Identification of HTRA4 as a Transcriptional Target of p63 in Trophoblast.

The placenta plays a crucial role in pregnancy success. ΔNp63α (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and is involved in CTB maintenance and differentiation. We examined the mechanisms of action of p63 by identifying its downstream targets. Gene expression changes were evaluated following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. High-temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, was identified as a gene reciprocally regulated by p63, and its expression was characterized in primary human placental tissues by RNA-sequencing and in situ hybridization. Potential p63 DNA-binding motifs were identified in the HTRA4 promoter, and p63 occupancy at some of these sites was confirmed using chromatin immunoprecipitation, followed by quantitative PCR in both JEG3 and trophoblast stem cells. These data begin to identify members of the transcriptional network downstream of p63, thus laying the groundwork for probing mechanisms by which this important transcription factor regulates trophoblast stemness and differentiation.

Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells.

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

Inflammation-driven deaminase deregulation fuels human pre-leukemia stem cell evolution.

Inflammation-dependent base deaminases promote therapeutic resistance in many malignancies. However, their roles in human pre-leukemia stem cell (pre-LSC) evolution to acute myeloid leukemia stem cells (LSCs) had not been elucidated. Comparative whole-genome and whole-transcriptome sequencing analyses of FACS-purified pre-LSCs from myeloproliferative neoplasm (MPN) patients reveal APOBEC3C upregulation, an increased C-to-T mutational burden, and hematopoietic stem and progenitor cell (HSPC) proliferation during progression, which can be recapitulated by lentiviral APOBEC3C overexpression. In pre-LSCs, inflammatory splice isoform overexpression coincides with APOBEC3C upregulation and ADAR1p150-induced A-to-I RNA hyper-editing. Pre-LSC evolution to LSCs is marked by STAT3 editing, STAT3ß isoform switching, elevated phospho-STAT3, and increased ADAR1p150 expression, which can be prevented by JAK2/STAT3 inhibition with ruxolitinib or fedratinib or lentiviral ADAR1 shRNA knockdown. Conversely, lentiviral ADAR1p150 expression enhances pre-LSC replating and STAT3 splice isoform switching. Thus, pre-LSC evolution to LSCs is fueled by primate-specific APOBEC3C-induced pre-LSC proliferation and ADAR1-mediated splicing deregulation.

Yy1 regulates Senp1 contributing to AMPA receptor GluR1 expression following neuronal depolarization.

BACKGROUND: Neuronal activity-induced changes in gene expression patterns are important mediators of neuronal plasticity. Many neuronal genes can be activated or inactivated in response to neuronal depolarization. Mechanisms that activate gene transcription are well established, but activity-dependent mechanisms that silence transcription are less understood. It is also not clear what is the significance of inhibiting these genes during neuronal activity. METHODS: Quantitative Real Time-PCR, western blot and immunofluorescence staining were performed to examine the expression of Senp1 and GluR1 in mouse cortical neurons. The alterations of Yy1 phosphorylation upon neuronal depolarization and the interaction of Yy1 with Brd4 were studied by protein co-immunoprecipitation. The regulators of Yy1 phosphorylation were identified by phosphatase inhibitors. Chromatin immunoprecipitation, in vitro DNA binding assay, luciferase assay and gene knockdown experiments were used to validate the roles of Yy1 and its phosphorylation as well as Brd4 in regulating Senp1 expression. RESULTS: We report that neuronal depolarization deactivates the transcription of the SUMO protease Senp1, an important component regulating synaptic transmission, scaling, and plasticity, through Yy1. In un-stimulated neurons, Senp1 transcription is activated by a Yy1-Brd4 transcription factor protein complex assembled on the Senp1 promoter. Upon membrane depolarization, however, Yy1 is dephosphorylated and the Yy1-Brd4 complex is evicted from the Senp1 promoter, reducing Senp1 transcription levels. Both Yy1 and Senp1 promote the expression of AMPA receptor subunit GluR1, a pivotal component in learning and memory. CONCLUSIONS: These results reveal an axis of Yy1/Brd4-Senp1 which regulates the expression of GluR1 during neuronal depolarization. This implicates a regulation mechanism in silencing gene expression upon neuronal activity.

