P63 targeted deletion under the FOXN1 promoter disrupts pre-and post-natal thymus development, function and maintenance as well as induces severe hair loss.

Progressive immune deficiency of aging is characterized by severe thymic atrophy, contracted T cell repertoire, and poor immune function. p63 is critical for the proliferative potential of embryonic and adult stem cells, as well as thymic epithelial cells (TECs). Because p63 null mice experience rapid post-natal lethality due to epidermal and limb morphogenesis defects, studies to define a role for p63 expression in TEC biology focused on embryonic thymus development and in vitro experiments. Since post-natal thymic stromal development and function differs from that of the embryo, we assessed the impact of lineage-restricted p63 loss on pre- and post-natal murine TEC function by generating mice with a loss of p63 function targeted to TEC, termed p63TECko mice. In adult p63TECko mice, severe thymic hypoplasia was observed with a lack in a discernable segregation into medullary and cortical compartments and peripheral T cell lymphopenia. This profound thymic defect was seen in both neonatal as well as embryonic p63TECko mice. In addition to TECs, p63 also plays in important role in the development of stratified epithelium of the skin; lack of p63 results in defects in skin epidermal stratification and differentiation. Interestingly, all adult p63TECko mice lacked hair follicles despite having normal p63 expression in the skin. Together our results show a critical role of TEC p63 in thymic development and maintenance and show that p63 expression is critical for hair follicle formation.

Spatiotemporal coordination of the RSF1-PLK1-Aurora B cascade establishes mitotic signaling platforms.

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.

San1 deficiency leads to cardiomyopathy due to excessive R-loop-associated DNA damage and cardiomyocyte hypoplasia.

R-loops are naturally occurring transcriptional intermediates containing RNA/DNA hybrids. Excessive R-loops cause genomic instability, DNA damage, and replication stress. Senataxin-associated exonuclease (San1) is a protein that interacts with Senataxin (SETX), a helicase resolving R-loops. It remains unknown if R-loops-induced DNA damage plays a role in the heart, especially in the proliferative neonatal cardiomyocytes (CMs). San1-/- mice were generated using the CRISPR/Cas9 technique. The newborn San1-/- mice show no overt phenotype, but their hearts were smaller with larger, yet fewer CMs. CM proliferation was impaired with reduced cell cycle-related transcripts and proteins. S9.6 staining revealed that excessive R-loops accumulated in the nucleus of neonatal San1-/- CMs. Increased γH2AX staining on newborn and adult heart sections exhibited increased DNA damage. Similarly, San1-/- AC16-cardiomyocytes showed cumulative R-loops and DNA damage, leading to the activation of cell cycle checkpoint kinase ATR and PARP1 hyperactivity, arresting G2/M cell-cycle and CM proliferation. Together, the present study uncovers an essential role of San1 in resolving excessive R-loops that lead to DNA damage and repressing CM proliferation, providing new insights into a novel biological function of San1 in the neonatal heart. San1 may serve as a novel therapeutic target for the treatment of hypoplastic cardiac disorders.

E3 ubiquitin ligase TRIM21 restricts hepatitis B virus replication by targeting HBx for proteasomal degradation.

As a cytosol ubiquitin ligase and antibody receptor, Tripartite motif-containing 21 (TRIM21) has been reported to mediate the restriction of hepatitis B virus (HBV) through an HBx-antibody-dependent intracellular neutralization (ADIN) mechanism. However, whether TRIM21 limits HBV replication by targeting viral proteins remains unclarified. In this study, we demonstrate that TRIM21 inhibits HBV gene transcription and replication in HBV plasmid transfected and HBV-infected hepatoma cells. RING and PRY-SPRY domains are involved in this activity. TRIM21 interacts with HBx protein and targets HBx for ubiquitination and proteasomal degradation, leading to impaired HBx-mediated degradation of structural maintenance of chromosomes 6 (Smc6) and suppression of HBV replication. TRIM21 fails to restrict the replication of an HBx-deficient HBV. And knock-down of Smc6 largely impairs the anti-HBV activity of TRIM21 in HepG2 cells. In a hydrodynamic injection (HDI)-based HBV mouse model, we confirm an in vivo anti-HBV and anti-HBx therapeutic effect of TRIM21 by over-expression or knocking-out strategy. Our findings reveal a novel mechanism that TRIM21 restricts HBV replication through targeting HBx-Smc5/6 pathway, which may have an implication in the future TRIM21-based therapeutic application.

