MRP5 and MRP9 play a concerted role in male reproduction and mitochondrial function.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry.

Primordial germ cells (PGCs) arise from cells of the post-implantation epiblast in response to cytokine signaling. PGC development can be recapitulated in vitro by differentiating epiblast-like cells (EpiLCs) into PGC-like cells (PGCLCs) through cytokine exposure. Interestingly, the cytokine requirement for PGCLC induction can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that NANOG mediates Otx2 downregulation in the absence of cytokines and that this is essential for PGCLC induction by NANOG. Moreover, the direct NANOG target gene Esrrb, which can substitute for several NANOG functions, does not downregulate Otx2 when overexpressed in EpiLCs and cannot promote PGCLC specification. However, expression of ESRRB in Otx2+/- EpiLCs rescues emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification.

Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry.

Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germline specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESC self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential, whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces the efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.

Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

Ventral tegmental area GABA, glutamate, and glutamate-GABA neurons are heterogeneous in their electrophysiological and pharmacological properties.

The ventral tegmental area (VTA) contains dopamine neurons intermixed with GABA-releasing (expressing vesicular GABA transporter, VGaT), glutamate-releasing (expressing vesicular glutamate transporter 2, VGluT2), and glutamate-GABA co-releasing (co-expressing VGluT2 and VGaT) neurons. By delivering INTRSECT viral vectors into the VTA of double vglut2-Cre/vgat-Flp transgenic mice, we targeted specific VTA cell populations for ex vivo recordings. We found that VGluT2+ VGaT- and VGluT2+ VGaT+ neurons on average had relatively hyperpolarized resting membrane potential, greater rheobase, and lower spontaneous firing frequency compared to VGluT2- VGaT+ neurons, suggesting that VTA glutamate-releasing and glutamate-GABA co-releasing neurons require stronger excitatory drive to fire than GABA-releasing neurons. In addition, we detected expression of Oprm1mRNA (encoding µ opioid receptors, MOR) in VGluT2+ VGaT- and VGluT2- VGaT+ neurons, and that the MOR agonist DAMGO hyperpolarized neurons with these phenotypes. Collectively, we demonstrate the utility of the double transgenic mouse to access VTA glutamate, glutamate-GABA, and GABA neurons to determine their electrophysiological properties. SIGNIFICANT STATEMENT: Some physiological properties of VTA glutamate-releasing and glutamate-GABA co-releasing neurons are distinct from those of VTA GABA-releasing neurons. µ-opioid receptor activation hyperpolarizes some VTA glutamate-releasing and some GABA-releasing neurons.

Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self-renewal.

The self-renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site-specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF-independent self-renewal. In contrast, phosphomimetic substitution of S131 enhances LIF-independent self-renewal, without influencing DNA binding. Modelling the DNA-homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self-renewal.

One ring to bring them all and in the darkness bind them: The trafficking of heme without deliverers.

Heme, as a hydrophobic iron-containing organic ring, is lipid soluble and can interact with biological membranes. The very same properties of heme that nature exploits to support life also renders heme potentially cytotoxic. In order to utilize heme, while also mitigating its toxicity, cells are challenged to tightly control the concentration and bioavailability of heme. On the bright side, it is reasonable to envision that, analogous to other transition metals, a combination of membrane-bound transporters, soluble carriers, and chaperones coordinate heme trafficking to subcellular compartments. However, given the dual properties exhibited by heme as a transition metal and lipid, it is compelling to consider the dark side: the potential role of non-proteinaceous biomolecules including lipids and nucleic acids that bind, sequester, and control heme trafficking and bioavailability. The emergence of inter-organellar membrane contact sites, as well as intracellular vesicles derived from various organelles, have raised the prospect that heme can be trafficked through hydrophobic channels. In this review, we aim to focus on heme delivery without deliverers - an alternate paradigm for the regulation of heme homeostasis through chaperone-less pathways for heme trafficking.

TET1 Interacts Directly with NANOG via Independent Domains Containing Hydrophobic and Aromatic Residues.

The DNA demethylase TET1 is highly expressed in embryonic stem cells and is important both for lineage commitment, and reprogramming to naïve pluripotency. TET1 interacts with the pluripotency transcription factor NANOG which may contribute to its biological activity in pluripotent cells. However, how TET1 interacts with other proteins is largely unknown. Here, we characterise the physical interaction between TET1 and NANOG using embryonic stem cells and bacterial expression systems. TET1 and NANOG interact through multiple binding sites that act independently. Critically, mutating conserved hydrophobic and aromatic residues within TET1 and NANOG abolishes the interaction. On chromatin, NANOG is predominantly localised at ESC enhancers. While TET1 binds to CpG dinucleotides in promoters using its CXXC domain, TET1 also binds to enhancers, though the mechanism involved is unknown. Comparative ChIP-seq analysis identifies genomic loci bound by both TET1 and NANOG, that correspond predominantly to pluripotency enhancers. Importantly, around half of NANOG transcriptional target genes are associated with TET1-NANOG co-bound sites. These results indicate a mechanism by which TET1 protein may be targeted to specific sites of action at enhancers by direct interaction with a transcription factor.

