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.

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis.

Thousands of epigenomic data sets have been generated in the past decade, but it is difficult for researchers to effectively use all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for validated systematic integration of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By using IDEAS as our integrative and discriminative epigenome annotation system, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of more than 200,000 candidate cis-regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website to aid research in genomics and hematopoiesis.

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of hematopoietic cells.

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for 20 cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions-along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for the regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

Prostate cancer susceptibility gene HIST1H1A is a modulator of androgen receptor signaling and epithelial to mesenchymal transition.

In 2018, approximately 165,000 new prostate cancer (PC) cases will be diagnosed, and over 29,000 men will succumb to PC in the U.S. alone. The means of assessing outcome in the clinic are inaccurate, and there is a pressing need to more precisely identify men at risk of aggressive PC. We previously identified HIST1H1A as a susceptibility gene for aggressive PC. HIST1H1A encodes H1.1, a member of the linker histone family that is involved in chromatin organization and compaction. To understand the molecular basis of aggressive PC, we have characterized how germline variation modulates susceptibility to neuroendocrine differentiation, which is a form of aggressive PC. Immunohistochemistry studies revealed that HIST1H1A is over-expressed in normal human prostate tissue compared to prostate adenocarcinoma. Functional characterization of HIST1H1A in prostate LNCaP cells indicated that HIST1HA over-expression increased cell growth, as well as the expression of neuroendocrine and epithelial-to-mesenchymal markers in vitro. Assay for Transposase-Accessible Chromatin (ATAC-seq), which is used to assess chromatin compaction and thus the transcriptional availability of individual genomic regions, demonstrated that H1.1 plays a prominent role in modulating Wnt signaling pathway genes, which are implicated in prostate tumorigenesis. These results demonstrate that HIST1H1A is a modulator of aggressive PC susceptibility.

Establishment of regulatory elements during erythro-megakaryopoiesis identifies hematopoietic lineage-commitment points.

BACKGROUND: Enhancers and promoters are cis-acting regulatory elements associated with lineage-specific gene expression. Previous studies showed that different categories of active regulatory elements are in regions of open chromatin, and each category is associated with a specific subset of post-translationally marked histones. These regulatory elements are systematically activated and repressed to promote commitment of hematopoietic stem cells along separate differentiation paths, including the closely related erythrocyte (ERY) and megakaryocyte (MK) lineages. However, the order in which these decisions are made remains unclear. RESULTS: To characterize the order of cell fate decisions during hematopoiesis, we collected primary cells from mouse bone marrow and isolated 10 hematopoietic populations to generate transcriptomes and genome-wide maps of chromatin accessibility and histone H3 acetylated at lysine 27 binding (H3K27ac). Principle component analysis of transcriptional and open chromatin profiles demonstrated that cells of the megakaryocyte lineage group closely with multipotent progenitor populations, whereas erythroid cells form a separate group distinct from other populations. Using H3K27ac and open chromatin profiles, we showed that 89% of immature MK (iMK)-specific active regulatory regions are present in the most primitive hematopoietic cells, 46% of which contain active enhancer marks. These candidate active enhancers are enriched for transcription factor binding site motifs for megakaryopoiesis-essential proteins, including ERG and ETS1. In comparison, only 64% of ERY-specific active regulatory regions are present in the most primitive hematopoietic cells, 20% of which containing active enhancer marks. These regions were not enriched for any transcription factor consensus sequences. Incorporation of genome-wide DNA methylation identified significant levels of de novo methylation in iMK, but not ERY. CONCLUSIONS: Our results demonstrate that megakaryopoietic profiles are established early in hematopoiesis and are present in the majority of the hematopoietic progenitor population. However, megakaryopoiesis does not constitute a "default" differentiation pathway, as extensive de novo DNA methylation accompanies megakaryopoietic commitment. In contrast, erythropoietic profiles are not established until a later stage of hematopoiesis, and require more dramatic changes to the transcriptional and epigenetic programs. These data provide important insights into lineage commitment and can contribute to ongoing studies related to diseases associated with differentiation defects.

