Publications

An immortalized neural cell line derived from the human ventral mesencephalon, called ReNCell, and its MeCP2 knock out were used. With it, we characterized the chromatin compositional transitions undergone during differentiation, with special emphasis on linker histones. While the WT cells displayed the development of dendrites and axons the KO cells did not, despite undergoing differentiation as monitored by NeuN. ReNCell expressed minimal amounts of histone H1.0 and their linker histone complement consisted mainly of histone H1.2, H1.4 and H1.5. The overall level of histone H1 exhibited a trend to increase during the differentiation of MeCP2 KO cells. The phosphorylation levels of histone H1 proteins decreased dramatically during ReNCell’s cell differentiation independently of the presence of MeCP2. Immunofluorescence analysis showed that MeCP2 exhibits an extensive co-localization with linker histones. Interestingly, the average size of the nucleus decreased during differentiation but in the MeCP2 KO cells, the smaller size of the nuclei at the start of differentiation increased by almost 40% after differentiation by 8 days (8 DIV). In summary, our data provide a compelling perspective on the dynamic changes of H1 histones during neural differentiation, coupled with the intricate interplay between H1 variants and MeCP2. <b>Abbreviations</b>: ACN, acetonitrile; A₂₃₀, absorbance at 230 nm; bFGF, basic fibroblast growth factor; CM, chicken erythrocyte histone marker; CNS, central nervous system; CRISPR, clustered regulated interspaced short palindromic repeatsDAPI, 4,’6-diaminidino-2-phenylindole; DIV, days <i>in vitro</i> (days after differentiation is induced); DMEM, Dulbecco’s modified Eagle medium; EGF, epidermal growth factor; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic proteinHPLC, high-performance liquid chromatography; IF, immunofluorescence; iPSCs, induced pluripotent stem cells; MAP2, microtubule-associated protein 2; MBD, methyl-binding domain; MeCP2, methyl-CpG binding protein 2; MS, mass spectrometry; NCP, nucleosome core particle; NeuN, neuron nuclear antigen; NPC, neural progenitor cellPAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PFA, paraformaldehyde; PTM, posttranslational modification; RP-HPLC, reversed phase HPLC; ReNCells, ReNCells VM; RPLP0, ribosomal protein lateral stalk subunit P0; RT-qPCR, reverse transcription quantitative polymerase-chain reaction; RTT, Rett Syndrome; SDS, sodium dodecyl sulphate; TAD, topologically associating domain; Triple KO, triple knockout.

&lt;div&gt;Abstract&lt;p&gt;Triple-negative breast cancer (TNBC) is the most heterogeneous and aggressive subtype of breast carcinoma, characterized by the absence of clinical biomarkers and the lack of targeted therapies. Despite numerous clinical trials, patient stratification remains suboptimal, limiting the identification of effective treatment strategies. In this study, we aimed to identify biomarkers exclusively expressed in the basal mammary epithelial compartment to refine TNBC subclassification. Computational analysis of single-cell RNA sequencing data defined a set of basal identity genes, which were subsequently validated by immunohistochemistry in two independent TNBC cohorts. This approach enabled the identification of a TNBC subgroup, termed true basal TNBC (tB-TNBC), which was associated with poorer prognosis. High-throughput screening of 3,200 FDA-approved compounds in breast cancer cell lines classified according to basal marker expression identified dasatinib as a promising candidate with selective activity against tB-TNBC models. In tB-TNBC patient-derived xenograft models, dasatinib treatment effectively suppressed tumor growth. Furthermore, &lt;i&gt;TAGLN&lt;/i&gt; emerged as a strong predictive biomarker of dasatinib response, with functional studies confirming its role in modulating drug sensitivity. Altogether, these findings support the clinical utility of basal markers for TNBC stratification and highlight a targeted treatment opportunity for patients with tB-TNBC.&lt;/p&gt;Significance:&lt;p&gt;Basal biomarkers enable subclassification of triple-negative breast cancer and reveal a specific tumor subgroup sensitive to dasatinib, providing an effective stratification and treatment strategy for patients.&lt;/p&gt;&lt;/div&gt;