Monitoring human papillomavirus prevalence among young Australian women undergoing routine chlamydia screening.

INTRODUCTION: Australia has recently implemented major changes in cervical cancer prevention policies including introduction of primary human papillomavirus (HPV) screening starting at age 25, and replacement of the quadrivalent HPV vaccine with the nonavalent vaccine in the national school-based program. We assessed the feasibility and utility of conducting HPV testing in residual clinical specimens submitted for routine Chlamydia trachomatis screening, as a means of tracking HPV vaccine program impact among young sexually active women. METHODS: De-identified residual specimens from women aged 16-24 years submitted for chlamydia testing were collected from three pathology laboratories in Victoria and New South Wales. Limited demographic information, and chlamydia test results were also collected. Patient identifiers were sent directly from the laboratories to the National HPV Vaccination Program Register, to obtain HPV vaccination histories. Samples underwent HPV genotyping using Seegene Anyplex II HPV 28 assay. RESULTS: Between April and July 2018, 362 residual samples were collected, the majority (60.2%) of which were cervical swabs. Demographic data and vaccination histories were received for 357 (98.6%) women (mean age 21.8, SD 2.0). Overall, 65.6% of women were fully vaccinated, 9.8% partially, and 24.7% unvaccinated. The majority (86.0%) resided in a major city, 35.9% were classified in the upper quintile of socioeconomic advantage and chlamydia positivity was 7.8%.The prevalence of quadrivalent vaccine-targeted types (HPV6/11/16/18) was 2.8% (1.5-5.1%) overall with no differences by vaccination status (p = 0.729). The prevalence of additional nonavalent vaccine-targeted types (HPV31/33/45/52/58) was 19.3% (15.6-23.8%). One or more oncogenic HPV types were detected in 46.8% (95% CI 41.6-52.0%) of women. CONCLUSIONS: HPV testing of residual chlamydia specimens provides a simple, feasible method for monitoring circulating genotypes. Applied on a larger scale this method can be utilised to obtain a timely assessment of nonavalent vaccine impact among young women not yet eligible for cervical screening.

Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naïve pluripotency marker.

Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2 Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

Segregation of the mouse germline and soma.

Mouse primordial germ cells (PGCs), originate from the early post-implantation epiblast in response to BMP4 secreted by the extraembryonic ectoderm. However, how BMP4 acts here has remained unclear. Recent work has identified the transcription factor (TF), OTX2 as a key determinant of the segregation of the germline from the soma. OTX2 is expressed ubiquitously in the early post-implantation epiblast, decreasing rapidly in cells that initiate the PGC programme. Otx2 mRNA is also rapidly repressed by BMP4 in vitro, in germline competent cells. Supporting a model in which BMP4 represses Otx2, enforcing sustained OTX2 expression in competent cells blocks germline entry. In contrast, Otx2-null epiblast cells enter the germline with increased efficiency in vitro and in vivo and can do so independently of BMP4. Also, Otx2-null cells can initiate germline entry even without the crucial PGC TF, BLIMP1. In this review, we survey recent advances and propose hypotheses concerning germline entry.

Routine cervical screening by primary HPV testing: early findings in the renewed National Cervical Screening Program.

OBJECTIVES: To report human papillomavirus (HPV) testing patterns and rates of oncogenic HPV-positivity for specimens submitted during the first 6 months after the National Cervical Screening Program switched from cytology- to primary HPV-based screening. DESIGN, PARTICIPANTS: Retrospective cross-sectional review of 195 606 specimens submitted for HPV testing, 1 December 2017 - 31 May 2018. SETTING: Large community-based general pathology laboratory in metropolitan Sydney. MAIN OUTCOME MEASURES: Prevalence of oncogenic HPV types (all, HPV16/18, non-HPV16/18) by reason for HPV test (primary screening, non-screening); for oncogenic HPV-positive women in the age band recommended for primary HPV screening (25-74 years), prevalence of cytologic abnormality and rates of 12-month follow-up and colposcopy recommendations. RESULTS: 195 606 samples were received: 157 700 (80.6%) for primary screening, 37 906 (19.4%) for non-screening tests. Oncogenic HPV was detected in 8.1% of screening tests (95% CI, 7.9-8.2%) and 20.9% of non-screening tests (95% CI, 20.5-21.3%); 35.5% (95% CI, 34.7-36.4%) of women of recommended screening age with positive oncogenic HPV screening test results also had a cytologic abnormality. The proportion of HPV16/18-positive samples with high grade abnormality was 15.3% (95% CI, 14.2-16.6%); for samples positive for other oncogenic HPV types, the proportion was 6.3% (95% CI, 5.8-6.8%). Repeat HPV testing after 12 months was recommended for 5.4% (95% CI, 5.3-5.5%) and direct colposcopy for 2.6% (95% CI, 2.5-2.7%) of screened women aged 25-74 years. CONCLUSIONS: High grade cytologic abnormalities were more common in women positive for HPV16/18, supporting their higher risk classification. Colposcopy referral rates were higher than during primary cytology-based testing, as predicted by clinical trial and modelling data. The prevalence of HPV was much higher in non-screening than in primary screening samples. Our findings indicate the renewed program is performing as expected during the initial HPV screening round.

OTX2 restricts entry to the mouse germline.

The successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast1. Induction requires BMP4 signalling to prospective PGCs2 and the intrinsic action of PGC transcription factors3-6. However, the molecular mechanisms that connect BMP4 to induction of the PGC transcription factors that are responsible for segregating PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Downregulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro markedly increases the efficiency of PGC-like cell differentiation and prolongs the period of PGC competence. In the absence of Otx2 activity, differentiation of PGC-like cells becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC transcription factor BLIMP1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that OTX2 functions repressively upstream of PGC transcription factors, acting as a roadblock to limit entry of epiblast cells to the germline to a small window in space and time, thereby ensuring correct numerical segregation of germline cells from the soma.

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation.

Self-renewal of embryonic stem cells (ESCs) cultured in LIF/fetal calf serum (FCS) is incomplete with some cells initiating differentiation. While this is reflected in heterogeneous expression of naive pluripotency transcription factors (TFs), the link between TF heterogeneity and differentiation is not fully understood. Here, we purify ESCs with distinct TF expression levels from LIF/FCS cultures to uncover early events during commitment from naïve pluripotency. ESCs carrying fluorescent Nanog and Esrrb reporters show Esrrb downregulation only in Nanoglow cells. Independent Esrrb reporter lines demonstrate that Esrrbnegative ESCs cannot effectively self-renew. Upon Esrrb loss, pre-implantation pluripotency gene expression collapses. ChIP-Seq identifies different regulatory element classes that bind both OCT4 and NANOG in Esrrbpositive cells. Class I elements lose NANOG and OCT4 binding in Esrrbnegative ESCs and associate with genes expressed preferentially in naïve ESCs. In contrast, Class II elements retain OCT4 but not NANOG binding in ESRRB-negative cells and associate with more broadly expressed genes. Therefore, mechanistic differences in TF function act cumulatively to restrict potency during exit from naïve pluripotency.

Hrg1 promotes heme-iron recycling during hemolysis in the zebrafish kidney.

Heme-iron recycling from senescent red blood cells (erythrophagocytosis) accounts for the majority of total body iron in humans. Studies in cultured cells have ascribed a role for HRG1/SLC48A1 in heme-iron transport but the in vivo function of this heme transporter is unclear. Here we present genetic evidence in a zebrafish model that Hrg1 is essential for macrophage-mediated heme-iron recycling during erythrophagocytosis in the kidney. Furthermore, we show that zebrafish Hrg1a and its paralog Hrg1b are functional heme transporters, and genetic ablation of both transporters in double knockout (DKO) animals shows lower iron accumulation concomitant with higher amounts of heme sequestered in kidney macrophages. RNA-seq analyses of DKO kidney revealed large-scale perturbation in genes related to heme, iron metabolism and immune functions. Taken together, our results establish the kidney as the major organ for erythrophagocytosis and identify Hrg1 as an important regulator of heme-iron recycling by macrophages in the adult zebrafish.

Functional Dissection of the Enhancer Repertoire in Human Embryonic Stem Cells.

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications functionally define active enhancers remains unclear. Here, we combine chromatin immunoprecipitation with a massively parallel reporter assay (ChIP-STARR-seq) to identify functional enhancers in human embryonic stem cells (ESCs) genome-wide in a quantitative unbiased manner. Although active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac, and H3K4me1 function as enhancers, with activity markedly changing under naive versus primed culture conditions. We identify an enhancer set associated with functions extending to non-ESC-specific processes. Moreover, although transposable elements associate with putative enhancers, only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalog of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.

Dynamic changes in Sox2 spatio-temporal expression promote the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos.

Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerize on adjacent cis regulatory motifs, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In the present study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy. We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast and primitive endoderm populations within the inner cell mass of the developing rodent blastocyst.

Esrrb Complementation Rescues Development of Nanog-Null Germ Cells.

The transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated in vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells.