LncRNA SOX1-OT V1 acts as a decoy of HDAC10 to promote SOX1-dependent hESC neuronal differentiation.

Long noncoding RNAs (lncRNAs) are abundantly expressed in the nervous system, but their regulatory roles in neuronal differentiation are poorly understood. Using a human embryonic stem cell (hESC)-based 2D neural differentiation approach and a 3D cerebral organoid system, we show that SOX1-OT variant 1 (SOX1-OT V1), a SOX1 overlapping noncoding RNA, plays essential roles in both dorsal cortical neuron differentiation and ventral GABAergic neuron differentiation by facilitating SOX1 expression. SOX1-OT V1 physically interacts with HDAC10 through its 5' region, acts as a decoy to block HDAC10 binding to the SOX1 promoter, and thus maintains histone acetylation levels at the SOX1 promoter. SOX1 in turn activates ASCL1 expression and promotes neuronal differentiation. Taken together, we identify a SOX1-OT V1/HDAC10-SOX1-ASCL1 axis, which promotes neurogenesis, highlighting a role for lncRNAs in hESC neuronal differentiation.

PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation.

Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.

Long noncoding RNA Q associates with Sox2 and is involved in the maintenance of pluripotency in mouse embryonic stem cells.

Large intergenic noncoding RNAs (lincRNAs) in ESCs may play an important role in the maintenance of pluripotency. The identification of stem cell-specific lincRNAs and their interacting partners will deepen our understanding of the maintenance of stem cell pluripotency. We identified a lincRNA, LincQ, which is specifically expressed in ESCs and is regulated by core pluripotent transcription factors. It was rapidly downregulated during the differentiation process. Knockdown of LincQ in ESCs led to differentiation, downregulation of pluripotency-related genes, and upregulation of differentiation-related genes. We found that exon 1 of LincQ can specifically bind to Sox2. The Soxp region in Sox2, rather than the high mobility group domain, is responsible for LincQ binding. Importantly, the interaction between LincQ and Sox2 is required for the maintenance of pluripotency in ESCs and the transcription of pluripotency genes. Esrrb and Tfcp2l1 are key downstream targets of LincQ and Sox2, since overexpression of Esrrb and Tfcp2l1 can restore the loss of ESC pluripotency that is induced by LincQ depletion. In summary, we found that LincQ specifically interacts with Sox2 and contributes to the maintenance of pluripotency, highlighting the critical role of lincRNA in the pluripotency regulatory network.

MicroRNA-153 improves the neurogenesis of neural stem cells and enhances the cognitive ability of aged mice through the notch signaling pathway.

Aging-related cognitive ability impairments are one of the main threats to public health, and impaired hippocampal neurogenesis is a major cause of cognitive decline during aging. However, the regulation of adult neurogenesis in the hippocampus requires further study. Here, we investigated the role of microRNA-153 (miR-153), a highly conserved microRNA in mice and humans, in adult neurogenesis. During the passaging of neural stem cells (NSCs) in vitro, endogenous miR-153 expression was downregulated, with a decrease in neuronal differentiation ability. In addition, miR-153 overexpression increased the neurogenesis of NSCs. Further studies showed that miR-153 regulated neurogenesis by precisely targeting the Notch signaling pathway through inhibition of Jagged1 and Hey2 translation. In vivo analysis demonstrated that miR-153 expression was decreased in the hippocampi of aged mice with impaired cognitive ability, and that miR-153 overexpression in the hippocampus promoted neurogenesis and markedly increased the cognitive abilities of the aged mice. Overall, our findings revealed that miR-153 affected neurogenesis by regulating the Notch signaling pathway and elucidated the function of miR-153 in aging-related, hippocampus-dependent cognitive ability impairments, and neurodegenerative diseases.

Pwp1 regulates telomere length by stabilizing shelterin complex and maintaining histone H4K20 trimethylation.

