Germ cells manifest a unique gene expression program and regain totipotency in the zygote. Here, we perform Hi-C analysis to examine 3D chromatin organization in male germ cells during spermatogenesis. We show that the highly compartmentalized 3D chromatin organization characteristic of interphase nuclei is attenuated in meiotic prophase. Meiotic prophase is predominated by short-range intrachromosomal interactions that represent a condensed form akin to that of mitotic chromosomes. However, unlike mitotic chromosomes, meiotic chromosomes display weak genomic compartmentalization, weak topologically associating domains, and localized point interactions in prophase. In postmeiotic round spermatids, genomic compartmentalization increases and gives rise to the strong compartmentalization seen in mature sperm. The X chromosome lacks domain organization during meiotic sex-chromosome inactivation. We propose that male meiosis occurs amid global reprogramming of 3D chromatin organization and that strengthening of chromatin compartmentalization takes place in spermiogenesis to prepare the next generation of life.

Pregnancy and parturition are intricately regulated to ensure successful reproductive outcomes. However, the factors that control gestational length in humans and other anthropoid primates remain poorly defined. Here, we show the endogenous retroviral long terminal repeat transposon-like human element 1B (THE1B) selectively controls placental expression of corticotropin-releasing hormone (CRH) that, in turn, influences gestational length and birth timing. Placental expression of CRH and subsequently prolonged gestational length were found in two independent strains of transgenic mice carrying a 180-kb human bacterial artificial chromosome (BAC) DNA that contained the full length of CRH and extended flanking regions, including THE1B. Restricted deletion of THE1B silenced placental CRH expression and normalized birth timing in these transgenic lines. Furthermore, we revealed an interaction at the 5' insertion site of THE1B with distal-less homeobox 3 (DLX3), a transcription factor expressed in placenta. Together, these findings suggest that retroviral insertion of THE1B into the anthropoid primate genome may have initiated expression of CRH in placental syncytiotrophoblasts via DLX3 and that this placental CRH is sufficient to alter the timing of birth.

The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.

The sex chromosomes are enriched with germline genes that are activated during the late stages of spermatogenesis. Due to meiotic sex chromosome inactivation (MSCI), these sex chromosome-linked genes must escape silencing for activation in spermatids, thereby ensuring their functions for male reproduction. RNF8, a DNA damage response protein, and SCML2, a germline-specific Polycomb protein, are two major, known regulators of this process. Here, we show that RNF8 and SCML2 cooperate to regulate ubiquitination during meiosis, an early step to establish active histone modifications for subsequent gene activation. Double mutants of Rnf8 and Scml2 revealed that RNF8-dependent monoubiquitination of histone H2A at Lysine 119 (H2AK119ub) is deubiquitinated by SCML2, demonstrating interplay between RNF8 and SCML2 in ubiquitin regulation. Additionally, we identify distinct functions of RNF8 and SCML2 in the regulation of ubiquitination: SCML2 deubiquitinates RNF8-independent H2AK119ub but does not deubiquitinate RNF8-dependent polyubiquitination. RNF8-dependent polyubiquitination is required for the establishment of H3K27 acetylation, a marker of active enhancers, while persistent H2AK119ub inhibits establishment of H3K27 acetylation. Following the deposition of H3K27 acetylation, H3K4 dimethylation is established as an active mark on poised promoters. Together, we propose a model whereby regulation of ubiquitin leads to the organization of poised enhancers and promoters during meiosis, which induce subsequent gene activation from the otherwise silent sex chromosomes in postmeiotic spermatids.

N6-methyladenosine (m6A), the most abundant internal modification in mRNA, has been implicated in tumorigenesis. As an m6A demethylase, ALKBH5 has been shown to promote the development of breast cancer and brain tumors. However, in acute myeloid leukemia (AML), ALKBH5 was reported to be frequently deleted, implying a tumor-suppressor role. Here, we show that ALKBH5 deletion is rare in human AML; instead, ALKBH5 is aberrantly overexpressed in AML. Moreover, its increased expression correlates with poor prognosis in AML patients. We demonstrate that ALKBH5 is required for the development and maintenance of AML and self-renewal of leukemia stem/initiating cells (LSCs/LICs) but not essential for normal hematopoiesis. Mechanistically, ALKBH5 exerts tumor-promoting effects in AML by post-transcriptional regulation of its critical targets such as TACC3, a prognosis-associated oncogene in various cancers. Collectively, our findings reveal crucial functions of ALKBH5 in leukemogenesis and LSC/LIC self-renewal/maintenance and highlight the therapeutic potential of targeting the ALKBH5/m6A axis.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrest and elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