Molecular convergence in ex vivo models of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor GATA1 Erythroid cells from patients with DBA have not been well characterized, and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an ex vivo culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation in comparison with controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translation is a common feature of DBA caused by both RP and GATA1 mutations. This trial was registered at www.clinicaltrials.gov as #NCT00106015.

Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways.

BACKGROUND: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by "bulk" assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. RESULTS: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: "Pre-MEP," enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; "E-MEP," strongly biased towards erythroid differentiation; and "MK-MEP," a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. CONCLUSIONS: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias.

SBR-Blood: systems biology repository for hematopoietic cells.

Extensive research into hematopoiesis (the development of blood cells) over several decades has generated large sets of expression and epigenetic profiles in multiple human and mouse blood cell types. However, there is no single location to analyze how gene regulatory processes lead to different mature blood cells. We have developed a new database framework called hematopoietic Systems Biology Repository (SBR-Blood), available online at http://sbrblood.nhgri.nih.gov, which allows user-initiated analyses for cell type correlations or gene-specific behavior during differentiation using publicly available datasets for array- and sequencing-based platforms from mouse hematopoietic cells. SBR-Blood organizes information by both cell identity and by hematopoietic lineage. The validity and usability of SBR-Blood has been established through the reproduction of workflows relevant to expression data, DNA methylation, histone modifications and transcription factor occupancy profiles.

Necdin is a breast cancer metastasis suppressor that regulates the transcription of c-Myc.

Metastasis is the primary cause of death in breast cancer. Earlier studies using a mammary tumorigenesis mouse model identified Necdin (Ndn)as a germline modifier of metastasis. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in a well-characterized breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.

Metastasis-associated protein ribosomal RNA processing 1 homolog B (RRP1B) modulates metastasis through regulation of histone methylation.

UNLABELLED: Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B-binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B-binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1), and heterochromatin protein 1-α (CBX5/HP1α). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1α complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation. IMPLICATIONS: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer.

Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites.

DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and erythroblasts (ERYs). Methyl binding domain protein 2 (MBD) enrichment of DNA followed by massively parallel sequencing (MBD-seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSCs, with a 40% reduction in CMPs, and a 67% reduction in ERYs. Only 3% of peaks arise during differentiation, demonstrating a genome-wide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20%-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSCs and CMPs. Elevated genome-wide DNA methylation in HSCs and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSCs. Using de novo motif discovery, we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPA and ELF1, are overrepresented in methylated regions. Our genome-wide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis.

The chloride channel inhibitor NS3736 [corrected] prevents bone resorption in ovariectomized rats without changing bone formation.

UNLABELLED: Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone formation. This study indicates that chloride channel inhibitors are highly promising for treatment of osteoporosis. INTRODUCTION: The chloride channel inhibitor, NS3736, blocked osteoclastic acidification and resorption in vitro with an IC50 value of 30 microM. When tested in the rat ovariectomy model for osteoporosis, daily treatment with 30 mg/kg orally protected bone strength and BMD by approximately 50% 6 weeks after surgery. Most interestingly, bone formation assessed by osteocalcin, mineral apposition rate, and mineralized surface index was not inhibited. MATERIALS AND METHODS: Analysis of chloride channels in human osteoclasts revealed that ClC-7 and CLIC1 were highly expressed. Furthermore, by electrophysiology, we detected a volume-activated anion channel on human osteoclasts. Screening 50 different human tissues showed a broad expression for CLIC1 and a restricted immunoreactivity for ClC-7, appearing mainly in osteoclasts, ovaries, appendix, and Purkinje cells. This highly selective distribution predicts that inhibition of ClC-7 should specifically target osteoclasts in vivo. We suggest that NS3736 is inhibiting ClC-7, leading to a bone-specific effect in vivo. RESULTS AND CONCLUSION: In conclusion, we show for the first time that chloride channel inhibitors can be used for prevention of ovariectomy-induced bone loss without impeding bone formation. We speculate that the coupling of bone resorption to bone formation is linked to the acidification of the resorption lacunae, thereby enabling compounds that directly interfere with this process to be able to positive uncouple this process resulting in a net bone gain.