Telomere maintenance is critical for chromosome stability. Here we report that periodic tryptophan protein 1 (PWP1) is involved in regulating telomere length homeostasis. Pwp1 appears to be essential for mouse development and embryonic stem cell (ESC) survival, as homozygous Pwp1-knockout mice and ESCs have never been obtained. Heterozygous Pwp1-knockout mice had shorter telomeres and decreased reproductive capacity. Pwp1 depletion induced rapid telomere shortening accompanied by reduced shelterin complex and increased DNA damage in telomeric regions. Mechanistically, PWP1 bound and stabilized the shelterin complex via its WD40 domains and regulated the overall level of H4K20me3. The rescue of telomere length in Pwp1-deficient cells by PWP1 overexpression depended on SUV4-20H2 co-expression and increased H4K20me3. Therefore, our study revealed a novel protein involved in telomere homeostasis in both mouse and human cells. This knowledge will improve our understanding of how chromatin structure and histone modifications are involved in maintaining telomere integrity.

Sin3a-Tet1 interaction activates gene transcription and is required for embryonic stem cell pluripotency.

Sin3a is a core component of histone-deacetylation-activity-associated transcriptional repressor complex, playing important roles in early embryo development. Here, we reported that down-regulation of Sin3a led to the loss of embryonic stem cell (ESC) self-renewal and skewed differentiation into mesendoderm lineage. We found that Sin3a functioned as a transcriptional coactivator of the critical Nodal antagonist Lefty1 through interacting with Tet1 to de-methylate the Lefty1 promoter. Further studies showed that two amino acid residues (Phe147, Phe182) in the PAH1 domain of Sin3a are essential for Sin3a-Tet1 interaction and its activity in regulating pluripotency. Furthermore, genome-wide analyses of Sin3a, Tet1 and Pol II ChIP-seq and of 5mC MeDIP-seq revealed that Sin3a acted with Tet1 to facilitate the transcription of a set of their co-target genes. These results link Sin3a to epigenetic DNA modifications in transcriptional activation and have implications for understanding mechanisms underlying versatile functions of Sin3a in mouse ESCs.

A Linc1405/Eomes Complex Promotes Cardiac Mesoderm Specification and Cardiogenesis.

Large intergenic non-coding RNAs (lincRNAs) play widespread roles in epigenetic regulation during multiple differentiation processes, but little is known about their mode of action in cardiac differentiation. Here, we identified the key roles of a lincRNA, termed linc1405, in modulating the core network of cardiac differentiation by functionally interacting with Eomes. Chromatin- and RNA-immunoprecipitation assays showed that exon 2 of linc1405 physically mediates a complex consisting of Eomes, trithorax group (TrxG) subunit WDR5, and histone acetyltransferase GCN5 binding at the enhancer region of Mesp1 gene and activates its expression during cardiac mesoderm specification of embryonic stem cells. Importantly, linc1405 co-localizes with Eomes, WDR5, and GCN5 at the primitive streak, and linc1405 depletion impairs heart development and function in vivo. In summary, linc1405 mediates a Eomes/WDR5/GCN5 complex that contributes to cardiogenesis, highlighting the critical roles of lincRNA-based complexes in the epigenetic regulation of cardiogenesis in vitro and in vivo.

N6-Methyladenosine modification of lincRNA 1281 is critically required for mESC differentiation potential.

Previous studies have revealed the critical roles of N6-methyladenosine (m6A) modification of mRNA in embryonic stem cells (ESCs), but the biological function of m6A in large intergenic noncoding RNA (lincRNA) is unknown. Here, we showed that the internal m6A modification of linc1281 mediates a competing endogenous RNA (ceRNA) model to regulate mouse ESC (mESC) differentiation. We demonstrated that loss of linc1281 compromises mESC differentiation and that m6A is highly enriched within linc1281 transcripts. Linc1281 with RRACU m6A sequence motifs, but not an m6A-deficient mutant, restored the phenotype in linc1281-depleted mESCs. Mechanistic analyses revealed that linc1281 ensures mESC identity by sequestering pluripotency-related let-7 family microRNAs (miRNAs), and this RNA-RNA interaction is m6A dependent. Collectively, these findings elucidated the functional roles of linc1281 and its m6A modification in mESCs and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns.

RTL1 promotes melanoma proliferation by regulating Wnt/ß-catenin signalling.

