Profiling the role of m6A effectors in the regulation of pluripotent reprogramming.

The N6-methyladenosine (m6A) RNA modification plays essential roles in multiple biological processes, including stem cell fate determination. To explore the role of the m6A modification in pluripotent reprogramming, we used RNA-seq to map m6A effectors in human iPSCs, fibroblasts, and H9 ESCs, as well as in mouse ESCs and fibroblasts. By integrating the human and mouse RNA-seq data, we found that 19 m6A effectors were significantly upregulated in reprogramming. Notably, IGF2BPs, particularly IGF2BP1, were among the most upregulated genes in pluripotent cells, while YTHDF3 had high levels of expression in fibroblasts. Using quantitative PCR and Western blot, we validated the pluripotency-associated elevation of IGF2BPs. Knockdown of IGF2BP1 induced the downregulation of stemness genes and exit from pluripotency. Proteome analysis of cells collected at both the beginning and terminal states of the reprogramming process revealed that the IGF2BP1 protein was positively correlated with stemness markers SOX2 and OCT4. The eCLIP-seq target analysis showed that IGF2BP1 interacted with the coding sequence (CDS) and 3'UTR regions of the SOX2 transcripts, in agreement with the location of m6A modifications. This study identifies IGF2BP1 as a vital pluripotency-associated m6A effector, providing new insight into the interplay between m6A epigenetic modifications and pluripotent reprogramming.

LncRNA Osilr9 coordinates promoter DNA demethylation and the intrachromosomal loop structure required for maintaining stem cell pluripotency.

Nuclear reprogramming of somatic cells into a pluripotent status has the potential to create patient-specific induced pluripotent stem cells for regenerative medicine. Currently, however, the epigenetic mechanisms underlying this pluripotent reprogramming are poorly understood. To delineate this epigenetic regulatory network, we utilized a chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to identify long noncoding RNAs (lncRNAs) embedded in the 3-dimensional intrachromosomal architecture of stem cell core factor genes. By combining CRIST-seq and RNA sequencing, we identified Oct4-Sox2 interacting lncRNA 9 (Osilr9) as a pluripotency-associated lncRNA. Osilr9 expression was associated with the status of stem cell pluripotency in reprogramming. Using short hairpin RNA (shRNA) knockdown, we showed that this lncRNA was required for the optimal maintenance of stem cell pluripotency. Overexpression of Osilr9 induced robust activation of endogenous stem cell core factor genes in fibroblasts. Osilr9 participated in the formation of the intrachromosomal looping required for the maintenance of pluripotency. After binding to the Oct4 promoter, Osilr9 recruited the DNA demethylase ten-eleven translocation 1, leading to promoter demethylation. These data demonstrate that Osilr9 is a critical chromatin epigenetic modulator that coordinates the promoter activity of core stem cell factor genes, highlighting the critical role of pluripotency-associated lncRNAs in stem cell pluripotency and reprogramming.

Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing.

Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

Oplr16 serves as a novel chromatin factor to control stem cell fate by modulating pluripotency-specific chromosomal looping and TET2-mediated DNA demethylation.

Formation of a pluripotency-specific chromatin network is a critical event in reprogramming somatic cells into pluripotent status. To characterize the regulatory components in this process, we used 'chromatin RNA in situ reverse transcription sequencing' (CRIST-seq) to profile RNA components that interact with the pluripotency master gene Oct4. Using this approach, we identified a novel nuclear lncRNA Oplr16 that was closely involved in the initiation of reprogramming. Oplr16 not only interacted with the Oct4 promoter and regulated its activity, but it was also specifically activated during reprogramming to pluripotency. Active expression of Oplr16 was required for optimal maintenance of pluripotency in embryonic stem cells. Oplr16 was also able to enhance reprogramming of fibroblasts into pluripotent cells. RNA reverse transcription-associated trap sequencing (RAT-seq) indicated that Oplr16 interacted with multiple target genes related to stem cell self-renewal. Of note, Oplr16 utilized its 3'-fragment to recruit the chromatin factor SMC1 to orchestrate pluripotency-specific intrachromosomal looping. After binding to the Oct4 promoter, Oplr16 recruited TET2 to induce DNA demethylation and activate Oct4 in fibroblasts, leading to enhanced reprogramming. These data suggest that Oplr16 may act as a pivotal chromatin factor to control stem cell fate by modulating chromatin architecture and DNA demethylation.

Long noncoding RNAs and their epigenetic function in hematological diseases.

Recent discoveries demonstrate the importance of long noncoding RNA (lncRNA) in the regulation of multiple major processes impacting development, differentiation, and metastasis of hematological diseases through epigenetic mechanisms. In contrast to genetic changes, epigenetic modification does not modify genes but is frequently reversible, thus providing opportunities for targeted treatment using specific inhibitors. In this review, we will summarize the function and epigenetic mechanism of lncRNA in malignant hematologic diseases.

Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.

BACKGROUND/AIMS: Adult T-cell leukemia/lymphoma (ATL) is a very aggressive T cell malignancy that carries a poor prognosis, primarily due to its resistance to chemotherapy and to life-threatening infectious complications. Interferon-alpha (IFNα) has been used in combination with the anti-retroviral drug zidovudine to treat patients with ATL. However, the efficacy of long-term therapy is significantly limited due to the systemic toxicity of IFNα. METHODS: We utilized phage display library screening to identify short peptides that specifically bind to Jurkat T lymphocyte leukemia cells. By fusing the Jurkat-binding peptide to the C-terminus of IFNα, we constructed an engineered chimeric IFNα molecule (IFNP) for the treatment of ATL. RESULTS: We found that IFNP exhibited significantly higher activity than wild type IFNα in inhibiting the growth of leukemia cells and inducing cell blockage at the G0/G1 phase. The synthetic IFNP molecule exerted its antitumor activity by upregulating the downstream genes involved in the STAT1 pathway and in apoptosis. Using a cell receptor binding assay, we showed that this Jurkat-binding peptide facilitated the binding affinity of IFNα to the cell surface type I IFN receptor. CONCLUSION: The isolated Jurkat-binding peptide significantly potentiates the therapeutic activity of IFNα in T lymphocyte leukemia cells. The engineered IFNP molecule may prove to a novel antitumor approach in the treatment of patients with ATL.

Valproic Acid Enhances iPSC Induction From Human Bone Marrow-Derived Cells Through the Suppression of Reprogramming-Induced Senescence.

Reprogramming of human somatic cells into pluripotent cells (iPSCs) by defined transcription factors is an extremely inefficient process. Treatment with the histone deacetylase inhibitor valproic acid (VPA) during reprogramming can improve the induction of iPSCs. To examine the specific mechanism underlying the role of VPA in reprogramming, we transfected human bone marrow-derived cells (HSC-J2 and HSC-L1) with lentiviruses carrying defined factors (OCT4, SOX2, KLF4, and c-MYC, OSKM) in the presence of VPA. We found that, OSKM lentiviruses caused significant senescence in transfected cells. Administration of VPA, however, significantly suppressed this reprogramming-induced stress. Notably, VPA treatment improved cell proliferation in the early stages of reprogramming, and this was related to the down-regulation of the activated p16/p21 pathway. In addition, VPA also released the G2/M phase blockade in lentivirus-transfected cells. This study demonstrates a new mechanistic role of the histone deacetylase inhibitor in enhancing the induction of pluripotency. J. Cell. Physiol. 231: 1719-1727, 2016. © 2015 Wiley Periodicals, Inc.

Converting Skin Fibroblasts into Hepatic-like Cells by Transient Programming.

Transplantation of hepatocytes is a promising therapy for end-stage liver disease, but the availability of functional cells currently precludes its clinical application. We now report a simple transient reprogramming approach to convert fibroblasts into hepatic-like cells. Human skin fibroblasts were treated with fish egg extracts to become the transiently remodeled cells (TRCs). After infected with retroviral EGFP, they were directly injected into the fetal monkey liver, where they underwent in situ differentiation in the hepatic niche. The hepatic-like cells were functional as shown by the synthesis of hepatic markers in vivo, including albumin, cytokeratin-18, and hepatic serum antigen. Similarly, when implanted in the mouse liver, the TRCs were differentiated into hepatic-like cells that synthesize albumin and CK18 and became completely integrated into the liver parenchyma. The potency of TRCs was mechanistically related to the activation of several signal pathways, which reactivate endogenous genes related to cell potency. This study demonstrates the feasibility of a simple and inexpensive epigenetic remodeling approach to convert human fibroblasts into therapeutic hepatic-like cells for the treatment of end-stage liver disease.

Restoration of IGF2 imprinting by polycomb repressive complex 2 docking factor SUZ12 in colon cancer cells.

The insulin-like growth factor II (IGF2) gene is aberrantly expressed in tumors as a result of loss of imprinting (LOI). Reactivation of the normally-suppressed maternal allele may lead to IGF2 upregulation and increased tumor growth, particularly in colon cancer. However, the mechanisms underlying IGF2 LOI in tumors are poorly defined. In this report, we identified polycomb repressive complex 2 (PRC2) docking factor SUZ12 as a critical factor in regulating IGF2 imprinting in tumors. Human colon cancer cell lines (HRT18 and HT29) show loss of IGF2 imprinting. Ectopic expression of SUZ12 restored normal monoallelic expression of IGF2 in these two colon cancer cell lines. Using chromatin immunoprecipitation (ChIP) and chromatin conformation capture (3C), we found that the virally-expressed SUZ12 bound to IGF2 promoters, coordinating with endogenous CTCF to orchestrate a long range intrachromosomal loop between the imprinting control region (ICR) and the IGF2 promoters. The histone methyltransferase EZH2 was recruited to the IGF2 promoters, where it induced H3K27 hypermethylation, suppressing one allele, leading to the restoration of IGF2 imprinting. These data demonstrate that SUZ12 is a key molecule in the regulation of monoallelic expression of IGF2, suggesting a novel epigenetic therapeutic strategy for modulating IGF2 production in human tumors.

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress.

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency.

A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies.

Dysregulation of the insulin-like growth factor type I receptor (IGF1R) has been implicated in the progression and therapeutic resistance of malignancies. In acute myeloid leukemia (AML) cells, IGF1R is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in cancer. We discovered a novel intragenic long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter. The IRAIN lncRNA was expressed exclusively from the paternal allele, with the maternal counterpart being silenced. Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction. In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients. These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.

An intragenic long noncoding RNA interacts epigenetically with the RUNX1 promoter and enhancer chromatin DNA in hematopoietic malignancies.

RUNX1, a master regulator of hematopoiesis, is the most commonly perturbed target of chromosomal abnormalities in hematopoietic malignancies. The t(8;21) translocation is found in 30-40% of cases of acute myeloid leukemia (AML). Recent whole-exome sequencing also reveals mutations and deletions of RUNX1 in some solid tumors. We describe a RUNX1-intragenic long noncoding RNA RUNXOR that is transcribed as unspliced transcript from an upstream overlapping promoter. RUNXOR was upregulated in AML samples and in response to Ara-C treatment in vitro. RUNXOR utilizes its 3'-terminal fragment to directly interact with the RUNX1 promoter and enhancers and participates in the orchestration of an intrachromosomal loop. The 3' region of RUNXOR also participates in long-range interchromosomal interactions with chromatin regions that are involved in multiple RUNX1 translocations. These data suggest that RUNXOR noncoding RNA may function as a previously unidentified candidate component that is involved in chromosomal translocation in hematopoietic malignancies.

Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure.

BACKGROUND AIMS: Acute liver failure (ALF), a life-threatening disease characterized by the sudden loss of hepatic function, can occur after an accidental or intentional acetaminophen overdose. METHODS: With the use of an ALF mouse model, we examined both the preventive and therapeutic potential of intravenously administered human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Primary hUCMSCs were purified from freshly collected full-term umbilical cords and intravenously transplanted into BALB/c mice either before and after ALF induced by acetaminophen intoxication. We found that hUCMSCs significantly improved survival rates and relative liver weight of mice in both pre-ALF and post-ALF animals. Correspondingly, serum levels of markers that reflect hepatic injury (ie, aspartate aminotransferase, alanine aminotransferase and total bilirubin) were significantly attenuated in the group receiving hUCMSC therapy. RESULTS: Mechanistically, we found that the protective potential of intravenously administered hUCMSCs was mediated by paracrine pathways that involved antioxidants (glutathione, superoxide dismutase), the reduction of inflammatory agents (tumor necrosis factor-α, interleukin-6) and elevated serum levels of hepatocyte growth factor. CONCLUSIONS: Through these paracrine effects, intravenously administered hUCMSCs reduced hepatic necrosis/apoptosis and enhanced liver regeneration. Thus, our data demonstrate that intravenously administered hUCMSCs may be useful in the prevention or treatment of acetaminophen-induced ALF.

LncRNAs-circRNAs as Rising Epigenetic Binary Superstars in Regulating Lipid Metabolic Reprogramming of Cancers.

As one of novel hallmarks of cancer, lipid metabolic reprogramming has recently been becoming fascinating and widely studied. Lipid metabolic reprogramming in cancer is shown to support carcinogenesis, progression, distal metastasis, and chemotherapy resistance by generating ATP, biosynthesizing macromolecules, and maintaining appropriate redox status. Notably, increasing evidence confirms that lipid metabolic reprogramming is under the control of dysregulated non-coding RNAs in cancer, especially lncRNAs and circRNAs. This review highlights the present research findings on the aberrantly expressed lncRNAs and circRNAs involved in the lipid metabolic reprogramming of cancer. Emphasis is placed on their regulatory targets in lipid metabolic reprogramming and associated mechanisms, including the clinical relevance in cancer through lipid metabolism modulation. Such insights will be pivotal in identifying new theranostic targets and treatment strategies for cancer patients afflicted with lipid metabolic reprogramming.

Mechanisms underlying therapeutic resistance of tyrosine kinase inhibitors in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a malignant clonal disease involving hematopoietic stem cells that is characterized by myeloid cell proliferation in bone marrow and peripheral blood, and the presence of the Philadelphia (Ph) chromosome with BCR-ABL fusion gene. Treatment of CML has dramatically improved since the advent of tyrosine kinase inhibitors (TKI). However, there are a small subset of CML patients who develop resistance to TKI. Mutations in the ABL kinase domain (KD) are currently recognized as the leading cause of TKI resistance in CML. In this review, we discuss the concept of resistance and summarize recent advances exploring the mechanisms underlying CML resistance. Overcoming TKI resistance appears to be the most successful approach to reduce the burden of leukemia and enhance cures for CML. Advances in new strategies to combat drug resistance may rapidly change the management of TKI-resistant CML and expand the prospects for available therapies.

Eukaryotic translation initiation factor eIF4G2 opens novel paths for protein synthesis in development, apoptosis and cell differentiation.

Protein translation is a critical regulatory event involved in nearly all physiological and pathological processes. Eukaryotic translation initiation factors are dedicated to translation initiation, the most highly regulated stage of protein synthesis. Eukaryotic translation initiation factor 4G2 (eIF4G2, also called p97, NAT1 and DAP5), an eIF4G family member that lacks the binding sites for 5' cap binding protein eIF4E, is widely considered to be a key factor for internal ribosome entry sites (IRESs)-mediated cap-independent translation. However, recent findings demonstrate that eIF4G2 also supports many other translation initiation pathways. In this review, we summarize the role of eIF4G2 in a variety of cap-independent and -dependent translation initiation events. Additionally, we also update recent findings regarding the role of eIF4G2 in apoptosis, cell survival, cell differentiation and embryonic development. These studies reveal an emerging new picture of how eIF4G2 utilizes diverse translational mechanisms to regulate gene expression.

Therapeutic restoration of female reproductive and endocrine dysfunction using stem cells.

Millions of women worldwide suffer from infertility associated with gynecologic disorders such as premature ovarian insufficiency, polycystic ovary syndrome, Asherman syndrome, endometriosis, preeclampsia, and fallopian tube obstruction. These disorders can lead to infertility and thereby affect the quality of life of the infertile couple because of their psychological impact and significant costs. In recent years, stem cell therapy has emerged as a therapeutic approach to repair or replace damaged tissues or organs. This review describes the recent development as well as the underlying mechanisms of stem cell therapy for a variety of female reproductive diseases, offering us new therapeutic options for the treatment of female reproductive and endocrine dysfunction.

Therapeutic potential of engineering the mitochondrial genome.

Mitochondrial diseases are a group of clinical disorders caused by mutations in the genes encoded by either the nuclear or the mitochondrial genome involved in mitochondrial oxidative phosphorylation. Disorders become evident when mitochondrial dysfunction reaches a cell-specific threshold. Similarly, the severity of disorders is related to the degree of gene mutation. Clinical treatments for mitochondrial diseases mainly rely on symptomatic management. Theoretically, replacing or repairing dysfunctional mitochondria to acquire and preserve normal physiological functions should be effective. Significant advances have been made in gene therapies, including mitochondrial replacement therapy, mitochondrial genome manipulation, nuclease programming, mitochondrial DNA editing, and mitochondrial RNA interference. In this paper, we review the recent progress in these technologies by focusing on advancements that overcome limitations.

Chromatin-RNA in situ Reverse Transcription Sequencing (CRIST-seq) Approach to Profile the Non-coding RNA Interaction Network.

Non-coding RNAs (ncRNAs) are defined as RNAs that do not encode proteins, but many ncRNAs do have the ability to regulate gene expression. These ncRNAs play a critical role in the epigenetic regulation of various physiological and pathological processes through diverse biochemical mechanisms. However, the existing screening methods to identify regulatory ncRNAs only focus on whole-cell expression levels and do not capture every ncRNA that targets certain genes. We describe a new method, chromatin-RNA in situ reverse transcription sequencing (CRIST-seq), that can identify all the ncRNAs that are associated with the regulation of any given gene. In this article, we targeted the ncRNAs that are associated with pluripotent gene Sox2, allowing us to catalog the ncRNA regulation network of pluripotency maintenance. This methodology is universally applicable for the study of epigenetic regulation of any genes by making simple changes on the CRISPR-dCas9 gRNAs. Key features This method provides a new technique for screening ncRNAs and establishing chromatin interaction networks. The target gene for this method can be any gene of interest and any site in the entire genome. This method can be further extended to detect RNAs, DNAs, and proteins that interact with target genes. Graphical overview.

Erratum: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer.

[This corrects the article on p. 714 in vol. 9, PMID: 31105998.].