Epigenetic regulation of gene silencing during stem cell commitment and differentiation

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Epigenetic regulation of gene silencing during stem cell commitment and differentiation

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Circular dichroism and thermal melting differentiation of Hoechst 33258 binding to the curved (A(4)T(4)) and straight (T(4)A(4)) DNA sequences

The ability of the B-DNA minor groove ligand Hoechst 33258 to discriminate between prototype curved and straight duplex DNA sequences was investigated by circular dichroism (CD) titrations at the wavelengths of absorbance of the ligand. The sequences were studied either within the framework of the ligated decamers (CA(4)T(4)G)(n) and (CT(4)A(4)G)(n), or within that of the single dodecamers GCA(4)T(4)GC and GCT(4)A(4)GC, to confirm and extend our earlier results based on fluorescence titrations of ligated decamers. A unique, strong binding site is invariantly present in both sequence units. The binding affinity of the drug for the site in the curved A(4)T(4) sequence was found 3- to 4-fold higher compared to the straight sequence. All these features hold true irrespective of the sequence framework, thus confirming that they reflect specific properties of the binding to the two sequences. Ligand binding increases the thermal stability of straight and curved duplex dodecamers to the same extent, thus maintaining the melting temperature differential between the two sequences. However, the different melting patterns and the difference between [total ligand]: [site] ratios needed for site saturation in the two duplexes are in agreement with the difference between binding constants derived from CD measurements, (C) 2002 Elsevier Science B.V. All rights reserved

Formation of an Active Tissue-Specific Chromatin Domain Initiated by Epigenetic Marking at the Embryonic Stem Cell Stage

The differentiation potential of stem cells is determined by the ability of these cells to establish and maintain developmentally regulated gene expression programs that are specific to different lineages. Although transcriptionally potentiated epigenetic states of genes have been described for haematopoietic progenitors, the developmental stage at which the formation of lineage-specific gene expression domains is initiated remains unclear. In this study, we show that an intergenic cis-acting element in the mouse λ5-VpreB1 locus is marked by histone H3 acetylation and histone H3 lysine 4 methylation at a discrete site in embryonic stem (ES) cells. The epigenetic modifications spread from this site toward the VpreB1 and λ5 genes at later stages of B-cell development, and a large, active chromatin domain is established in pre-B cells when the genes are fully expressed. In early B-cell progenitors, the binding of haematopoietic factor PU.1 coincides with the expansion of the marked region, and the region becomes a center for the recruitment of general transcription factors and RNA polymerase II. In pre-B cells, E2A also binds to the locus, and general transcription factors are distributed across the active domain, including the gene promoters and the intergenic region. These results suggest that localized epigenetic marking is important for establishing the transcriptional competence of the λ5 and VpreB1 genes as early as the pluripotent ES cell stage

SAGA complex and Gcn5 are necessary for respiration in budding yeast

In budding yeast, growth through fermentation and/or respiration is dependent on the type of carbon source present in the medium. SAGA complex is themain acetylation complex and is required, togetherwith Rtg factors, for nucleus-mitochondria communication and transcriptional activation of specific nuclear genes. Even though acetylation is necessary formitochondria activity and respiratory pathways the direct role of histone acetyltransferases and SAGA complex has never been investigated directly. In this study we demonstrate, for the first time, that Gcn5 and SAGA are needed for respiratory metabolismand oxygen consumption. According to a central role for acetylation in respirationwe find that the Gcn5 inhibitor CPTH2 had higher efficacy on cells grown in glycerol containing media. We also demonstrated that the opposing activities of Gcn5 and Hda1 modify selectively H3- AcK18 and are essential for respiration. Taken together our results suggest a novel paradigm coupling acetyltransferase activity to respiratory metabolism. Correspondingly we propose the selective utilization of KAT inhibitor CPTH2, combined to the modulation of the respiratory metabolism of the cell, as a promising novel tool of intervention in cancer cell

Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role

KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2

Abstract Background Kidney cancer and clear cell renal carcinoma (ccRCC) are the 16th most common cause of death worldwide. ccRCC is often metastasized at diagnosis, and surgery remains the main treatment; therefore, early diagnosis and new therapeutic strategies are highly desirable. KAT inhibitor CPTH2 lowers histone H3 acetylation and induces apoptosis in colon cancer and cultured cerebellar granule neurons. In this study, we have evaluated the effects of CPTH2 on ccRCC 786-O cell line and analyzed drug targets expressed in ccRCC tumor tissues at different grade. Results CPTH2 decreases cell viability, adhesion, and invasiveness in ccRCC cell line 786-O. It shows preferential inhibition for KAT3B-p300 with hypoacetilating effects on histone H3 at specific H3-K18. Immunohistochemical analysis of 70 ccRCC tumor tissues compared with peritumoral normal epithelium showed a statistical significant reduction of p300/H3AcK18 paralleled by an increase of H3AcK14 in G1 grade and an opposed trend during tumor progression to worst grades. In this study, we demonstrate that these marks are CPTH2 targets and significative prognosticators of low-grade ccRCC tumor. Conclusions ccRCC is substantially insensitive to current therapies, and the efficacy of clinical treatment is dependent on the dissemination stage of the tumor. The present study shows that CPTH2 is able to induce apoptosis and decrease the invasiveness of a ccRCC cell line through the inhibition of KAT3B. In a tumor tissue analysis, we identified new prognosticator marks in grade G1 ccRCC tumors. Low KAT3B/H3AcK18 vs. high H3AcK14 were found in G1 while an opposed trend characterized tumor progression to worst grades. Our collected results suggest that CPTH2 reducing KAT3B and H3AcK18 can be considered a promising candidate for counteracting the progression of ccRCC tumors

Identification of neuroblastoma cell lines with uncommon TAZ+/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells

Background Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells.Methods We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset.Results Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105+/CD90+/CD73+/CD29+/CD146+/GD2+/TAZ+). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105+/CD73+/TAZ+). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells.Conclusions We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells

Additional file 4: of KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2

Immunostaining of tissue sections from ccRCC tumor and normal tissues with p300, H3AcK18, and H3AcK14 antibodies. Two opposite cases are shown, patient no. 1 with low p300/H3AcK18 vs. high H3AcK14. Patient no. 41, the opposite, high p300/H3AcK18 vs. low H3AcK14. (TIFF 37242 kb