Cutaneous melanoma is a highly malignant and metastatic skin cancer with high mortality. However, its underlying mechanisms remain largely unclear. Here, we found that retrotransposon-like 1 (RTL1) is highly enriched in melanoma tissue, especially in early and horizontal growth tissues. Knockdown of RTL1 in melanoma cells resulted in cell proliferation suppression; cell cycle arrest at G1 phase; and down-regulation of E2F1, CYCLIN D1, cyclin-dependent kinase 6 (CDK6) and c-MYC. Moreover, overexpression of RTL1 in melanoma cells accelerated cell proliferation, promoted passage of the cell cycle beyond G1 phase, and increased the expression of cell cycle related genes. Mechanistically, we found that knockdown of RTL1 inhibited the Wnt/ß-Catenin pathway by regulating the expression of genes specifically involved in ß-CATENIN stabilization. Furthermore, the overexpression and knockdown of ß-CATENIN rescued the effects of RTL1 on melanoma cell proliferation and the cell cycle. These findings were also confirmed via tumour xenografts in nude mice. Together, our results demonstrated that RTL1 promotes melanoma cell proliferation by regulating the Wnt/ß-Catenin signalling pathway.

HDAC10 promotes angiogenesis in endothelial cells through the PTPN22/ERK axis.

Angiogenesis is crucially involved in many physiological and pathological processes including tumor growth, but the molecular mechanisms regulating angiogenesis are incompletely understood. In this study, we investigated the functions and mechanism of histone deacetylase 10 (HDAC10), a member of the HDAC II family, in regulation of angiogenesis. HDAC10 overexpression in human umbilical vein endothelial cells (HUVECs) promoted tube formation, whereas depletion of HDAC10 from HUVECs inhibited tube formation in vitro and in vivo. Mechanistically, HDAC10 overexpression increased extracellular-regulated kinase 1/2 (ERK1/2) activation, whereas depletion of HDAC10 inhibited ERK1/2 activation. Finally, HDAC10 promoted ERK1/2 phosphorylation by deacetylating the promoter of protein tyrosine phosphatase, non-receptor type 22 (PTPN22) and inhibiting the expression of PTPN22, which is a negative regulator of ERK phosphorylation. Collectively, our results identify HDAC10 as a key regulator of angiogenesis and reveal that HDAC10 functions in this process by binding and deacetylating the PTPN22 promoter and subsequently inhibiting PTPN22 expression, which in turn increases ERK1/2 phosphorylation. Our studies suggest that HDAC10 is a potential target for therapeutic intervention to inhibit angiogenesis and tumor growth.

Motifs in the amino-terminus of CENP-A are required for its accumulation within the nucleus and at the centromere.

Centromere protein A (CENP-A) is a variant of core histone H3 that marks the centromere's location on the chromosome. The mechanisms that target the protein to the nucleus and the centromere have not been defined. In this study, we found that deletion of the first 53 but not the first 29 residues of CENP-A from the amino-terminus, resulted in its cytoplasmic localization. Two motifs, R42R43R44 and K49R52K53K56, which are reported to be required for DNA contact in the centromere nucleosome, were found to be critical for CENP-A nuclear accumulation. These two motifs potentially mediated its interaction with Importin-ß but were not involved in CENP-A centromeric localization. A third novel motif, L60L61I62R63K64, was found to be essential for the centromeric accumulation of CENP-A. The nonpolar hydrophobic residues L60L61I62, but not the basic residues R63K64, were found to be the most important residues. A protein interaction assay suggested that this motif is not involved in the interaction of CENP-A with its deposition factors but potentially mediates its interaction with core histone H4 and CENP-B. Our study uncovered the role of the amino-terminus of CENP-A in localization.

Dysregulation of the SIRT1/OCT6 Axis Contributes to Environmental Stress-Induced Neural Induction Defects.

Environmental stresses are increasingly acknowledged as core causes of abnormal neural induction leading to neural tube defects (NTDs). However, the mechanism responsible for environmental stress-triggered neural induction defects remains unknown. Here, we report that a spectrum of environmental stresses, including oxidative stress, starvation, and DNA damage, profoundly activate SIRT1, an NAD+-dependent lysine deacetylase. Both mouse embryos and in vitro differentiated embryonic stem cells (ESCs) demonstrated a negative correlation between the expression of SIRT1 and that of OCT6, a key neural fate inducer. Activated SIRT1 radically deacetylates OCT6, triggers an OCT6 ubiquitination/degradation cascade, and consequently increases the incidence of NTD-like phenotypes in mice or hinders neural induction in both human and mouse ESCs. Together, our results suggest that early exposure to environmental stresses results in the dysregulation of the SIRT1/OCT6 axis and increases the risk of NTDs.

Sirt6 Promotes DNA End Joining in iPSCs Derived from Old Mice.

