation, thereby ameliorating vascular calcification.

Homeobox D3, A Novel Link Between Bone Morphogenetic Protein 9 and Transforming Growth Factor Beta 1 Signaling.

AIMS: Several signaling pathways contribute to endothelial-mesenchymal transitions and vascular calcification, including bone morphogenetic protein (BMP) and transforming growth factor (TGF) ß signaling. The transcription factor homeobox D3 (Hoxd3) is known to regulate an invasive endothelial phenotype, and the aim of the study is to determine if HOXD3 modulates BMP and TGFß signaling in the endothelium. METHODS AND RESEARCH: We report that the endothelium with high BMP activity due to the loss of BMP inhibitor matrix Gla protein (MGP) shows induction of Hoxd3. HOXD3 is part of a BMP-triggered cascade. When activated by BMP9, activin receptor-like kinase (ALK) 1 induces HOXD3 expression. Hoxd3 promoter is a direct target of phosphorylated (p) SMAD1, a mediator of BMP signaling. High BMP activity further results in enhanced TGFß signaling due to induction of TGFß1 and its receptor, ALK5. This is mediated by HOXD3, which directly targets the Tgfb1 promoter. Finally, TGFß1 and BMP9 stimulate the expression of MGP, which limits the enhanced ALK1 induction by counteracting BMP4. The cascade of BMP9-HOXD3-TGFß also affects Notch signaling and angiogenesis through induction of Notch ligand Jagged 2 and suppression of Notch ligand delta-like 4 (Dll4). CONCLUSION: The results suggest that HOXD3 is a novel link between BMP9/ALK1 and TGFß1/ALK5 signaling. TRANSLATIONAL PERSPECTIVE: BMP and TGFß signaling are instrumental in vascular disease such as vascular calcification and atherosclerosis. This study demonstrated a novel type of cross talk between endothelial BMP and TGFß signaling as mediated by HOXD3. The results provide a possible therapeutic approach to control dysfunctional BMP and TGFß signaling by regulating HOXD3.

Skip is essential for Notch signaling to induce Sox2 in cerebral arteriovenous malformations.

Notch signaling and Sry-box (Sox) family transcriptional factors both play critical roles in endothelial cell (EC) differentiation in vascularization. Recent studies have shown that excessive Notch signaling induces Sox2 to cause cerebral arteriovenous malformations (AVMs). Here, we examine human pulmonary AVMs and find no induction of Sox2. Results of epigenetic studies also show less alteration of Sox2-DNA binding in pulmonary AVMs than in cerebral AVMs. We identify high expression of ski-interacting protein (Skip) in brain ECs, a Notch-associated chromatin-modifying protein that is lacking in lung ECs. Knockdown of Skip abolished Notch-induction of Sox2 in brain ECs, while restoration of Skip in lung ECs enabled Notch-mediated Sox2 induction. The results suggest that Skip is a key factor for induction of Sox2 in cerebral AVMs.

Elevated endothelial Sox2 causes lumen disruption and cerebral arteriovenous malformations.

Lumen integrity in vascularization requires fully differentiated endothelial cells (ECs). Here, we report that endothelial-mesenchymal transitions (EndMTs) emerged in ECs of cerebral arteriovenous malformation (AVMs) and caused disruption of the lumen or lumen disorder. We show that excessive Sry-box 2 (Sox2) signaling was responsible for the EndMTs in cerebral AVMs. EC-specific suppression of Sox2 normalized endothelial differentiation and lumen formation and improved the cerebral AVMs. Epigenetic studies showed that induction of Sox2 altered the cerebral-endothelial transcriptional landscape and identified jumonji domain-containing protein 5 (JMJD5) as a direct target of Sox2. Sox2 interacted with JMJD5 to induce EndMTs in cerebral ECs. Furthermore, we utilized a high-throughput system to identify the ß-adrenergic antagonist pronethalol as an inhibitor of Sox2 expression. Treatment with pronethalol stabilized endothelial differentiation and lumen formation, which limited the cerebral AVMs.

Crosstalk between BMP and Notch Induces Sox2 in Cerebral Endothelial Cells.

Bone morphogenetic protein (BMP) and Notch signaling are critical for endothelial cell (EC) differentiation in vascular development. Recent studies have shown that excess BMP activity induces Notch signaling in cerebral ECs resulting in arteriovenous malformation (AVMs). However, it is unclear how the crosstalk between BMP and Notch signaling affects cerebral EC differentiation at the gene regulatory level. Here, we report that BMP6 activates the activin receptor-like kinase (ALK) 3, a BMP type 1 receptor, to induce Notch1 receptor and Jagged1 and Jagged2 ligands. We show that increased expression of the Notch components alters the transcriptional regulatory complex in the SRY-Box 2 (Sox2) promoter region so as to induce its expression in cerebral ECs. Together, our results identify Sox2 as a direct target of BMP and Notch signaling and provide information on how altered BMP and Notch signaling affects the endothelial transcriptional landscape.

The transcription factor SOX30 is a key regulator of mouse spermiogenesis.

The postmeiotic development of male germ cells, also known as spermiogenesis, features the coordinated expression of a large number of spermatid-specific genes. However, only a limited number of key transcription factors have been identified and the underlying regulatory mechanisms remain largely unknown. Here, we report that SOX30, the most-divergent member of the Sry-related high-motility group box (SOX) family of transcription factors, is essential for mouse spermiogenesis. The SOX30 protein was predominantly expressed in spermatids, while its transcription was regulated by retinoic acid and by MYBL1 before and during meiosis. Sox30 knockout mice arrested spermiogenesis at step 3 round spermatids, which underwent apoptosis and abnormal chromocenter formation. We also determined that SOX30 regulated the expression of hundreds of spermatid-specific protein-coding and long non-coding RNA genes. SOX30 bound to the proximal promoter of its own gene and activated its transcription. These results reveal SOX30 as a novel key regulator of spermiogenesis that regulates its own transcription to enforce and activate this meiotic regulatory pathway.