Deficiency of CXXC finger protein 1 leads to small changes in heart rate but moderate epigenetic alterations and significant protein downregulation of hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) ion channels in mice.

BACKGROUND: The normal cardiac rhythm is generated in the sinoatrial node (SAN). Changes in ionic currents of the SAN may cause sinus arrhythmia. CXXC finger protein 1 (Cfp1) is an epigenetic regulator that is involved in transcriptional regulation of multiple genes. OBJECTIVE: The purpose of this study was to explore whether Cfp1 controls SAN function through regulation of ion channel-related genes. METHODS: Electrophysiological study, patch clamp recording, reverse transcriptase polymerase chain reaction, optical mapping, chromatin immunoprecipitation, and immunofluorescence staining were performed to evaluate the function of SAN and underlying mechanism on Cfp1 heterozygous knockout (Cfp1+/-) mice. RESULTS: Heart rate was slower slightly and SAN recovery time was longer in Cfp1+/- mice than controls. Whole-cell patch-clamp recording showed that the firing rate of action potentials was reduced in Cfp1+/- mice. The density of If current was reduced by 66% in SAN cells of Cfp1+/- mice but the densities of ICa, ICa-L, and ICa-T were not changed. The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) mRNA level in SAN tissue of Cfp1+/- mice was reduced. The HCN4 protein was significantly decreased in SAN cells and tissues after heterozygous deletion of Cfp1. Chromatin immunoprecipitation assay on cultured HL-1 cells demonstrated that Cfp1 was enriched in the promoter regions of HCN4. Knockdown of Cfp1 reduced H3K4 trimethylation, H3K9 acetylation, and H3K27 acetylation of HCN4 promoter region. CONCLUSION: Deficiency of Cfp1 leads to small changes in heart rate by moderate epigenetic modification alterations and significant protein downregulation of HCN4 ion channels in mice.

Loss of MYSM1 inhibits the oncogenic activity of cMYC in B cell lymphoma.

MYSM1 is a chromatin-binding protein, widely investigated for its functions in haematopoiesis in human and mouse; however, its role in haematologic malignancies remains unexplored. Here, we investigate the cross-talk between MYSM1 and oncogenic cMYC in the transcriptional regulation of genes encoding ribosomal proteins, and the implications of these mechanisms for cMYC-driven carcinogenesis. We demonstrate that in cMYC-driven B cell lymphoma in mouse models, MYSM1-loss represses ribosomal protein gene expression and protein synthesis. Importantly, the loss of MYSM1 also strongly inhibits cMYC oncogenic activity and protects against B cell lymphoma onset and progression in the mouse models. This advances the understanding of the molecular and transcriptional mechanisms of lymphomagenesis, and suggests MYSM1 as a possible drug target for cMYC-driven malignancies.

B Lymphocyte Specification Is Preceded by Extensive Epigenetic Priming in Multipotent Progenitors.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

Targeting hyperactive TGFBR2 for treating MYOCD deficient lung cancer.

Purpose: Clinical success of cancer therapy is severely limited by drug resistance, attributed in large part to the loss of function of tumor suppressor genes (TSGs). Developing effective strategies to treat those tumors is challenging, but urgently needed in clinic. Experimental Design: MYOCD is a clinically relevant TSG in lung cancer patients. Our in vitro and in vivo data confirm its tumor suppressive function. Further analysis reveals that MYOCD potently inhibits stemness of lung cancer stem cells. Mechanistically, MYOCD localizes to TGFBR2 promoter region and thereby recruits PRMT5/MEP50 complex to epigenetically silence its transcription. Conclusions: NSCLC cells deficient of MYOCD are particularly sensitive to TGFBR kinase inhibitor (TGFBRi). TGFBRi and stemness inhibitor synergize with existing drugs to treat MYOCD deficient lung cancers. Our current work shows that loss of function of MYOCD creates Achilles' heels in lung cancer cells, which might be exploited in clinic.

uhrf1 and dnmt1 Loss Induces an Immune Response in Zebrafish Livers Due to Viral Mimicry by Transposable Elements.

Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, uhrf1 and dnmt1. We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify tnfa as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of tnfa activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors sting and mavs reduced expression of interferon response genes and tnfa depletion dramatically reduced cell death in uhrf1 mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that tnfa mediates cell death as a potential mechanism to eliminate these damaged cells.

Neonatal diabetes mutations disrupt a chromatin pioneering function that activates the human insulin gene.

Despite the central role of chromosomal context in gene transcription, human noncoding DNA variants are generally studied outside of their genomic location. This limits our understanding of disease-causing regulatory variants. INS promoter mutations cause recessive neonatal diabetes. We show that all INS promoter point mutations in 60 patients disrupt a CC dinucleotide, whereas none affect other elements important for episomal promoter function. To model CC mutations, we humanized an ∼3.1-kb region of the mouse Ins2 gene. This recapitulated developmental chromatin states and cell-specific transcription. A CC mutant allele, however, abrogated active chromatin formation during pancreas development. A search for transcription factors acting through this element revealed that another neonatal diabetes gene product, GLIS3, has a pioneer-like ability to derepress INS chromatin, which is hampered by the CC mutation. Our in vivo analysis, therefore, connects two human genetic defects in an essential mechanism for developmental activation of the INS gene.

Impaired Peyer's patch development in BOB.1/OBF.1-deficient mice.

BOB.1/OBF.1 expression regulates the transcription of direct and indirect target genes. We propose that BOB.1/OBF.1 affects CXCL13-CXCR5 signaling of LTinducer and LTorganizer cells during embryonic Peyer's patch organogenesis as well as of B cells and follicular dendritic cells during lymphocyte homing at postnatal stages of secondary lymphoid organ development.

Overall survival of pancreatic ductal adenocarcinoma is doubled by Aldh7a1 deletion in the KPC mouse.

Rationale: The activity of aldehyde dehydrogenase 7A1 (ALDH7A1), an enzyme that catalyzes the lipid peroxidation of fatty aldehydes was found to be upregulated in pancreatic ductal adenocarcinoma (PDAC). ALDH7A1 knockdown significantly reduced tumor formation in PDAC. We raised a question how ALDH7A1 contributes to cancer progression. Methods: To answer the question, the role of ALDH7A1 in energy metabolism was investigated by knocking down and knockdown gene in mouse model, because the role of ALDH7A1 has been reported as a catabolic enzyme catalyzing fatty aldehyde from lipid peroxidation to fatty acid. Oxygen consumption rate (OCR), ATP production, mitochondrial membrane potential, proliferation assay and immunoblotting were performed. In in vivo study, two human PDAC cell lines were used for pre-clinical xenograft model as well as spontaneous PDAC model of KPC mice was also employed for anti-cancer therapeutic effect. Results:ALDH7A1 knockdown significantly reduced tumor formation with reduction of OCR and ATP production, which was inversely correlated with increase of 4-hydroxynonenal. This implies that ALDH7A1 is critical to process fatty aldehydes from lipid peroxidation. Overall survival of PDAC is doubled by cross breeding of KPC (KrasG12D; Trp53R172H; Pdx1-Cre) and Aldh7a1-/- mice. Conclusion: Inhibitions of ALDH7A1 and oxidative phosphorylation using gossypol and phenformin resulted in a regression of tumor formation in xenograft mice model and KPC mice model.

P63 Deficiency and CDX2 Overexpression Lead to Barrett's-Like Metaplasia in Mouse Esophageal Epithelium.

BACKGROUND: The cellular origin and molecular mechanisms of Barrett's esophagus (BE) are still controversial. Trans-differentiation is a mechanism characterized by activation of the intestinal differentiation program and inactivation of the squamous differentiation program. AIMS: Renal capsule grafting (RCG) was used to elucidate whether CDX2 overexpression on the basis of P63 deficiency in the esophageal epithelium may generate intestinal metaplasia. METHODS: P63-/-;Villin-Cdx2 embryos were generated by crossing P63+/- mice with Villin-Cdx2 mice. E18.5 esophagus was xenografted in a renal capsule grafting (RCG) model. At 1, 2, or 4 weeks after RCG, the mouse esophagus was immunostained for a proliferation marker (BrdU), squamous transcription factors (SOX2, PAX9), squamous differentiation markers (CK5, CK4, and CK1), intestinal transcription factors (CDX1, HNF1α, HNF4α, GATA4, and GATA6), intestinal columnar epithelial cell markers (A33, CK8), goblet cell marker (MUC2, TFF3), Paneth cell markers (LYZ and SOX9), enteroendocrine cell marker (CHA), and Tuft cell marker (DCAMKL1). RESULTS: The P63-/-;Villin-Cdx2 RCG esophagus was lined with proliferating PAS/AB+ cuboidal cells and formed an intestinal crypt-like structure. The goblet cell markers (TFF3 and MUC2) and intestinal transcription factors (CDX1, HNF1α, HNF4α, GATA4, and GATA6) were expressed although no typical morphology of goblet cells was observed. Other intestinal cell markers including enteroendocrine cell marker (CHA), Paneth cell markers (LYZ and Sox9), and intestinal secretory cell marker (UEA/WGA) were also expressed in the P63-/-;Villin-Cdx2 RCG esophagus. Squamous cell markers (PAX9 and SOX2) were also expressed, suggesting a transitional phenotype. CONCLUSION: CDX2 overexpression on the basis of P63 deficiency in esophageal epithelial cells induces Barrett's-like metaplasia in vivo. Additional factors may be needed to drive this transitional phenotype into full-blown BE.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

Targeting transcriptional coregulator OCA-B/Pou2af1 blocks activated autoreactive T cells in the pancreas and type 1 diabetes.

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

Characterization of a LuxR repressor for 3,17ß-HSD in Comamonas testosteroni ATCC11996.

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.

YAP and TAZ protect against white adipocyte cell death during obesity.

The expansion of the white adipose tissue (WAT) in obesity goes along with increased mechanical, metabolic and inflammatory stress. How adipocytes resist this stress is still poorly understood. Both in human and mouse adipocytes, the transcriptional co-activators YAP/TAZ and YAP/TAZ target genes become activated during obesity. When fed a high-fat diet (HFD), mice lacking YAP/TAZ in white adipocytes develop severe lipodystrophy with adipocyte cell death. The pro-apoptotic factor BIM, which is downregulated in adipocytes of obese mice and humans, is strongly upregulated in YAP/TAZ-deficient adipocytes under HFD, and suppression of BIM expression reduces adipocyte apoptosis. In differentiated adipocytes, TNFα and IL-1ß promote YAP/TAZ nuclear translocation via activation of RhoA-mediated actomyosin contractility and increase YAP/TAZ-mediated transcriptional regulation by activation of c-Jun N-terminal kinase (JNK) and AP-1. Our data indicate that the YAP/TAZ signaling pathway may be a target to control adipocyte cell death and compensatory adipogenesis during obesity.

Cellular kinetics of hematopoietic cells with Sfpi1 deletion are present at different frequencies in bone-marrow and spleen in X-irradiated mice.

PURPOSE: Several past studies using a mouse model of radiation-induced AML (rAML) have shown that hemizygous deletion of the Sfpi1 gene (HDSG) is an initiating event for the development of rAML. In this study, we examined the difference in frequency of HDSG in hematopoietic stem cells (HSCs) Rich hematopoietic Cell population (HRCs) from bone marrow (BM) and spleen of C3H mice irradiated with 3 Gy X-rays. MATERIALS AND METHODS: 8-weeks old male C3H mice were irradiated 3Gy of whole body X-ray (1 Gy/min) and mice were sacrificed at 1, 4, 8, and 26 weeks. Then, HSPCs were isolated from BM of femur and spleen, the frequency of HRCs with Sfpi1 gene deletion was analyzed by fluorescence in situ hybridization (FISH). RESULTS AND CONCLUSIONS: The frequency of HRCs with HDSG in both BM and spleen was increased 1 week after X-irradiation. Then, the frequency of HRCs with HDSG in BM showed a gradual decrease from 4 to 26 weeks, whereas HRCs with HDSG in spleen remained high, even at 26 weeks after X-irradiation. HDSG is less likely to be eliminated, particularly in the spleen, after X-irradiation. The spleen as well as BM of the femur may be major sites of rAML development.

Retinoic acid signaling within pancreatic endocrine progenitors regulates mouse and human ß cell specification.

Retinoic acid (RA) signaling is essential for multiple developmental processes, including appropriate pancreas formation from the foregut endoderm. RA is also required to generate pancreatic progenitors from human pluripotent stem cells. However, the role of RA signaling during endocrine specification has not been fully explored. In this study, we demonstrate that the disruption of RA signaling within the NEUROG3-expressing endocrine progenitor population impairs mouse ß cell differentiation and induces ectopic expression of crucial δ cell genes, including somatostatin. In addition, the inhibition of the RA pathway in hESC-derived pancreatic progenitors downstream of NEUROG3 induction impairs insulin expression. We further determine that RA-mediated regulation of endocrine cell differentiation occurs through Wnt pathway components. Together, these data demonstrate the importance of RA signaling in endocrine specification and identify conserved mechanisms by which RA signaling directs pancreatic endocrine cell fate.