Epigenetic defects in stem cells deficient in polycomb group function

During development, the expression states of many genes must be maintained through cell divisions in order to ensure lineage-, time-, and dose-appropriate patterns of gene expression. This transcriptional memory is independent of permanent DNA sequences changes and instead involves reversible epigenetic mechanisms. Polycomb Group (PcG) proteins represent a conserved family of developmental regulators that mediate heritable transcriptional silencing by covalently modifying histone proteins. Here, we demonstrate that mutations in the PcG gene Embryonic ectoderm development (Eed) produce a variety of epigenetic defects in mouse embryos and stem cells. EED is a noncatalytic subunit of Polycomb Repressive Complex 2, a 600 kDa complex containing a number of proteins, including the histone H3-lysine 27 (H3K27) methyltransferase EZH2. Consistent with the role of PcG genes in transcriptional memory, Eed mutant embryos and trophoblast stem cells have defects in genomic imprinting, a process by which an allele's expression is dependent upon the gender of the parent from which it was inherited. To determine whether these gene expression defects revealed a required role for EED in PRC2 function, we characterized the status of H3K27 methylation in Eed mutant stem cells. H3K7 can be mono-, di-, or trimethylated (H3K27me1, H3K27me2, H3K27me3, respectively), but it has been unclear which of these marks are mediated by PRC2. Here, we demonstrate that EED is required for all three methylation states. Additionally, although EED is present as four distinct isoforms in mammalian cells, these isoforms are not necessary for H3K27 methylation. Instead, EED's core WD-40 motifs and histone binding domain alone are sufficient to mediate histone methylation. Finally, although the histone methylation defects in Eed mutant stem cells appear to be global, the imprinted expression defects are restricted to DNA hypomethylated, extraembryonic tissues and to genes that are imprinted normally in DNA methyltransferase 1 (Dnmt1) mutant placentas. Together, these results suggest that histone methylation and DNA methylation may have non-overlapping roles in imprinted gene regulation

Molecular and Functional Mapping of EED Motifs Required for PRC2-Dependent Histone Methylation

Polycomb Group (PcG) proteins represent a conserved family of developmental regulators that mediate heritable transcriptional silencing by modifying chromatin states. One PcG complex, the PRC2 complex, is composed of several proteins, including the histone H3 lysine 27 (H3K27) methyltransferase EZH2 and the WD-repeat protein EED. Histone H3K27 can be mono- (H3K27me1), di- (H3K27me2), or trimethylated (H3K27me3). However, it remains unclear what regulates the number of methyl groups added to H3K27 in a particular nucleosome. In mammalian cells, EED is present as four distinct isoforms, which are believed to be produced by utilizing four distinct, in-frame translation start sites in a common Eed mRNA. A mutation that disables all four EED isoforms produces defects in H3K27 methylation.1 To assess the roles of individual EED isoforms in H3K27 methylation, we first characterized three of the four EED isoform start sites and then demonstrated that individual isoforms are not necessary for H3K27me1, H3K27me2, or H3K27me3. Instead, we show that the core WD-40 motifs and the histone binding region of EED alone are sufficient for the generation of all three marks, demonstrating that EED isoforms do not control the number of methyl groups added to H3K27

Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

BACKGROUND:Metazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3' end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3' end processing. The 3' end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS:In Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3' end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells. CONCLUSIONS/SIGNIFICANCE:These data are consistent with a model in which changing the 3' end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development

Lymphoma and Pathology in Sub-Saharan Africa Current Approaches and Future Directions

The care of patients with lymphoma relies heavily on accurate tissue diagnosis and classification. In sub-Saharan Africa, where lymphoma burden is increasing because of population growth, aging, and continued epidemic levels of human immunodeficiency virus infection, diagnostic pathology services are limited. This article summarizes lymphoma epidemiology, current diagnostic capacity, and obstacles and opportunities for improving practice in the region

Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice

ABSTRACT BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo . Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice. IMPORTANCE Epstein-Barr virus infects approximately 90% of the world’s population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBV’s BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV

Practical Successes in Telepathology Experiences in Africa

Across much of Africa, there is a critical shortage of pathology services necessary for clinical care. Even in settings where specialty-level clinical care, such as medical oncology, is available, access to anatomic pathology services has often lagged behind. Pathology laboratories in the region are challenging to establish and maintain. This article describes the successful implementation of telepathology services in Malawi and reviews other successful programs developed to support diagnostic pathology in resource-limited settings

Identification of Human Papillomavirus Infection in Cancer Tissue by Targeted Next-generation Sequencing

Human papillomaviruses (HPV) are oncogenic DNA viruses implicated in squamous cell carcinomas of several anatomic sites, as well as endocervical adenocarcinomas. Identification of HPV is an actionable finding in some carcinomas, potentially influencing tumor classification, prognosis, and management. We incorporated capture probes for oncogenic HPV strains 16 and 18 into a broader next-generation sequencing (NGS) panel designed to identify actionable mutations in solid malignancies. A total of 21 head and neck, genitourinary and gynecological squamous cell carcinomas and endocervical adenocarcinomas were sequenced as part of the UNCSeq project. Using p16 immunohistochemical results as the gold standard, we set a cutoff for proportion of aligned HPV reads that maximized performance of our NGS assay (92% sensitive, 100% specific for HPV). These results suggest that sequencing of oncogenic pathogens can be incorporated into targeted NGS panels, extending the clinical utility of genomic assays

The Murine Polycomb Group Protein Eed Is Required for Global Histone H3 Lysine-27 Methylation

PcG proteins mediate heritable transcriptional silencing by generating and recognizing covalent histone modifications. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase (HMTase) Ezh2, the WD-repeat protein Eed, and the Zn-finger protein Suz12. Ezh2 methylates histone H3 on lysine 27 (H3K27) [1, 2, 3 and 4], which serves as an epigenetic mark mediating silencing. H3K27 can be mono-, di-, or trimethylated (1mH3K27, 2mH3K27, and 3mH3K27, respectively) [5]. Hence, either PRC2 must be regulated so as to add one methyl group to certain nucleosomes but two or three to others, or distinct complexes must be responsible for 1m-, 2m-, and 3mH3K27. Consistent with the latter possibility, 2mH3K27 and 3mH3K27, but not 1mH3K27, are absent in Suz12¿/¿ embryos, which lack both Suz12 and Ezh2 protein [6]. Mammalian proteins required for 1mH3K27 have not been identified. Here, we demonstrate that unlike Suz12 and Ezh2, Eed is required not only for 2m- and 3mH3K27 but also global 1mH3K27. These results provide a functionally important distinction between PRC2 complex components and implicate Eed in PRC2-independent histone methylation

Discovering' primary effusion lymphoma in Malawi

Primary effusion lymphoma (PEL) is a rare non-Hodgkin lymphoma (NHL) associated with human herpesvirus 8 (HHV8) and increased incidence among HIV-positive individuals. PEL rarely presents as solid tumor masses, or extracavitary PEL (EPEL). We found one case of PEL and no EPEL reported from sub-Saharan Africa (SSA), despite high prevalence of HIV and HHV8. With improved lymphoma diagnostic infrastructure in Malawi, we recently diagnosed the first two cases of EPEL, out of 85 HIV-associated aggressive NHL cases during 2013–2017

A prospective description of HIV-associated multicentric Castleman disease in Malawi

Despite high burden of Kaposi sarcoma (KS) and HIV, multicentric Castleman disease (MCD) is rarely reported in sub-Saharan Africa (SSA). A pathology series in Uganda demonstrated latency-associated nuclear antigen (LANA) and viral interleukin-6 positivity by immunohistochemistry (IHC) in 3 of 64 reactive-appearing lymph nodes, consistent with missed MCD diagnoses,1 but this was before antiretroviral therapy (ART) scale-up when MCD may have been relatively infrequent. In South Africa, a pathology series reported many HIV+ B-cell lymphoproliferations without specifically mentioning MCD.2 A retrospective series from South Africa reported 35 MCD cases during 1990–2014, with most cases in the most recent years within this time period