Brd4's Bromodomains Mediate Histone H3 Acetylation and Chromatin Remodeling in Pluripotent Cells through P300 and Brg1.

The pluripotent state of embryonic stem cells (ESCs) is defined by its transcriptome and epigenome. The chromatin reader Brd4 determines ESC identity. Although Brd4 regulation in gene transcription has been well described, its contribution to the chromatin landscape is less known. Here, we show that Brd4's bromodomains partner with the histone acetyltransferase P300, increasing its enzymatic activities. Augmenting histone acetylation by Brd4-P300 interaction recruits the chromatin remodeler Brg1 altering chromatin structure. This pathway is important for maintaining the expression and chromatin patterns of pluripotency-associated genes, such as Oct4, Nanog, and the X chromosome regulatory long noncoding RNAs Tsix and Xite. Furthermore, we show that the Brd4-P300 interaction regulates the de novo formation of chromatin marks during ESC differentiation, as exemplified by controlling the master regulators of mesoderm formation. Collectively, we delineate the function of Brd4 in organizing the chromatin structure that contributes to gene transcriptional regulation and cell fate determination.

Long noncoding RNA Sox2ot and transcription factor YY1 co-regulate the differentiation of cortical neural progenitors by repressing Sox2.

Long noncoding RNAs (lncRNAs) are emerging as key regulators of crucial cellular processes. However, the molecular mechanisms of many lncRNA functions remain uncharacterized. Sox2ot is an evolutionarily conserved lncRNA that transcriptionally overlaps the pluripotency gene Sox2, which maintains the stemness of embryonic stem cells and tissue-specific stem cells. Here, we show that Sox2ot is expressed in the developing mouse cerebral cortex, where it represses neural progenitor (NP) proliferation and promotes neuronal differentiation. Sox2ot negatively regulates self-renewal of neural stem cells, and is predominately expressed in the nucleus and inhibits Sox2 levels. Sox2ot forms a physical interaction with a multifunctional transcriptional regulator YY1, which binds several CpG islands in the Sox2 locus in a Sox2ot-dependent manner. Similar to Sox2ot, YY1 represses NP expansion in vivo. These results demonstrate a regulatory role of Sox2ot in promoting cortical neurogenesis, possibly by repressing Sox2 expression in NPs, through interacting with YY1.

CIRM Alpha Stem Cell Clinics: Collaboratively Addressing Regenerative Medicine Challenges.

The California Institute for Regenerative Medicine (CIRM) Alpha Stem Cell Clinic (ASCC) Network was launched in 2015 to address a compelling unmet medical need for rigorous, FDA-regulated, stem cell-related clinical trials for patients with challenging, incurable diseases. Here, we describe our multi-center experiences addressing current and future challenges.

Sequential chromatin immunoprecipitation to detect SUMOylated MeCP2 in neurons.

The small ubiquitin-like modifier (SUMO) is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM). Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SUMOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2) is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP) for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chromatin factors at specific genomic regions.

Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition.

Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output.

Histone demethylation maintains Prdm14 and Tsix expression and represses xIst in embryonic stem cells.

Epigenetic reprogramming is exemplified by the remarkable changes observed in cellular differentiation and X-chromosome inactivation (XCI) in mammalian female cells. Histone 3 lysine 27 trimethylation (H3K27me3) is a modification that suppresses gene expression in multiple contexts including embryonic stem cells (ESCs) and decorates the entire inactive X-chromosome. The conversion of female somatic cells to induced pluripotency is accompanied by X-chromosome reactivation (XCR) and H3K27me3 erasure. Here, we show that the H3K27-specific demethylase Utx regulates the expression of the master regulators for XCI and XCR: Prdm14, Tsix, and Xist. Female ESC transcriptome analysis using a small molecule inhibitor for H3K27 demethylases, GSK-J4, identifies novel targets of H3K27 demethylation. Consistent with a recent report that GSK-J4 can inhibit other histone demethylase, we found that elevated H3K4me3 levels are associated with increased gene expression including Xist. Our data suggest multiple regulatory mechanisms for XCI via histone demethylation.

