RCOR2 is a subunit of the LSD1 complex that regulates ESC property and substitutes for SOX2 in reprogramming somatic cells to pluripotency

Histone demethylase LSD1 can form complex with different Rcor family corepressors in different cell types. It remains unknown if cell-specific Rcor proteins function specifically in distinct cell types. Here, we report that Rcor2 is predominantly expressed in ESCs and forms a complex with LSD1 and facilitates its nucleosomal demethylation activity. Knockdown of Rcor2 in ESCs inhibited ESC proliferation and severely impaired the pluripotency. Moreover, knockdown of Rcor2 greatly impaired the formation of induced pluripotent stem (iPS) cells. In contrast, ectopic expression of Rcor2 in somatic cells together with Oct4, Sox2, and Klf4 promoted the formation of iPS cells. Most interestingly, ectopic expression of Rcor2 in both mouse and human somatic cells effectively substituted the requirement for exogenous Sox2 expression in somatic cell reprogramming.     

DNA hypermethylation in somatic cells correlates with higher reprogramming efficiency

The efficiency of somatic cell reprogramming to pluripotency using defined factors is dramatically affected by the cell type of origin. Here, we show that human keratinocytes, which can be reprogrammed at a higher efficiency than fibroblast [Nat Biotechnol 2008;26:1276-1284], share more genes hypermethylated at CpGs with human embryonic stem cells (ESCs) than other somatic cells frequently used for reprogramming. Moreover, pluripotent cells reprogrammed from keratinocytes (KiPS) are more similar to ESCs than those reprogrammed from fibroblasts (FiPS) in regard to DNA methylation levels, mostly due to the presence of genes that fail to acquire high levels of DNA methylation in FiPS cells. We propose that higher reprogramming efficiency correlates with the hypermethylation of tissue-specific genes rather than with a more permissive pluripotency gene network.     

Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle

The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4-green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4-GFP activation has been found to occur 40-48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4-GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4-GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (approximately 22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion.     

Epigenetic modification is central to genome reprogramming in somatic cell nuclear transfer

The recent high-profile reports of the derivation of human embryonic stem cells (ESCs) from human blastocysts produced by somatic cell nuclear transfer (SCNT) have highlighted the possibility of making autologous cell lines specific to individual patients. Cell replacement therapies have much potential for the treatment of diverse conditions, and differentiation of ESCs is highly desirable as a means of producing the ranges of cell types required. However, given the range of immunophenotypes of ESC lines currently available, rejection of the differentiated cells by the host is a potentially serious problem. SCNT offers a means of circumventing this by producing ESCs of the same genotype as the donor. However, this technique is not without problems because it requires resetting of the gene expression program of a somatic cell to a state consistent with embryonic development. Some remodeling of parental DNA does occur within the fertilized oocyte, but the somatic genome presented in a radically different format to those of the gametes. Hence, it is perhaps unsurprising that many genes are expressed aberrantly within "cloned" embryos and the ESCs derived from them. Epigenetic modification of the genome through DNA methylation and covalent modification of the histones that form the nucleosome is the key to the maintenance of the differentiated state of the cell, and it is this that must be reset during SCNT. This review focuses on the mechanisms by which this is achieved and how this may account for its partial failure in the "cloning" process. We also highlight the potential dangers this may introduce into ESCs produced by this technology.     

Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency

Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.     

Chromosome-wide regulation of euchromatin-specific 5mC to 5hmC conversion in mouse ES cells and female human somatic cells

DNA cytosine methylation (5mC) is indispensable for a number of cellular processes, including retrotransposon silencing, genomic imprinting, and X chromosome inactivation in mammalian development. Recent studies have focused on 5-hydroxymethylcytosine (5hmC), a new epigenetic mark or intermediate in the DNA demethylation pathway. However, 5hmC itself has no role in pluripotency maintenance in mouse embryonic stem cells (ESCs) lacking Dnmt1, 3a, and 3b. Here, we demonstrated that 5hmC accumulated on euchromatic chromosomal bands that were marked with di- and tri-methylated histone H3 at lysine 4 (H3K4me2/3) in mouse ESCs. By contrast, heterochromatin enriched with H3K9me3, including mouse chromosomal G-bands, pericentric repeats, human satellite 2 and 3, and inactive X chromosomes, was not enriched with 5hmC. Therefore, enzymes that hydroxylate the methyl group of 5mC belonging to the Tet family might be excluded from inactive chromatin, which may restrict 5mC to 5hmC conversion in euchromatin to prevent nonselective de novo DNA methylation.     

Immunohistochemical expression of p53, bcl-2, and p21waf1 in 48 Argentinean children with non-Hodgkin lymphoma.

BACKGROUND: Lymphoma accounts for 10% of all childhood cancers in developed countries. In Argentina, the incidence is 13.6%; of these, 53% are non-Hodgkin lymphomas (NHL) and 47% are Hodgkin lymphomas. Overexpression of p53, p21, and bcl-2 has been studied mainly in adults with NHL and correlated with clinical features and survival. The authors' aim was to analyze overexpression of these cellular oncogenes in children with NHL. METHODS: Formalin-fixed paraffin-embedded lymph node biopsies from 48 children with NHL were studied by immunohistochemistry with monoclonal antibodies for p53, bcl-2, and p21. RESULTS: Overexpression of p53 was detected in 81% of cases; bcl-2 positivity was 46% and p21 positivity was 42%. The p53/p21 expression patterns (types IV and V patterns), which correspond to the overexpression of nonfunctional p53, accounted for 46% of NHL. None of the studied oncogene patterns correlated with poor clinical patient outcome or histologic subtype. CONCLUSIONS: Of 81% of cases with p53 overexpression, 46% exhibited a nonfunctional p53 pattern, and this may contribute to dysregulation of proliferation, bcl-2 expression in up to 46% of cases may reflect a failure of bcl-2 downregulation in pre-B and immature B cells.     

Detection in primary chronic myeloid leukaemia cells of p210BCR-ABL1 in complexes with adaptor proteins CBL, CRKL, and GRB2

Chronic myeloid leukemia (CML) arises as a consequence of the expression of a chimeric fusion protein, p210BCR-ABL1, which is localized to the cytoplasm and has constitutive protein tyrosine kinase activity. Extensive publications report that p210BCR-ABL1 complexed with multiple cytoplasmic proteins can modulate several cell signaling pathways. However, while altered signaling states can be demonstrated in primary CML material, most of the reported analytical work on complexed proteins has been done in cell lines expressing p210BCR-ABL1. This has been necessary because primary hemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR-ABL1, precluding accurate p210BCR-ABL1 quantification by Western blotting or investigation of coimmunoprecipitating proteins in primary cells. This degradative activity has proven intractable to inhibition by conventional protease inhibitors. We show here that the degradative activity in primary cells is associated with cell lysosomes and is most likely to be an acid-dependent hydrolase. By lysing primary hemopoietic cells at high pH, we have demonstrated substantial inhibition of the p210BCR-ABL1-degradative activity and now report, to the best of our knowledge, the first published demonstration by coimmunoprecipitation of the association between p210BCR-ABL1 and cytoplasmic effector proteins in primary CML material.     

Clinical and pathological impact of VHL,PBRM1, BAP1, SETD2, KDM6A,and JARID1c in clear cell renal cell carcinoma

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1‐mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted. © 2013 Wiley Periodicals, Inc.     

Jarid2 and PRC2, partners in regulating gene expression

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.     

Antibody feedback and somatic mutation in B cells: regulation of mutation by immune complexes with IgG antibody

Summary: In response to an appropriate antigenic stimulus, and with help from T lymphocytes, naive B cells differentiate into plasmacytes which produce the primary (germline-encoded) IgM and IgG antibody with low affinity for the antigen. The isotype switch from IgM to IgG coincides with the burst of germinal center reaction and the onset of "somatic hypermutation. Here we propose that formation of immune complexes between the residual antigen and the primary IgG antibody, which activate complement and localize specifically in the network of follicular dendritic cells, provides an important signal for triggering the mutation mechanism in germinal center B cells. This hypothesis has been supported by studies on immunogenicity of immune complexes in vivo. The experiments have included an immunization with pre-formed antigen/IgG antibody complex and/or an administration of IgG and body shortly after the antigen injection. Either of these strategies, which are known to augment the germinal center formation, resulted in earlier onset of somatic mutation and increased mutation frequency in VDJ rearrangements in antigen-reactive B cells, provided that help from T cells was also present. It is presumed that the antigen/antibody/complement complex is able to deliver this important signal by cross-linking of antigen receptor with the CD21/CD19/CD81 molecules on B cells. As a corollary, the signaling by immune complexes may lower the threshold of cell activation determined by receptor affinity for antigen and stimulate diverse V-gene repertoire of B-cell clones in germinal centers.