| 1. |
- Frank, Stefan, et al.
(författare)
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yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.
- 2019
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Ingår i: Cell stem cell. - : Elsevier BV. - 1875-9777 .- 1934-5909. ; 24:2, s. 318-327.e8
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Tidskriftsartikel (refereegranskat)abstract
- Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.
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| 2. |
- Kopparapu, Pradeep Kumar, et al.
(författare)
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Gene-body hypermethylation controlled cryptic promoter and miR26A1-dependent EZH2 regulation of TET1 gene activity in chronic lymphocytic leukemia
- 2017
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 8:44, s. 77595-77608
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Tidskriftsartikel (refereegranskat)abstract
- The Ten-eleven-translocation 1 (TET1) protein is a member of dioxygenase protein family that catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. TET1 is differentially expressed in many cancers, including leukemia. However, very little is known about mechanism behind TET1 deregulation. Previously, by characterizing global methylation patterns in CLL patients using MBD-seq, we found TET1 as one of the differentially methylated regions with gene-body hypermethylation. Herein, we characterize mechanisms that control TET1 gene activity at the transcriptional level. We show that treatment of CLL cell lines with 5-aza 2'-deoxycytidine (DAC) results in the activation of miR26A1, which causes decrease in both mRNA and protein levels of EZH2, which in turn results in the decreased occupancy of EZH2 over the TET1 promoter and consequently the loss of TET1 expression. In addition, DAC treatment also leads to the activation of antisense transcription overlapping the TET1 gene from a cryptic promoter, located in the hypermethylated intronic region. Increased expression of intronic transcripts correlates with decreased TET1 promoter activity through the loss of RNA Pol II occupancy. Thus, our data demonstrate that TET1 gene activation in CLL depends on miR26A1 regulated EZH2 binding at the TET1 promoter and silencing of novel cryptic promoter by gene-body hypermethylation.
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| 3. |
- Kopparapu, Pradeep Kumar, et al.
(författare)
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MCPH1 maintains long-term epigenetic silencing of ANGPT2 in chronic lymphocytic leukemia.
- 2015
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Ingår i: The FEBS journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 282:10
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Tidskriftsartikel (refereegranskat)abstract
- The microcephalin gene (MCPH1) [also known as inhibitor of human telomerase reverse transcriptase (hTERT) expression] is a tumor suppressor gene that is functionally involved in the DNA damage response. Angiopoietin 2 (ANGPT2) is a crucial factor regulating tumor angiopoiesis. Deregulation of angiogenesis is one of the hallmarks of many cancers, including chronic lymphocytic leukemia (CLL). In CLL, ANGPT2 is a well-studied potential prognostic marker. As MCPH1 overlaps with the ANGPT2 transcription unit on the same chromosome but in the opposite orientation, we wanted to study the functional role of MCPH1 in regulation of ANGPT2 in CLL. The mRNA expression levels of MCPH1 and ANGPT2, including the MCPH1 target gene hTERT, showed significant differences between two prognostic groups, i.e. IGHV-mutated and IGHV-unmutated (P = 0.007 for MCPH1, P = 0.0002 for ANGPT2, and P = 0.00001 for hTERT), in which the expression level of MCPH1 was inversely correlated with the expression levels of hTERT and ANGPT2. Downregulation of MCPH1 resulted in upregulation of ANGPT2, accompanied by loss of its promoter methylation. Using chromatin immunoprecipitation and coimmunoprecipitation assays, we found that MCPH1 binds to the ANGPT2 promoter and recruits DNA methyltransferases, thereby silencing ANGPT2. Thus, our data suggest a novel function for MCPH1 in regulating and maintaining ANGPT2 silencing in CLL through regulation of promoter DNA methylation.
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| 4. |
- Kosalai, Subazini Thankaswamy, 1980, et al.