Two separate molecular pathways have been proposed for the early carcinogenic events observed in the oral cavity and pharynx: one is associated with chemical etiological factors such as smoking and drinking, and the other one is associated with HPV insertion.A proof-of-principle study was performed to ascertain if global DNA methylation could be used to distinguish between the early molecular changes in premalignant oral lesions.Personal histories of tobacco and alcohol use were obtained by questionnaire. HPV insertion in tumor tissue was detected by polymerase chain reaction (PCR). Global DNA methylation levels were obtained using HPLC for fraction separation and mass spectrometry for quantification. Predictive simulations were performed to explore potential associations between different etiological factors and the global DNA methylation index. Significance of results was ascertained using Pearson's Chi-squared test.The global methylation index was found to be 4.28 (95% CI, 4.1, 4.4) in an oral cancer case series. Pearson's chi squared test showed no statistically significant difference between cases that had smoking (p = 0.21), drinking (p = 0.31) or HPV insertion (p = 0.34) as etiologic risk factors, when compared to cases that did not. An inverse significant association between smoking and DNA methylation was observed. As the smoking effect increases, the global methylation index decreases, In addition, no associations between the probability of DNA methylation and drinking, or DNA methylation and HPV insertion were observed in simulations.The global DNA methylation index was shown to vary for oral cancer cases with different etiologies. Smoking was inversely correlated with DNA methylation levels when generalized linear model simulations were performed. Future studies should look at global DNA methylation alterations associated to the progression from normal to premalignant oral epithelium tissue in a cohort of smokers and nonsmokers.

&lt;p&gt;Representative recurrent imbalanced regions in different orthoxenografts with acquired CDDP resistance&lt;/p&gt;

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&lt;p&gt;DNA methylation (M-values) of the CpG island probes located within six PCC/PGL susceptibility genes encoding Krebs cycle enzymes (SDHA, SDHAF2, SDHB, SDHD, FH, and MDH2) in the 11 analyzed tumors, compared to in vitro methylated DNA (IVD).&lt;/p&gt;

Supplementary Figures 1-4 from Chromosomal Instability Correlates with Genome-wide DNA Demethylation in Human Primary Colorectal Cancers

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

&lt;div&gt;Abstract&lt;p&gt;CD137 (4-1BB) costimulation imprints long-term changes that instruct the ultimate behavior of T cells that have previously experienced CD137 ligation. Epigenetic changes could provide a suitable mechanism for these long-term consequences. Genome-wide DNA methylation arrays were carried out on human peripheral blood CD8&lt;sup&gt;+&lt;/sup&gt; T lymphocytes stimulated with agonist monoclonal antibody to CD137, including urelumab, which is in phase I/II clinical trials for cancer immunotherapy. Several genes showed consistent methylation patterns in response to CD137 costimulation, which were confirmed by pyrosequencing in a series of healthy donors. &lt;i&gt;CD96, HHLA2, CCR5, CXCR5&lt;/i&gt;, and &lt;i&gt;CCL5&lt;/i&gt; were among the immune-related genes regulated by differential DNA methylation, leading to changes in mRNA and protein expression. These genes are also differentially methylated in naïve versus antigen-experienced CD8&lt;sup&gt;+&lt;/sup&gt; T cells. The transcription factor TCF1 and the microRNA miR-21 were regulated by DNA methylation upon CD137 costimulation. Such gene-expression regulatory factors can, in turn, broaden the effects of DNA methylation by controlling expression of their target genes. Overall, chromatin remodeling is postulated to leave CD137-costimulated T lymphocytes poised to differentially respond upon subsequent antigen recognition. Accordingly, CD137 connects costimulation during priming to genome-wide DNA methylation and chromatin reprogramming. &lt;i&gt;Cancer Immunol Res; 6(1); 69–78. ©2017 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro–B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development.

Aberrant methylation of 5'CpG islands is a key epigenetic event in many human cancers. A PCR-based technique of methylated CpG island amplification followed by representational difference analysis was used to identify genes methylated in cancer. Two of the CpG islands identified mapped to the 5' untranslated region of the PAX5 alpha and beta genes. These genes, located on chromosome 9p13, are transcribed from two distinct promoters and form two alternative first exons that are subsequently spliced to the common exons 2-10. The resulting splice variants encode two distinct transcription factors important in cell differentiation and embryonic development. Examination of the methylation status of each gene using methylation-specific PCR revealed that both genes are methylated in approximately 65% of breast and lung tumors. Bisulfite sequencing revealed dense methylation patterns within each 5'CpG island, strongly correlating with transcriptional silencing. Expression in cell lines with dense methylation of either the PAX5 alpha or beta promoter region was restored after treatment with the demethylating agent 5-Aza-2'-deoxycytidine. The PAX5 beta gene encodes for the transcription factor B cell-specific activating protein that, in turn, directly regulates CD19, a gene shown to negatively control cell growth. A strong association was observed between PAX5 beta methylation and loss of expression of the CD19 gene demonstrating that inactivation of the PAX5 beta gene likely contributes to neoplastic development by inhibiting growth regulation through effects on CD19 gene expression. Recent studies have demonstrated the importance of PAX5 gene alterations in human cancer. Our results are the first to identify aberrant promoter methylation as a common mechanism for dysregulation of these genes in solid tumors.