Reprogramming p53-Deficient Germline Stem Cells Into Pluripotent State by Nanog.

Cultured mouse spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), revert back to pluripotent state either spontaneously or upon being modified genetically. However, the reprogramming efficiencies are low, and the underlying mechanism remains poorly understood. In the present study, we conducted transcriptomic analysis and found that many transcription factors and epigenetic modifiers were differentially expressed between GSCs and embryonic stem cells. We failed in reprogramming GSCs to pluripotent state using the Yamanaka 4 Factors, but succeeded when Nanog and Tet1 were included. More importantly, reprogramming was also achieved with Nanog alone in a p53-deficient GSC line with an efficiency of 0.02‰. These GSC-derived-induced pluripotent stem cells possessed in vitro and in vivo differentiation abilities despite the low rate of chimera formation, which might be caused by abnormal methylation in certain paternally imprinted genes. Together, these results show that GSCs can be reprogrammed to pluripotent state via multiple avenues and contribute to our understanding of the mechanisms of GSC reprogramming.

Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint.

ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.

MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins.

miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.

Retinoic Acid Is Sufficient for the In Vitro Induction of Mouse Spermatocytes.

Meiosis is the key step in gametogenesis. However, the mechanism of mammalian meiosis remains poorly understood due to the lack of an in vitro model. Here, we report that retinoic acid (RA) is sufficient for inducing leptotene/zygotene spermatocytes from cultured mouse spermatogonial stem cells. Multiple genes regulated by RA were identified by RNA sequencing. RA in combination with pup Sertoli cell co-culture resulted in a higher induction efficiency of 28%. Comparisons in the transcriptomic profiles of the induced spermatogenic cells and the isolated ones revealed the progressive induction of the germ cells. Using this model, we showed that Stra8, Agpat3, Fam57a, Wdr91, and Sox30 contributed to the proliferation and meiosis initiation differentially. In conclusion, we have efficiently generated spermatocytes using an RA/pup Sertoli cell-based in vitro model and provided proof-of-concept evidence for its application in identifying genes involved in mammalian meiosis.

[Effects of acupuncture intervention on expression of glucose-regulated protein 78 and C/EBP homologous protein in hippocampal CA 1 region in rats with hyperspasmia].

OBJECTIVE: To observe the effect of acupuncture intervention on expression of glucose-regulated protein 78 (Grp 78) and C/EBP homologous protein (CHOP) in the hippocampus in epilepsy rats so as to explore its mechanism underlying improvement of hyperspasmia-induced brain injury. METHODS: Forty-two SD rats were randomly divided into normal control group (n = 6), model group (n = 18), and acupuncture group (n = 18). The epileptic seizure model was established by intraperitonel injection of Pentylenetetrazol (50 mg/kg, 2 mL). Manual acupuncture stimulation of "Baihui" (GV 20) and "Dazhui" (GV 14) was conducted for rats of the acupuncture group for 30 min. Two hours (h), 12 h and 48 h after acupuncture intervention, the hippocampal tissue was sampled (6 rats at each time-point). The expression levels of Grp 78 and CHOP proteins in the hippocampal CA 1 region were detected by immunohistochemistry. RESULTS: Compared with the normal group, the expression levels of Grp 78 protein at time-points of 2 h and 12 h, and those of CHOP protein at 2 h, 12 h and 48 h after epilpeptic seizure were significantly increased in the model group (P < 0.01). After acupuncture treatment, the expression levels of Grp 78 at 12 and 48 h were significantly increased, and those of CHOP protein at 2 h, 12 h and 24 h in the acupuncture group were considerably downregulated (P < 0.05, P < 0.01). CONCLUSION: Acupuncture treatment can up-regulate Grp 78 protein expression and down-regulate CHOP protein expression level in epilepsy rats , which may contribute to its protective effect on seizure-induced brain injury.

Generation of male germ cells from mouse induced pluripotent stem cells in vitro.

Germ cells are the only cell type that passes genetic information to the next generation. In most metazoan species, primordial germ cells (PGCs) were induced from epiblasts by signals from the neighboring tissues. In vitro derivation of germ cells from the pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced PSCs (iPSCs) are of great values for the treatment of infertility, for animal breeding, and for studying the mechanism of germ cell development. Although the derivations of male germ cells from PSCs have been previously reported, most of the studies failed to conduct the induction in a well-controlled and highly efficient manner. Here, we report the derivation of induced PGC-like cells (iPGCLCs) from mouse iPSCs via induced epiblast-like cells (iEpiLCs) as being monitored by the expression of enhanced green fluorescent protein gene under the control of the promoter of stimulated by retinoic acid 8 (Stra8-EGFP). The identity of iPGCLCs was characterized by examining the expression of multiple marker genes as well as by the recovery of spermatogenesis after they were transplanted to the testis of infertile W/W(v) mice. Furthermore, iPGCLCs were either induced to germline stem cell-like cells (iGSCLCs) or reverted back to embryonic germ cell-like cells (iEGCLCs). In conclusion, we have established an efficient procedure for inducing iPSCs into iPGCLCs that can be further expanded and induced to more developed germ cells. This work indicates that the technology of in vitro germ cell induction is becoming more sophisticated and can be further improved.