(författare)
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EZH2 upregulates the PI3K/AKT pathway through IGF1R and MYC in clinically aggressive chronic lymphocytic leukaemia
- 2019
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 14:11, s. 1125-1140
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Tidskriftsartikel (refereegranskat)abstract
- EZH2 is overexpressed in poor-prognostic chronic lymphocytic leukaemia (CLL) cases, acting as an oncogene; however, thus far, the EZH2 target genes in CLL have not been disclosed. In this study, using ChIP-sequencing, we identified EZH2 and H3K27me3 target genes in two prognostic subgroups of CLL with distinct prognosis and outcome, i.e., cases with unmutated (U-CLL, n = 6) or mutated IGHV genes (M-CLL, n = 6). While the majority of oncogenic pathways were equally enriched for EZH2 target genes in both prognostic subgroups, PI3K pathway genes were differentially bound by EZH2 in U-CLL versus M-CLL. The occupancy of EZH2 for selected PI3K pathway target genes was validated in additional CLL samples (n = 16) and CLL cell lines using siRNA-mediated EZH2 downregulation and ChIP assays. Intriguingly, we found that EZH2 directly binds to the IGF1R promoter along with MYC and upregulates IGF1R expression in U-CLL, leading to downstream PI3K activation. By investigating an independent CLL cohort (n = 96), a positive correlation was observed between EZH2 and IGF1R expression with higher levels in U-CLL compared to M-CLL. Accordingly, siRNA-mediated downregulation of either EZH2, MYC or IGF1R and treatment with EZH2 and MYC pharmacological inhibitors in the HG3 CLL cell line induced a significant reduction in PI3K pathway activation. In conclusion, we characterize for the first time EZH2 target genes in CLL revealing a hitherto unknown implication of EZH2 in modulating the PI3K pathway in a non-canonical, PRC2-independent way, with potential therapeutic implications considering that PI3K inhibitors are effective therapeutic agents for CLL.
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| 5. |
- Mondal, Tanmoy, 1981, et al.
(författare)
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Sense-antisense lncRNA pair encoded by locus 6p22.3 determines neuroblastoma susceptibility via the USP36-CHD7-SOX9 regulatory axis
- 2018
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Ingår i: Cancer Cell. - : Elsevier BV. - 1535-6108 .- 1878-3686. ; 33:3, s. 417-434.e7
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Tidskriftsartikel (refereegranskat)abstract
- Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.
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| 6. |
- Subhash, Santhilal, 1987, et al.
(författare)
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Global DNA methylation profiling reveals new insights into epigenetically deregulated protein coding and long noncoding RNAs in CLL.
- 2016
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Ingår i: Clinical Epigenetics. - : Springer Science and Business Media LLC. - 1868-7083 .- 1868-7075. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background: Methyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq) is a robust and powerful method for analyzing methylated CpG-rich regions with complete genome-wide coverage. In chronic lymphocytic leukemia (CLL), the role of CpG methylated regions associated with transcribed long noncoding RNAs (lncRNA) and repetitive genomic elements are poorly understood. Based on MBD-Seq, we characterized the global methylation profile of high CpG-rich regions in different CLL prognostic subgroups based on IGHV mutational status. Results: Our study identified 5800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal controls. From cllDMGs, 40 % of hypermethylated and 60 % of hypomethylated genes were mapped to noncoding RNAs. In addition, we found that the major repetitive elements such as short interspersed elements (SINE) and long interspersed elements (LINE) have a high percentage of cllDMRs (differentially methylated regions) in IGHV subgroups compared to normal controls. Finally, two novel lncRNAs (hypermethylated CRNDE and hypomethylated AC012065.7) were validated in an independent CLL sample cohort (48 samples) compared with 6 normal sorted B cell samples using quantitative pyrosequencing analysis. The methylation levels showed an inverse correlation to gene expression levels analyzed by real-time quantitative PCR. Notably, survival analysis revealed that hypermethylation of CRNDE and hypomethylation of AC012065.7 correlated with an inferior outcome. Conclusions: Thus, our comprehensive methylation analysis by MBD-Seq provided novel hyper and hypomethylated long noncoding RNAs, repetitive elements, along with protein coding genes as potential epigenetic-based CLL-signature genes involved in disease pathogenesis and prognosis.
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| 7. |
- Subhash, Santhilal, 1987, et al.
(författare)
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H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units
- 2018
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:18
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Tidskriftsartikel (refereegranskat)abstract
- Recently lncRNAs have been implicated in the sub-compartmentalization of eukaryotic genome via genomic targeting of chromatin remodelers. To explore the function of lncRNAs in the maintenance of active chromatin, we characterized lncRNAs from the chromatin enriched with H3K4me2 and WDR5 using chromatin RNA immunoprecipitation (ChRIP). Significant portion of these enriched lncRNAs were arranged in antisense orientation with respect to their protein coding partners. Among these, 209 lncRNAs, commonly enriched in H3K4me2 and WDR5 chromatin fractions, were named as active chromatin associated lncRNAs (active lncCARs). Interestingly, 43% of these active lncCARs map to divergent transcription units. Divergent transcription (XH) units were overrepresented in the active lncCARs as compared to the inactive lncCARs. ChIP-seq analysis revealed that active XH transcription units are enriched with H3K4me2, H3K4me3 and WDR5. WDR5 depletion resulted in the loss of H3K4me3 but not H3K4me2 at the XH promoters. Active XH CARs interact with and recruit WDR5 to XH promoters, and their depletion leads to decrease in the expression of the corresponding protein coding genes and loss of H3K4me2, H3K4me3 and WDR5 at the active XH promoters. This study unravels a new facet of chromatin-based regulation at the divergent XH transcription units by this newly identified class of H3K4me2/WDR5 chromatin enriched lncRNAs.
