Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications.

DNA replication fork speed underlies cell fate changes and promotes reprogramming.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.

Intersection of regulatory pathways controlling hemostasis and hemochorial placentation.

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.

Collective regulation of chromatin modifications predicts replication timing during cell cycle.

Replication timing (RT) associates with genome architecture, while having a mixed relationship to histone marks. By profiling replication at high resolution and assessing broad histone marks across the cell cycle at the resolution of RT with and without genetic perturbation, we address the causal relationship between histone marks and RT. Four primary chromatin states, including an uncharacterized H3K36me2 state, emerge and define 97% of the mappable genome. RT and local replication patterns (e.g., initiation zones) quantitatively associate with chromatin states, histone mark dynamics, and spatial chromatin structure. Manipulation of broad histone marks and enhancer elements by overexpressing the histone H3 lysine 9/36 tri-demethylase KDM4A impacts RT across 11% of the genome. Broad histone modification changes were strong predictors of the observed RT alterations. Lastly, replication within H3K36me2-enriched neighborhoods is sensitive to KDM4A overexpression and is controlled at a megabase scale. These studies establish a role for collective chromatin mark regulation in modulating RT.

Simultaneous Inhibition of LSD1 and TGFß Enables Eradication of Poorly Immunogenic Tumors with Anti-PD-1 Treatment.

Epigenetic regulators are a class of promising targets in combination with immune checkpoint inhibitors for cancer treatment, but the impact of the broad effects of perturbing epigenetic regulators on tumor immunotherapy remains to be fully explored. Here we show that ablation of the histone demethylase LSD1 in multiple tumor cells induces TGFß expression, which exerts an inhibitory effect on T-cell immunity through suppressing the cytotoxicity of intratumoral CD8+ T cells and consequently dampens the antitumor effect of LSD1 ablation-induced T-cell infiltration. Importantly, concurrent depletion of LSD1 and TGFß in combination with PD-1 blockade significantly increases both CD8+ T-cell infiltration and cytotoxicity, leading to eradication of poorly immunogenic tumors and a long-term protection from tumor rechallenge. Thus, combining LSD1 inhibition with blockade of TGFß and PD-1 may represent a promising triple combination therapy for treating certain refractory tumors. SIGNIFICANCE: Cotargeting LSD1 and TGFß cooperatively elevates intratumoral CD8+ T-cell infiltration and unleashes their cytotoxicity, leading to tumor eradication upon anti-PD-1 treatment. Our findings illustrate a duality of epigenetic perturbations in immunotherapy and implicate the combination of LSD1 inhibition with dual PD-1/TGFß blockade in treating certain poorly immunogenic tumors.This article is highlighted in the In This Issue feature, p. 1861.

SUV39H2 controls trophoblast stem cell fate.

BACKGROUND: The placenta is formed by the coordinated expansion and differentiation of trophoblast stem (TS) cells along a multi-lineage pathway. Dynamic regulation of histone 3 lysine 9 (H3K9) methylation is pivotal to cell differentiation for many cell lineages, but little is known about its involvement in trophoblast cell development. METHODS: Expression of H3K9 methyltransferases was surveyed in rat TS cells maintained in the stem state and following differentiation. The role of suppressor of variegation 3-9 homolog 2 (SUV39H2) in the regulation of trophoblast cell lineage development was investigated using a loss-of-function approach in rat TS cells and ex vivo cultured rat blastocysts. RESULTS: Among the twelve-known H3K9 methyltransferases, only SUV39H2 exhibited robust differential expression in stem versus differentiated TS cells. SUV39H2 transcript and protein expression were high in the stem state and declined as TS cells differentiated. Disruption of SUV39H2 expression in TS cells led to an arrest in TS cell proliferation and activation of trophoblast cell differentiation. SUV39H2 regulated H3K9 methylation status at loci exhibiting differentiation-dependent gene expression. Analyses of SUV39H2 on ex vivo rat blastocyst development supported its role in regulating TS cell expansion and differentiation. We further identified SUV39H2 as a downstream target of caudal type homeobox 2, a master regulator of trophoblast lineage development. CONCLUSIONS: Our findings indicate that SUV39H2 contributes to the maintenance of TS cells and restrains trophoblast cell differentiation. GENERAL SIGNIFICANCE: SUV39H2 serves as a contributor to the epigenetic regulation of hemochorial placental development.

The lysine demethylase KDM4A controls the cell-cycle expression of replicative canonical histone genes.