The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression.

Embryonic stem cell (ESC) pluripotency is controlled by defined transcription factors. During cellular differentiation, ESCs undergo a global epigenetic reprogramming. Female ESCs exemplify this process as one of the two X-chromosomes is globally silenced during X chromosome inactivation (XCI) to balance the X-linked gene disparity with XY males. The pluripotent factor OCT4 regulates XCI by triggering X chromosome pairing and counting. OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist. To control its activity as a master regulator in pluripotency and XCI, OCT4 must have chromatin protein partners. Here we show that BRD4, a member of the BET protein subfamily, interacts with OCT4. BRD4 occupies the regulatory regions of pluripotent genes and the lncRNAs of XCI. BET inhibition or depletion of BRD4 reduces the expression of many pluripotent genes and shifts cellular fate showing that BRD4 is pivotal for transcription in ESCs.

The localization of histone H3K27me3 demethylase Jmjd3 is dynamically regulated.

Jmjd3 is required for cellular differentiation and senescence, and inhibits the induction of pluripotent stem cells by demethylating histone 3 lysine 27 trimethylation (H3K27me3). Although recent studies reveal crucial biological roles for Jmjd3, it is unclear how its demethylase activity is controlled. Here, we show that nuclear localization of Jmjd3 is required for effective demethylation of H3K27me3. Our subcellular localization analysis of Jmjd3 shows that the N-terminal region of the protein is responsible for its nuclear placement, whereas the C-terminal region harboring the catalytic Jumonji C (JmjC) domain cannot situate into the nucleus. We identify two classical nuclear localization signals (cNLSs) in the N-terminal domain of Jmjd3. Forced nuclear emplacement of the catalytic domain of Jmjd3 by fusion with a heterologous cNLS significantly enhances its H3K27me3 demethylation activity. A dynamic nucleocytoplasmic shuttling of endogenous Jmjd3 occurs in mouse embryonic fibroblasts. Jmjd3 is localized both into the cytoplasm and the nucleus, and its nuclear export is dependent on Exportin-1, as treatment with leptomycin B triggers nuclear accumulation of Jmjd3. These results suggest that the subcellular localization of Jmjd3 is dynamically regulated and has pivotal roles for H3K27me3 status.

The dynamics of X-chromosome inactivation in mouse development.

Mammals utilize chromosomes to determine sex, but this leads to a problem with gene inequality. In the mouse, gene dosage disparity is evident by the presence of two X chromosomes in the female and a single X and Y chromosome in the male. To balance the X-linked transcriptional dose difference between the sexes, one of the two female X-chromosomes is silenced to equal the dose of XY males-a crucial developmental process known as X-chromosome inactivation. Here we highlight dosage compensation in the mouse, and detail the known mechanisms to set and erase these epigenetic marks during development.

Balancing the dose in the mouse.

Organisms that use a chromosomal basis of sex determination have a problem of gene inequality. In the mouse, this dimorphism is evident by the presence of two X-chromosomes in females, while males have a single X- and a single Y-chromosome. To balance this disparity, one of the two female X-chromosomes is transcriptionally silenced to neutralize the gene dose with the XY male. Dosage compensation in mammals is known as X-chromosome inactivation (XCI) and is a crucial early developmental process. XCI is an example of epigenetics: a phenotype resulting in changes on a chromosome without a change in nucleic acid sequence. Studies in mouse embryology and genetics have answered many questions about the process of balancing the dose. In this chapter, I highlight how the mouse dosage compensates the gene disparity between XX females and XY males in a crucial epigenetic process called X-chromosome inactivation (XCI).

Understanding the molecular circuitry of cell lineage specification in the early mouse embryo.

Pluripotent stem cells hold great promise for cell-based therapies in regenerative medicine. However, critical to understanding and exploiting mechanisms of cell lineage specification, epigenetic reprogramming, and the optimal environment for maintaining and differentiating pluripotent stem cells is a fundamental knowledge of how these events occur in normal embryogenesis. The early mouse embryo has provided an excellent model to interrogate events crucial in cell lineage commitment and plasticity, as well as for embryo-derived lineage-specific stem cells and induced pluripotent stem (iPS) cells. Here we provide an overview of cell lineage specification in the early (preimplantation) mouse embryo focusing on the transcriptional circuitry and epigenetic marks necessary for successive differentiation events leading to the formation of the blastocyst.

The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting.

Pluripotency of embryonic stem (ES) cells is controlled by defined transcription factors. During differentiation, mouse ES cells undergo global epigenetic reprogramming, as exemplified by X-chromosome inactivation (XCI) in which one female X chromosome is silenced to achieve gene dosage parity between the sexes. Somatic XCI is regulated by homologous X-chromosome pairing and counting, and by the random choice of future active and inactive X chromosomes. XCI and cell differentiation are tightly coupled, as blocking one process compromises the other and dedifferentiation of somatic cells to induced pluripotent stem cells is accompanied by X chromosome reactivation. Recent evidence suggests coupling of Xist expression to pluripotency factors occurs, but how the two are interconnected remains unknown. Here we show that Oct4 (also known as Pou5f1) lies at the top of the XCI hierarchy, and regulates XCI by triggering X-chromosome pairing and counting. Oct4 directly binds Tsix and Xite, two regulatory noncoding RNA genes of the X-inactivation centre, and also complexes with XCI trans-factors, Ctcf and Yy1 (ref. 17), through protein-protein interactions. Depletion of Oct4 blocks homologous X-chromosome pairing and results in the inactivation of both X chromosomes in female cells. Thus, we have identified the first trans-factor that regulates counting, and ascribed new functions to Oct4 during X-chromosome reprogramming.

Putting the 'HAT' back on survival signalling: the promises and challenges of HDAC inhibition in the treatment of neurological conditions.

Decreased histone acetyltransferase activity and transcriptional dysfunction have been implicated in almost all neurodegenerative conditions. Increasing net histone acetyltransferase activity through inhibition of the histone deacetylases (HDACs) has been shown to be an effective strategy to delay or halt progression of neurological disease in cellular and rodent models. These findings have provided firm rationale for Phase I and Phase II clinical trials of HDAC inhibitors in Huntington's disease, spinal muscular atrophy, and Freidreich's ataxia. In this review, we discuss the current findings and promise of HDAC inhibition as a strategy for treating neurological disorders. Despite the fact that HDAC inhibitors are in an advanced stage of development, we suggest other approaches to modulating HDAC function that may be less toxic and more efficacious than the canonical agents developed so far.

Antagonism between DNA and H3K27 methylation at the imprinted Rasgrf1 locus.

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.

Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein.

X-chromosome inactivation (XCI) ensures the equality of X-chromosome dosages in male and female mammals by silencing one X in the female. To achieve the mutually exclusive designation of active X (Xa) and inactive X (Xi), the process necessitates that two Xs communicate in trans through homologous pairing. Pairing depends on a 15-kb region within the genes Tsix and Xite. Here, we dissect molecular requirements and find that pairing can be recapitulated by 1- to 2-kb subfragments of Tsix or Xite with little sequence similarity. However, a common denominator among them is the presence of the protein Ctcf, a chromatin insulator that we find to be essential for pairing. By contrast, the Ctcf-interacting partner, Yy1 (ref. 8), is not required. Pairing also depends on transcription. Transcriptional inhibition prevents new pair formation but does not perturb existing pairs. The kinetics suggest a pairing half-life of <1 h. We propose that pairing requires Ctcf binding and co-transcriptional activity of Tsix and Xite.