To facilitate long-term survival, cells must exit the cell cycle and enter quiescence, a reversible non-replicative state. Budding yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence is unknown. By investigating the nascent transcriptome, we identified over a thousand noncoding RNAs in quiescent and G1 yeast cells, and found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. Additionally, both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that, in quiescence, the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs. RNA sequencing through quiescent entry revealed two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, thereby facilitating efficient quiescence entry. Our work identified a previously unknown key biological role for the nuclear exosome-NNS pathway in mRNA regulation and uncovered a novel layer of gene-expression control in quiescence.

In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge.

Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we use CRISPR/Cas9 gene editing to create a conditional GR knockdown in Sprague Dawley rats. To test the impact of cell- and region-specific GR knockdown on physiology and behavior, we targeted GR knockdown to output neurons of the prelimbic cortex. Prelimbic knockdown of GR in females caused deficits in acquisition and extinction of fear memory during auditory fear conditioning, whereas males exhibited enhanced active-coping behavior during forced swim. Our data support the utility of this conditional knockdown rat to afford high-precision knockdown of GR across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging the behavioral tractability and enhanced brain size of the rat as a model organism.

The voltage-gated potassium channel Kv4.2 is a critical regulator of dendritic excitability in the hippocampus and is crucial for dendritic signal integration. Kv4.2 mRNA and protein expression as well as function are reduced in several genetic and pharmacologically induced rodent models of epilepsy and autism. It is not known, however, whether reduced Kv4.2 is just an epiphenomenon or a disease-contributing cause of neuronal hyperexcitability and behavioral impairments in these neurological disorders. To address this question, we used male and female mice heterozygous for a Kv.2 deletion and adult-onset manipulation of hippocampal Kv4.2 expression in male mice to assess the role of Kv4.2 in regulating neuronal network excitability, morphology and anxiety-related behaviors. We observed a reduction in dendritic spine density and reduced proportions of thin and stubby spines but no changes in anxiety, overall activity, or retention of conditioned freezing memory in Kv4.2 heterozygous mice compared with wildtype littermates. Using EEG analyses, we showed elevated theta power and increased spike frequency in Kv4.2 heterozygous mice under basal conditions. In addition, the latency to onset of kainic acid-induced seizures was significantly shortened in Kv4.2 heterozygous mice compared with wildtype littermates, which was accompanied by a significant increase in theta power. By contrast, overexpressing Kv4.2 in wildtype mice through intrahippocampal injection of Kv4.2-expressing lentivirus delayed seizure onset and reduced EEG power. These results suggest that Kv4.2 is an important regulator of neuronal network excitability and dendritic spine morphology, but not anxiety-related behaviors. In the future, manipulation of Kv4.2 expression could be used to alter seizure susceptibility in epilepsy.

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disease in humans, characterized by abnormal deposition of cholesterol and phospholipids in cornea caused by mutations in the UbiA prenyltransferase domain containing 1 (UBIAD1) gene. In this study, we generated a mouse line carrying Ubiad1 N100S point mutation using the CRISPR/Cas9 technique to investigate the pathogenesis of SCD. In vivo confocal microscopy revealed hyper-reflective dot-like deposits in the anterior cornea in heterozygotes and homozygotes. No significant change was found in corneal epithelial barrier function or wound healing. Electron microscopy revealed abnormal mitochondrial morphology in corneal epithelial, stromal, and endothelial cells. Mitochondrial DNA copy number assay showed 1.27 ± 0.07 fold change in homozygotes versus 0.98 ± 0.05 variation in wild type mice (P < 0.05). Lipidomic analysis indicated abnormal metabolism of glycerophosphoglycerols, a lipid class found in mitochondria. Four (34:1, 34:2, 36:2, and 44:8) of the 11 glycerophosphoglycerols species identified by mass spectrometry showed a significant increase in homozygous corneas compared with heterozygous and wild-type mouse corneas. Unexpectedly, we did not find a difference in the corneal cholesterol level between different genotypes by filipin staining or lipidomic analysis. The Ubiad1N100S mouse provides a promising animal model of SCD revealing that mitochondrial dysfunction is a prominent component of the disease. The different phenotype in human and mouse may due to difference in cholesterol metabolism between species.

Dendritic spines are small, dynamic protrusions along the dendrite that comprise more than 90% of excitatory connections in the brain, making them essential sites for neuronal communication. These synaptic sites change throughout the process of development, reducing in density and shifting morphology as synapses are refined. One important class of dendritic spine regulators is microRNA (miRNA), small-noncoding RNAs that post-transcriptionally regulate gene expression. Several studies suggest that miRNA-324-5p regulates dendritic spine formation. In addition, we have previously shown that miR-324-5p plays a role in seizure and long-term potentiation, both of which involve dendritic spine changes. In this study, we aimed to characterize the role of miRNA-324-5p in developmental spine regulation by assessing the effect of Mir324 knockout (KO) on dendritic spine density and expression of a subset of dendritic proteins at select developmental time points. We show that miR-324-5p expression is developmentally regulated and peaks at 4 weeks of age. We demonstrate that loss of miR-324-5p expression leads to differential changes in both target protein expression and spine density at different time points during development, disrupting the pattern of spine density changes and leading to a premature loss of dendritic spines in KO mice, which is compensated later. Our findings indicate that miR-324-5p plays a role in synaptic refinement across development. Additionally, our data illustrate the importance of context in the study of miRNA, as regulation by and/or of miRNA can vary dramatically across development and in disease.

MicroRNAs are an emerging class of synaptic regulators. These small noncoding RNAs post-transcriptionally regulate gene expression, thereby altering neuronal pathways and shaping cell-to-cell communication. Their ability to rapidly alter gene expression and target multiple pathways makes them interesting candidates in the study of synaptic plasticity. Here, we demonstrate that the proconvulsive microRNA miR-324-5p regulates excitatory synapse structure and function in the hippocampus of mice. Both Mir324 knockout (KO) and miR-324-5p antagomir treatment significantly reduce dendritic spine density in the hippocampal CA1 subregion, and Mir324 KO, but not miR-324-5p antagomir treatment, shift dendritic spine morphology, reducing the proportion of thin, "unstable" spines. Western blot and quantitative Real-Time PCR revealed changes in protein and mRNA levels for potassium channels, cytoskeletal components, and synaptic markers, including MAP2 and Kv4.2, which are important for long-term potentiation (LTP). In line with these findings, slice electrophysiology revealed that LTP is severely impaired in Mir324 KO mice, while neurotransmitter release probability remains unchanged. Overall, this study demonstrates that miR-324-5p regulates dendritic spine density, morphology, and plasticity in the hippocampus, potentially via multiple cytoskeletal and synaptic modulators.

The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.

Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.

Phenotype analysis of female mice lacking androgen receptor (AR) deficient (AR-/-) indicates that the development of mammary glands is retarded with reduced ductal branching in the prepubertal stages, and fewer Cap cells in the terminal end buds, as well as decreased lobuloalveolar development in adult females, and fewer milk-producing alveoli in the lactating glands. The defective development of AR-/- mammary glands involves the defects of insulin-like growth factor I-insulin-like growth factor I receptor and mitogen-activated protein kinase (MAPK) signals as well as estrogen receptor (ER) activity. Similar growth retardation and defects in growth factor-mediated Ras/Raf/MAPK cascade and ER signaling are also found in AR-/- MCF7 breast cancer cells. The restoration assays show that AR NH2-terminal/DNA-binding domain, but not the ligand-binding domain, is essential for normal MAPK function in MCF7 cells, and an AR mutant (R608K), found in male breast cancer, is associated with the excessive activation of MAPK. Together, our data provide the first in vivo evidence showing that AR-mediated MAPK and ER activation may play important roles for mammary gland development and MCF7 breast cancer cell proliferation.

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonad.

The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency.