One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

TET1 is a tumor suppressor of hematopoietic malignancy.

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

The DNA dioxygenase Tet1 regulates H3K27 modification and embryonic stem cell biology independent of its catalytic activity.

Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1m/m) and knockout (Tet1-/-) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3+; H3K27me3+) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1-/-, but not Tet1m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.

Resolution of sister centromeres requires RanBP2-mediated SUMOylation of topoisomerase IIalpha.

RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis. However, the biological relevance of RanBP2 and the in vivo targets of its E3 ligase activity are unknown. Here we show that animals with low amounts of RanBP2 develop severe aneuploidy in the absence of overt transport defects. The main chromosome segregation defect in cells from these mice is anaphase-bridge formation. Topoisomerase IIalpha (Topo IIalpha), which decatenates sister centromeres prior to anaphase onset to prevent bridges, fails to accumulate at inner centromeres when RanBP2 levels are low. We find that RanBP2 sumoylates Topo IIalpha in mitosis and that this modification is required for its proper localization to inner centromeres. Furthermore, mice with low amounts of RanBP2 are highly sensitive to tumor formation. Together, these data identify RanBP2 as a chromosomal instability gene that regulates Topo IIalpha by sumoylation and suppresses tumorigenesis.

Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion.

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.

Altered hydroxymethylation is seen at regulatory regions in pancreatic cancer and regulates oncogenic pathways.

Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4 Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.

TET1 plays an essential oncogenic role in MLL-rearranged leukemia.

The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.

Catalytic-dependent and -independent roles of TET3 in the regulation of specific genetic programs during neuroectoderm specification.

The ten-eleven-translocation family of proteins (TET1/2/3) are epigenetic regulators of gene expression. They regulate genes by promoting DNA demethylation (i.e., catalytic activity) and by partnering with regulatory proteins (i.e., non-catalytic functions). Unlike Tet1 and Tet2, Tet3 is not expressed in mouse embryonic stem cells (ESCs) but is induced upon ESC differentiation. However, the significance of its dual roles in lineage specification is less defined. By generating TET3 catalytic-mutant (Tet3m/m) and knockout (Tet3-/-) mouse ESCs and differentiating them to neuroectoderm (NE), we identify distinct catalytic-dependent and independent roles of TET3 in NE specification. We find that the catalytic activity of TET3 is important for activation of neural genes while its non-catalytic functions are involved in suppressing mesodermal programs. Interestingly, the vast majority of differentially methylated regions (DMRs) in Tet3m/m and Tet3-/- NE cells are hypomethylated. The hypo-DMRs are associated to aberrantly upregulated genes while the hyper-DMRs are linked to downregulated neural genes. We find the maintenance methyltransferase Dnmt1 as a direct target of TET3, which is downregulated in TET3-deficient NE cells and may contribute to the increased DNA hypomethylation. Our findings establish that the catalytic-dependent and -independent roles of TET3 have distinct contributions to NE specification with potential implications in development.

Tet2 regulates Sin3a recruitment at active enhancers in embryonic stem cells.

Tet2 is a member of the Ten-eleven translocation (Tet1/2/3) family of enzymes and is expressed in embryonic stem cells (ESCs). It demethylates DNA (catalytic functions) and partners with chromatin modifiers (noncatalytic functions) to regulate genes. However, the significance of these functions in ESCs is less defined. Using Tet2 catalytic mutant (Tet2m/m) and knockout (Tet2-/-) ESCs, we identified Tet2 target genes regulated by its catalytic dependent versus independent roles. Tet2 was enriched at their active enhancers and promoters to demethylate them. We also identified the histone deacetylase component Sin3a as a Tet2 partner, co-localizing at promoters and active enhancers. Tet2 deficiency diminished Sin3a at these regions. Tet2 and Sin3a co-occupancy overlapped with Tet1. Combined loss of Tet1/2, but not of their catalytic activities, reduced Sin3a at active enhancers. These findings establish Tet2 catalytic and noncatalytic functions as regulators of DNA demethylation and Sin3a recruitment at active enhancers in ESCs.

Comparative analysis of Tet2 catalytic-deficient and knockout bone marrow over time.