Induced pluripotent stem cells (iPSCs) have great potential for treating age-related diseases, but the genome integrity of iPSCs is critically important. Here, we demonstrate that non-homologous end joining (NHEJ), rather than homologous recombination (HR), is less efficient in iPSCs from old mice than young mice. We further find that Sirt6 is downregulated in iPSCs from old mice. Sirt6 directly binds to Ku80 and facilitates the Ku80/DNA-PKcs interaction, thus promoting DNA-PKcs phosphorylation at residue S2056, leading to efficient NHEJ. Rescue experiments show that introducing a combination of Sirt6 and the Yamanaka factors during reprogramming significantly promotes DNA double-strand break (DSB) repair by activating NHEJ in iPSCs derived from old mice. Thus, our study suggests a strategy to improve the quality of iPSCs derived from old donors by activating NHEJ and stabilizing the genome.

HDAC10 promotes lung cancer proliferation via AKT phosphorylation.

Histone deacetylase 10 (HDAC10) is a member of the class II HDACs, and its role in cancer is emerging. In this study, we found that HDAC10 is highly expressed in lung cancer tissues. It resides mainly in the cytoplasm of lung cancer cells but resides in the nucleus of adjacent normal cells. Further examinations revealed that HDAC10 resides in the cytoplasm in multiple lung cancer cell lines, including the A549, H358 and H460 cell lines, but mainly resides in the nucleus of normal lung epithelial 16HBE cells. A leucine-rich motif, R505L506L507C508V509A510L511, was identified as its nuclear localization signal (NLS), and a mutant (Mut-505-511) featuring mutations to A at each of its original R and L positions was found to be nuclear-localization defective. Functional analysis revealed that HDAC10 promoted lung cancer cell growth and that its knockdown induced cell cycle arrest and apoptosis. Mechanistic studies showed that HDAC10 knockdown significantly decreased the phosphorylation of AKT at Ser473 and that AKT expression significantly rescued the cell cycle arrest and apoptosis elicited by HDAC10 knockdown. A co-immunoprecipitation assay suggested that HDAC10 interacts with AKT and that inhibition of HDAC10 activity decreases its interaction with and phosphorylation of AKT. Finally, we confirmed that HDAC10 promoted lung cancer proliferation in a mouse model. Our study demonstrated that HDAC10 localizes and functions in the cytoplasm of lung cancer cells, thereby underscoring its potential role in the diagnosis and treatment of lung cancer.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells.

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation.

A motif from Lys216 to Lys222 in human BUB3 protein is a nuclear localization signal and critical for BUB3 function in mitotic checkpoint.

Human BUB3 is a key mitotic checkpoint factor that recognizes centromeric components and recruits other mitotic checkpoint molecules to the unattached kinetochore. The key amino acid residues responsible for its localization are not yet defined. In this study, we identified a motif from Lys(216) to Lys(222) in BUB3 as its nuclear localization signal. A BUB3 mutant with deletion of this motif (Del216-222) was found to localize to both the cytoplasm and the nucleus, distinct from the exclusively nuclear distribution of wild-type BUB3. Further analysis revealed that residues Glu(213), Lys(216), Lys(217), Lys(218), Tyr(219), and Phe(221), but not Lys(222), contribute to nuclear localization. Interestingly, the nuclear localization signal was also critical for the kinetochore localization of BUB3. The deletion mutant Del216-222 and a subtle mutant with four residue changes in this region (E213Q/K216E/K217E/K218E (QE)) did not localize to the kinetochore efficiently or mediate mitotic checkpoint arrest. Protein interaction data suggested that the QE mutant was able to interact with BUB1, MAD2, and BubR1 but that its association with the centromeric components CENP-A and KNL1 was impaired. A motif from Leu(61) to Leu(65) in CENP-A was found to be involved in the association of BUB3 and CENP-A in cells; however, further assays suggested that CENP-A does not physically interact with BUB3 and does not affect BUB3 localization. Our findings help to dissect the mechanisms of BUB3 in mitotic checkpoint signaling.

Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling.

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self-renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-40 repeat-containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self-renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation-seq results revealed that the PWP1-occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs.

MiR-495 suppresses mesendoderm differentiation of mouse embryonic stem cells via the direct targeting of Dnmt3a.