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| 8. |
- Wernig-Zorc, Sara, et al.
(författare)
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Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia.
- 2019
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Ingår i: Epigenetics & chromatin. - : Springer Science and Business Media LLC. - 1756-8935. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis.We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression.Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.
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| 9. |
- Kopparapu, Pradeep Kumar, et al.
(författare)
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Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma : Impact on EZH2 expression
- 2016
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 11:5, s. 335-343
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Tidskriftsartikel (refereegranskat)abstract
- Downregulation of miR26A1 has been reported in various B-cell malignancies; however, the mechanism behind its deregulation remains largely unknown. We investigated miR26A1 methylation and expression levels in a well-characterized series of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). From 450K methylation arrays, we first observed miR26A1 (cg26054057) as uniformly hypermethylated in MCL (n = 24) (all >75%), while CLL (n = 18) showed differential methylation between prognostic subgroups. Extended analysis using pyrosequencing confirmed our findings and real-time quantitative PCR verified low miR26A1 expression in both CLL (n = 70) and MCL (n = 38) compared to normal B-cells. Notably, the level of miR26A1 methylation predicted outcome in CLL, with higher levels seen in poor-prognostic, IGHV-unmutated CLL. Since EZH2 was recently reported as a target for miR26A1, we analyzed the expression levels of both miR26A1 and EZH2 in primary CLL samples and observed an inverse correlation. By overexpression of miR26A1 in CLL and MCL cell lines, reduced EZH2 protein levels were observed using both Western blot and flow cytometry. In contrast, methyl-inhibitor treatment led to upregulated miR26A1 expression with a parallel decrease of EZH2 expression. Finally, increased levels of apoptosis were observed in miR26A1-overexpressing cell lines, further underscoring the functional relevance of miR26A1. In summary, we propose that epigenetic silencing of miR26A1 is required for the maintenance of increased levels of EZH2, which in turn translate into a worse outcome, as shown in CLL, highlighting miR26A1 as a tumor suppressor miRNA.
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| 10. |
- Kopparapu, Pradeep Kumar, et al.
(författare)
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Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma: Impact on EZH2 expression. : Epigenetic inactivation of miR - 26A1 in CLL and MCL
- 2016
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 11:5, s. 335-343
-
Tidskriftsartikel (refereegranskat)abstract
- Downregulation of miR26A1 has been reported in various B-cell malignancies; however, the mechanism behind its deregulation remains largely unknown. We investigated miR26A1 methylation and expression levels in a well-characterized series of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). From 450K methylation arrays, we first observed miR26A1 (cg26054057) as uniformly hypermethylated in MCL (n=24) (all >75%), while CLL (n=18) showed differential methylation between prognostic subgroups. Extended analysis using pyrosequencing confirmed our findings and real-time quantitative PCR verified low miR26A1 expression in both CLL (n=70) and MCL (n=38) compared to normal B-cells. Notably, the level of miR26A1 methylation predicted outcome in CLL, with higher levels seen in poor-prognostic, IGHV-unmutated CLL. Since EZH2 was recently reported as a target for miR26A1, we analyzed the expression levels of both miR26A1 and EZH2 in primary CLL samples and observed an inverse correlation. By overexpression of miR26A1 in CLL and MCL cell lines, reduced EZH2 protein levels were observed using both Western blot and flow cytometry. In contrast, methyl-inhibitor treatment led to upregulated miR26A1 expression with a parallel decrease of EZH2 expression. Finally, increased levels of apoptosis were observed in miR26A1-overexpressing cell lines, further underscoring the functional relevance of miR26A1. In summary, we propose that epigenetic silencing of miR26A1 is required for the maintenance of increased levels of EZH2, which in turn translate into a worse outcome, as shown in CLL, highlighting miR26A1 as a tumor suppressor miRNA.
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