TET2 is a member of the Ten-eleven translocation (Tet) family of DNA dioxygenases that regulate gene expression by promoting DNA demethylation (enzymatic activity) and partnering with chromatin regulatory complexes (nonenzymatic functions). TET2 is highly expressed in the hematopoietic lineage, where its molecular functions are the subject of continuous investigations because of the prevalence of TET2 mutations in hematologic malignancies. Previously, we have implicated Tet2 catalytic and noncatalytic functions in the regulation of myeloid and lymphoid lineages, respectively. However, the impact of these functions of Tet2 on hematopoiesis as the bone marrow ages remains unclear. Here, we conducted comparative transplantations and transcriptomic analyses of 3-, 6-, 9-, and 12-month-old Tet2 catalytic mutant (Mut) and knockout (KO) bone marrow. Tet2 Mut bone marrow of all ages exclusively caused hematopoietic disorders of the myeloid lineage. In contrast, young Tet2 KO bone marrow developed both lymphoid and myeloid diseases, whereas older Tet2 KO bone marrow predominantly elicited myeloid disorders with shorter latency than age-matched Tet2 Mut bone marrow. We identified robust gene dysregulation in Tet2 KO Lin- cells at 6 months that involved lymphoma and myelodysplastic syndrome and/or leukemia-causing genes, many of which were hypermethylated early in life. There was a shift from lymphoid to myeloid gene deregulation in Tet2 KO Lin- cells with age, underpinning the higher incidence of myeloid diseases. These findings expand on the dynamic regulation of bone marrow by Tet2 and show that its catalytic-dependent and -independent roles have distinct impacts on myeloid and lymphoid lineages with age.

Reprogramming of postnatal neurons into induced pluripotent stem cells by defined factors.

Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression.

Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Ten eleven translocation 1 (Tet1) is a DNA dioxygenase that promotes DNA demethylation by oxidizing 5-methylcytosine. It can also partner with chromatin-activating and repressive complexes to regulate gene expressions independent of its enzymatic activity. Tet1 is highly expressed in embryonic stem cells (ESCs) and regulates pluripotency and differentiation. However, its roles in ESC cell cycle progression and proliferation have not been investigated. Using a series of Tet1 catalytic mutant (Tet1m/m), knockout (Tet1-/-) and wild type (Tet1+/+) mouse ESCs (mESCs), we identified a non-catalytic role of Tet1 in the proper cell cycle progression and proliferation of mESCs. Tet1-/-, but not Tet1m/m, mESCs exhibited a significant reduction in proliferation and delayed progression through G1. We found that the cyclin-dependent kinase inhibitor p21/Cdkn1a was uniquely upregulated in Tet1-/- mESCs and its knockdown corrected the slow proliferation and delayed G1 progression. Mechanistically, we found that p21 was a direct target of Tet1. Tet1 occupancy at the p21 promoter overlapped with the repressive histone mark H3K27me3 as well as with the H3K27 trimethyl transferase PRC2 component Ezh2. A loss of Tet1, but not loss of its catalytic activity, significantly reduced the enrichment of Ezh2 and H3K27 trimethylation at the p21 promoter without affecting the DNA methylation levels. We also found that the proliferation defects of Tet1-/- mESCs were independent of their differentiation defects. Together, these findings established a non-catalytic role for Tet1 in suppressing p21 in mESCs to ensure a rapid G1-to-S progression, which is a key hallmark of ESC proliferation. It also established Tet1 as an epigenetic regulator of ESC proliferation in addition to its previously defined roles in ESC pluripotency and differentiation.

Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis.

Ten-eleven translocation (Tet) enzymes promote DNA demethylation by oxidizing 5-methylcytosine. They are expressed during development and are essential for mouse gastrulation. However, their postgastrulation functions are not well established. We find that global or endothelial-specific loss of all three Tet enzymes immediately after gastrulation leads to reduced number of hematopoietic stem and progenitor cells (HSPCs) and lethality in mid-gestation mouse embryos. This is due to defects in specification of HSPCs from endothelial cells (ECs) that compromise primitive and definitive hematopoiesis. Mechanistically, loss of Tet enzymes in ECs led to hypermethylation and down-regulation of NFκB1 and master hematopoietic transcription factors (Gata1/2, Runx1, and Gfi1b). Restoring Tet catalytic activity or overexpression of these factors in Tet-deficient ECs rescued hematopoiesis defects. This establishes Tet enzymes as activators of hematopoiesis programs in ECs for specification of HSPCs during embryogenesis, which is distinct from their roles in adult hematopoiesis, with implications in deriving HSPCs from pluripotent cells.

Idax and Rinf facilitate expression of Tet enzymes to promote neural and suppress trophectodermal programs during differentiation of embryonic stem cells.