Chromatin modulation provides a key checkpoint for controlling cell cycle regulated gene networks. The replicative canonical histone genes are one such gene family under tight regulation during cell division. These genes are most highly expressed during S phase when histones are needed to chromatinize the new DNA template. While this fact has been known for a while, limited knowledge exists about the specific chromatin regulators controlling their temporal expression during cell cycle. Since histones and their associated mutations are emerging as major players in diseases such as cancer, identifying the chromatin factors modulating their expression is critical. The histone lysine tri-demethylase KDM4A is regulated over cell cycle and plays a direct role in DNA replication timing, site-specific rereplication, and DNA amplifications during S phase. Here, we establish an unappreciated role for the catalytically active KDM4A in directly regulating canonical replicative histone gene networks during cell cycle. Of interest, we further demonstrate that KDM4A interacts with proteins controlling histone expression and RNA processing (i.e., hnRNPUL1 and FUS/TLS). Together, this study provides a new function for KDM4A in modulating canonical histone gene expression.

Histone Lysine Methylation Dynamics Control EGFR DNA Copy-Number Amplification.

Acquired chromosomal DNA copy gains are a feature of many tumors; however, the mechanisms that underpin oncogene amplification are poorly understood. Recent studies have begun to uncover the importance of epigenetic states and histone lysine methyltransferases (KMT) and demethylases (KDM) in regulating transient site-specific DNA copy-number gains (TSSG). In this study, we reveal a critical interplay between a myriad of lysine methyltransferases and demethylases in modulating H3K4/9/27 methylation balance to control extrachromosomal amplification of the EGFR oncogene. This study further establishes that cellular signals (hypoxia and EGF) are able to directly promote EGFR amplification through modulation of the enzymes controlling EGFR copy gains. Moreover, we demonstrate that chemical inhibitors targeting specific KMTs and KDMs are able to promote or block extrachromosomal EGFR amplification, which identifies potential therapeutic strategies for controlling EGFR copy-number heterogeneity in cancer, and, in turn, drug response. SIGNIFICANCE: This study identifies a network of epigenetic factors and cellular signals that directly control EGFR DNA amplification. We demonstrate that chemical inhibitors targeting enzymes controlling this amplification can be used to rheostat EGFR copy number, which uncovers therapeutic opportunities for controlling EGFR DNA amplification heterogeneity and the associated drug response.This article is highlighted in the In This Issue feature, p. 161.

Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.

Hypoxia Signaling and Placental Adaptations.

Oxygen is an essential nutrient for cells. Oxygen is delivered to tissues via red blood cells through the vasculature. Molecular mechanisms mediating cellular responses to low oxygen tension have been identified. Hypoxia-inducible factors (HIFs) are activated by low oxygen and promote transcriptional regulation of downstream effector genes, which lead to cellular adaptations. Controlled hypoxia exposure is utilized as an experimental tool to investigate biological processes, regulating cellular adaptations. Here we describe detailed protocols for hypoxia exposure of pregnant rodent models and low oxygen exposure of trophoblast stem cells, utilizing gas-regulated chamber systems. The presentation also includes phenotypic analyses of the manipulated animal models and cells.

Origin of a rapidly evolving homeostatic control system programming testis function.

Mammals share common strategies for regulating reproduction, including a conserved hypothalamic-pituitary-gonadal axis; yet, individual species exhibit differences in reproductive performance. In this report, we describe the discovery of a species-restricted homeostatic control system programming testis growth and function. Prl3c1 is a member of the prolactin gene family and its protein product (PLP-J) was discovered as a uterine cytokine contributing to the establishment of pregnancy. We utilized mouse mutagenesis of Prl3c1 and revealed its involvement in the regulation of the male reproductive axis. The Prl3c1-null male reproductive phenotype was characterized by testiculomegaly and hyperandrogenism. The larger testes in the Prl3c1-null mice were associated with an expansion of the Leydig cell compartment. Prl3c1 locus is a template for two transcripts (Prl3c1-v1 and Prl3c1-v2) expressed in a tissue-specific pattern. Prl3c1-v1 is expressed in uterine decidua, while Prl3c1-v2 is expressed in Leydig cells of the testis. 5'RACE, chromatin immunoprecipitation and DNA methylation analyses were used to define cell-specific promoter usage and alternative transcript expression. We examined the Prl3c1 locus in five murid rodents and showed that the testicular transcript and encoded protein are the result of a recent retrotransposition event at the Mus musculus Prl3c1 locus. Prl3c1-v1 encodes PLP-J V1 and Prl3c1-v2 encodes PLP-J V2. Each protein exhibits distinct intracellular targeting and actions. PLP-J V2 possesses Leydig cell-static actions consistent with the Prl3c1-null testicular phenotype. Analysis of the biology of the Prl3c1 gene has provided insight into a previously unappreciated homeostatic setpoint control system programming testicular growth and function.

Defining the Role of Estrogen Receptor ß in the Regulation of Female Fertility.

Estrogens are essential hormones for the regulation of fertility. Cellular responses to estrogens are mediated by estrogen receptor α (ESR1) and estrogen receptor ß (ESR2). In mouse and rat models, disruption of Esr1 causes infertility in both males and females. However, the role of ESR2 in reproductive function remains undecided because of a wide variation in phenotypic observations among Esr2-mutant mouse strains. Regulatory pathways independent of ESR2 binding to its cognate DNA response element have also been implicated in ESR2 signaling. To clarify the regulatory roles of ESR2, we generated two mutant rat models: one with a null mutation (exon 3 deletion, Esr2ΔE3) and the other with an inframe deletion selectively disrupting the DNA binding domain (exon 4 deletion, Esr2ΔE4). In both models, we observed that ESR2-mutant males were fertile. ESR2-mutant females exhibited regular estrous cycles and could be inseminated by wild-type (WT) males but did not become pregnant or pseudopregnant. Esr2-mutant ovaries were small and differed from WT ovaries by their absence of corpora lutea, despite the presence of follicles at various stages of development. Esr2ΔE3- and Esr2ΔE4-mutant females exhibited attenuated preovulatory gonadotropin surges and did not ovulate in response to a gonadotropin regimen effective in WT rats. Similarities of reproductive deficits in Esr2ΔE3 and Esr2ΔE4 mutants suggest that DNA binding-dependent transcriptional function of ESR2 is critical for preovulatory follicle maturation and ovulation. Overall, the findings indicate that neuroendocrine and ovarian deficits are linked to infertility observed in Esr2-mutant rats.

Natural killer-cell deficiency alters placental development in rats.

Natural killer (NK) cells are the most prevalent leukocyte population in the uterus during early pregnancy. Natural killer cells contribute to uterine vascular (spiral artery) remodeling in preparation for the increased demand on these vessels later in pregnancy. A second wave of spiral artery modification is directed by invasive trophoblast cells. The significance of the initial wave of NK-cell-mediated vascular remodeling in species exhibiting deep trophoblast invasion such as humans and rats is not known. The purpose of this study was to generate a genetic model of NK-cell deficiency in rats, and determine the consequences of NK-cell deficiency on spiral artery remodeling and reproductive outcomes. To accomplish this task, we utilized zinc finger nuclease-mediated genome editing of the rat interleukin-15 (Il15) gene. Il15 encodes a cytokine required for NK-cell lineage development. Using this strategy, a founder rat was generated containing a frameshift deletion in Il15. Uteri of females harboring a homozygous mutation at the Il15 locus contained no detectable NK cells. NK-cell deficiency did not impact fetal growth or viability. However, NK-cell deficiency caused major structural changes to the placenta, including expansion of the junctional zone and robust, early-onset activation of invasive trophoblast-guided spiral artery remodeling. In summary, we successfully generated an NK-cell-deficient rat and showed, using this model, that NK cells dampen the extent of trophoblast invasion and delay trophoblast-directed spiral artery remodeling. This study furthers our understanding of the role of NK cells on uterine vascular remodeling, trophoblast invasion, and placental development.

Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery.

In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of 'gain-of-function' and 'loss-of-function' blastocyst manipulations can be evaluated with in vitro outgrowth assays or following transfer to pseudopregnant rats.

HIF-KDM3A-MMP12 regulatory circuit ensures trophoblast plasticity and placental adaptations to hypoxia.

The hemochorial placenta develops from the coordinated multilineage differentiation of trophoblast stem (TS) cells. An invasive trophoblast cell lineage remodels uterine spiral arteries, facilitating nutrient flow, failure of which is associated with pathological conditions such as preeclampsia, intrauterine growth restriction, and preterm birth. Hypoxia plays an instructive role in influencing trophoblast cell differentiation and regulating placental organization. Key downstream hypoxia-activated events were delineated using rat TS cells and tested in vivo, using trophoblast-specific lentiviral gene delivery and genome editing. DNA microarray analyses performed on rat TS cells exposed to ambient or low oxygen and pregnant rats exposed to ambient or hypoxic conditions showed up-regulation of genes characteristic of an invasive/vascular remodeling/inflammatory phenotype. Among the shared up-regulated genes was matrix metallopeptidase 12 (MMP12). To explore the functional importance of MMP12 in trophoblast cell-directed spiral artery remodeling, we generated an Mmp12 mutant rat model using transcription activator-like nucleases-mediated genome editing. Homozygous mutant placentation sites showed decreased hypoxia-dependent endovascular trophoblast invasion and impaired trophoblast-directed spiral artery remodeling. A link was established between hypoxia/HIF and MMP12; however, evidence did not support Mmp12 as a direct target of HIF action. Lysine demethylase 3A (KDM3A) was identified as mediator of hypoxia/HIF regulation of Mmp12 Knockdown of KDM3A in rat TS cells inhibited the expression of a subset of the hypoxia-hypoxia inducible factor (HIF)-dependent transcripts, including Mmp12, altered H3K9 methylation status, and decreased hypoxia-induced trophoblast cell invasion in vitro and in vivo. The hypoxia-HIF-KDM3A-MMP12 regulatory circuit is conserved and facilitates placental adaptations to environmental challenges.

CITED2 modulation of trophoblast cell differentiation: insights from global transcriptome analysis.

Trophoblast stem (TS) cells possess the capacity to differentiate along a multi-lineage pathway yielding several specialized cell types. The regulatory network controlling trophoblast cell differentiation is poorly understood. Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) has been implicated in the regulation of placentation; however, we know little about how CITED2 acts to influence trophoblast cells. Rat Rcho-1 TS cells can be manipulated to proliferate or differentiate into specialized trophoblast lineages and are an excellent model for investigating trophoblast differentiation. CITED2 transcript and protein showed a robust induction during Rcho-1 TS cell differentiation. We used an shRNA knockdown approach to disrupt CITED2 expression in order to investigate its involvement in trophoblast cell differentiation. RNA-sequencing was used to examine the impact of CITED2 on trophoblast cell differentiation. CITED2 disruption affected the differentiating trophoblast cell transcriptome. CITED2 possessed a prominent role in the regulation of cell differentiation with links to several signal transduction pathways and to hypoxia-regulated and coagulation processes. In summary, our findings indicate that CITED2 contributes to the regulation of trophoblast cell differentiation.

OVO-like 1 regulates progenitor cell fate in human trophoblast development.

Epithelial barrier integrity is dependent on progenitor cells that either divide to replenish themselves or differentiate into a specialized epithelium. This paradigm exists in human placenta, where cytotrophoblast cells either propagate or undergo a unique differentiation program: fusion into an overlying syncytiotrophoblast. Syncytiotrophoblast is the primary barrier regulating the exchange of nutrients and gases between maternal and fetal blood and is the principal site for synthesizing hormones vital for human pregnancy. How trophoblast cells regulate their differentiation into a syncytium is not well understood. In this study, we show that the transcription factor OVO-like 1 (OVOL1), a homolog of Drosophila ovo, regulates the transition from progenitor to differentiated trophoblast cells. OVOL1 is expressed in human placenta and was robustly induced following stimulation of trophoblast differentiation. Disruption of OVOL1 abrogated cytotrophoblast fusion and inhibited the expression of a broad set of genes required for trophoblast cell fusion and hormonogenesis. OVOL1 was required to suppress genes that maintain cytotrophoblast cells in a progenitor state, including MYC, ID1, TP63, and ASCL2, and bound specifically to regions upstream of each of these genes. Our results reveal an important function of OVOL1 as a regulator of trophoblast progenitor cell fate during human trophoblast development.

Neonatal Progesterone Programs Adult Uterine Responses to Progesterone and Susceptibility to Uterine Dysfunction.

In this report, we investigated the consequences of neonatal progesterone exposure on adult rat uterine function. Female pups were subcutaneously injected with vehicle or progesterone from postnatal days 3 to 9. Early progesterone exposure affected endometrial gland biogenesis, puberty, decidualization, and fertility. Because decidualization and pregnancy success are directly linked to progesterone action on the uterus, we investigated the responsiveness of the adult uterus to progesterone. We first identified progesterone-dependent uterine gene expression using RNA sequencing and quantitative RT-PCR in Holtzman Sprague-Dawley rats and progesterone-resistant Brown Norway rats. The impact of neonatal progesterone treatment on adult uterine progesterone responsiveness was next investigated using quantitative RT-PCR. Progesterone resistance affected the spectrum and total number of progesterone-responsive genes and the magnitude of uterine responses for a subset of progesterone targets. Several progesterone-responsive genes in adult uterus exhibited significantly dampened responses in neonatally progesterone-treated females compared with those of vehicle-treated controls, whereas other progesterone-responsive transcripts did not differ between female rats exposed to vehicle or progesterone as neonates. The organizational actions of progesterone on the uterus were dependent on signaling through the progesterone receptor but not estrogen receptor 1. To summarize, neonatal progesterone exposure leads to disturbances in endometrial gland biogenesis, progesterone resistance, and uterine dysfunction. Neonatal progesterone effectively programs adult uterine responsiveness to progesterone.