Embryonic stem cells (ESCs) are promising resources for clinical therapies due to their potential to generate multiple cell types. The dynamic expression of de novo methyltransferases (Dnmt3a and Dnmt3b) is essential to ESCs; however, the regulatory mechanism of Dnmt3a or Dnmt3b expression in ESCs is still poorly understood. Here, we reported that decreased expression of microRNA-495 (miR-495) in the first 2days of embryoid body (EB) formation was required for mouse embryonic stem cell (mESC) differentiation because repressed mesoderm and endoderm lineages were detected in ectopic miR-495 expression mESCs. This effect was reversed by the function blockade of miR-495. We identified Dnmt3a as a functional target of miR-495 and showed that endogenous miR-495 repressed the expression of Dnmt3a in mESCs. Furthermore, the effect of miR-495 on mESCs could be eliminated by Dnmt3a overexpression. Moreover, miR-495 had no effect on the expression of Dnmt3b despite the findings obtained from previous studies that mainly focused on the common characteristics of the regulatory mechanisms of Dnmt3a and Dnmt3b expression. Thus, our studies not only uncovered a previously uncharacterized function of miR-495 in mESC differentiation but also generated a new idea to explore the mechanisms governing the functional difference between Dnmt3a and Dnmt3b.

Overexpression of miR-126 inhibits the activation and migration of HSCs through targeting CRK.

BACKGROUND & AIMS: MicroRNAs (miRNAs) have been shown to play essential roles in HSCs activation which contributes to hepatic fibrosis. Our previous miRNA microarray results suggested that miR-126 might be decreased during HSCs activation as other studies. The aim of this study is to investigate the role of miR-126 during HSCs activation. METHODS: In this study, the expression of miR-126 during HSCs activation was measured and confirmed by qRT-PCR. Then, miR-126 expression was restored by transfection of lentivirus vector encoding miR-126. Futhermore, cell proliferation was assayed by the cell counting kit-8 (CCK-8), cell migration was assayed by transwell assay, and the markers of activation of HSCs, α-SMA and collagen type I, were assayed by qRT-PCR, Western Blotting, Immunostaining and ELISA. Luciferase reporter assay was used to find the target of miR-126, and Western Blotting and Immunostaining was used to validate the target of miR-126. Then, the expression and the role of the target of miR-126 during HSCs activation was further assessed. RESULTS: The expression of miR-126 was confirmed to be significantly decreased during HSCs activation. Overexpression of miR-126 significantly inhibited HSCs migration but did not affect HSCs proliferation. The expression of α-SMA and collagen type I were both obviously decreased by miR-126 restoration. CRK was found to be the target of miR-126 and overexpression of miR-126 significantly inhibited CRK expression. And it was found that overexpression of CRK also significantly decreased miR-126 expression and promoted HSCs activation. CONCLUSIONS: Our study showed that overexpression of miR-126 significantly inhibited the activation and migration of HSCs through targeting CRK which can also decrease miR-126 expression and promote HSCs activation.

Histone deacetylase (HDAC) 10 suppresses cervical cancer metastasis through inhibition of matrix metalloproteinase (MMP) 2 and 9 expression.

Aberrant expression of histone deacetylases (HDACs) is associated with carcinogenesis. Some HDAC inhibitors are widely considered as promising anticancer therapeutics. A major obstacle for development of HDAC inhibitors as highly safe and effective anticancer therapeutics is that our current knowledge on the contributions of different HDACs in various cancer types remains scant. Here we report that the expression level of HDAC10 was significantly lower in patients exhibiting lymph node metastasis compared with that in patients lacking lymph node metastasis in human cervical squamous cell carcinoma. Forced expression of HDAC10 in cervical cancer cells significantly inhibited cell motility and invasiveness in vitro and metastasis in vivo. Mechanistically, HDAC10 suppresses expression of matrix metalloproteinase (MMP) 2 and 9 genes, which are known to be critical for cancer cell invasion and metastasis. At the molecular level, HDAC10 binds to MMP2 and -9 promoter regions, reduces the histone acetylation level, and inhibits the binding of RNA polymerase II to these regions. Furthermore, an HDAC10 mutant lacking histone deacetylase activity failed to mimic the functions of full-length protein. These results identify a critical role of HDAC10 in suppression of cervical cancer metastasis, underscoring the importance of developing isoform-specific HDAC inhibitors for treatment of certain cancer types such as cervical squamous cell carcinoma.