The Inhibitor of disheveled and axin (Idax) and its ortholog the Retinoid inducible nuclear factor (Rinf) are DNA binding proteins with nuclear and cytoplasmic functions. Rinf is expressed in embryonic stem cells (ESCs) where it regulates transcription of the Ten-eleven translocation (Tet) enzymes, promoting neural and suppressing mesendoderm/trophectoderm differentiation. Here, we find that Idax, which is not expressed in ESCs, is induced upon differentiation. Like Rinf, Idax facilitates neural and silences trophectodermal programs. Individual or combined loss of Idax and Rinf led to downregulation of neural and upregulation of trophectoderm markers during differentiation of ESCs to embryoid bodies as well as during directed differentiation of ESCs to neural progenitor cells (NPCs) and trophoblast-like cells. These defects resemble those of Tet-deficient ESCs. Consistently, Tet genes are direct targets of Idax and Rinf, and loss of Idax and Rinf led to downregulation of Tet enzymes during ESC differentiation to NPCs and trophoblast-like cells. While Idax and Rinf single and double knockout (DKO) mice were viable and overtly normal, DKO embryos had reduced expression of several NPC markers in embryonic forebrains and deregulated expression of selected trophoblast markers in placentas. NPCs derived from DKO forebrains had reduced self-renewal while DKO placentas had increased junctional zone and reduced labyrinth layers. Together, our findings establish Idax and Rinf as regulators of Tet enzymes for proper differentiation of ESCs.

TET Enzymes and 5-Hydroxymethylcytosine in Neural Progenitor Cell Biology and Neurodevelopment.

Studies of tissue-specific epigenomes have revealed 5-hydroxymethylcytosine (5hmC) to be a highly enriched and dynamic DNA modification in the metazoan nervous system, inspiring interest in the function of this epigenetic mark in neurodevelopment and brain function. 5hmC is generated by oxidation of 5-methylcytosine (5mC), a process catalyzed by the ten-eleven translocation (TET) enzymes. 5hmC serves not only as an intermediate in DNA demethylation but also as a stable epigenetic mark. Here, we review the known functions of 5hmC and TET enzymes in neural progenitor cell biology and embryonic and postnatal neurogenesis. We also discuss how TET enzymes and 5hmC regulate neuronal activity and brain function and highlight their implications in human neurodevelopmental and neurodegenerative disorders. Finally, we present outstanding questions in the field and envision new research directions into the roles of 5hmC and TET enzymes in neurodevelopment.

Roles and Regulations of TET Enzymes in Solid Tumors.

The mechanisms governing the methylome profile of tumor suppressors and oncogenes have expanded with the discovery of oxidized states of 5-methylcytosine (5mC). Ten-eleven translocation (TET) enzymes are a family of dioxygenases that iteratively catalyze 5mC oxidation and promote cytosine demethylation, thereby creating a dynamic global and local methylation landscape. While the catalytic function of TET enzymes during stem cell differentiation and development have been well studied, less is known about the multifaceted roles of TET enzymes during carcinogenesis. This review outlines several tiers of TET regulation and overviews how TET deregulation promotes a cancer phenotype. Defining the tissue-specific and context-dependent roles of TET enzymes will deepen our understanding of the epigenetic perturbations that promote or inhibit carcinogenesis.

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus.

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.

Non-catalytic Roles of Tet2 Are Essential to Regulate Hematopoietic Stem and Progenitor Cell Homeostasis.

The Ten-eleven translocation (TET) enzymes regulate gene expression by promoting DNA demethylation and partnering with chromatin modifiers. TET2, a member of this family, is frequently mutated in hematological disorders. The contributions of TET2 in hematopoiesis have been attributed to its DNA demethylase activity, and the significance of its nonenzymatic functions has remained undefined. To dissect the catalytic and non-catalytic requirements of Tet2, we engineered catalytically inactive Tet2 mutant mice and conducted comparative analyses of Tet2 mutant and Tet2 knockout animals. Tet2 knockout mice exhibited expansion of hematopoietic stem and progenitor cells (HSPCs) and developed myeloid and lymphoid disorders, while Tet2 mutant mice predominantly developed myeloid malignancies reminiscent of human myelodysplastic syndromes. HSPCs from Tet2 knockout mice exhibited distinct gene expression profiles, including downregulation of Gata2. Overexpression of Gata2 in Tet2 knockout bone marrow cells ameliorated disease phenotypes. Our results reveal the non-catalytic roles of TET2 in HSPC homeostasis.