Supplementary MaterialsAdditional file 1. analysis shown represents one of three independent

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Supplementary MaterialsAdditional file 1. analysis shown represents one of three independent experiments. 13072_2019_253_MOESM2_ESM.pdf (123K) GUID:?96329CAF-6D0F-4BB1-AEDD-2FAD770B1165 Additional file 3. Kcnq1ot1 expression does not decrease during differentiation. RT-qPCR analysis of Kcnq1ot1, expression in undifferentiated (and the muscle-specific gene (value ?0.05 (*); value ?0.01 (**). 13072_2019_253_MOESM3_ESM.pdf (1.8M) GUID:?D63D3CA9-5353-47EB-9FF8-6AB3B73904D8 Additional file 4. Differential epigenetic status of the maternal and paternal p57 intragenic regions. Left: Allele-specific ChIP-qPCR analysis of H3K4me3 accumulation at Maternal and Paternal intragenic regions (M-and P-promoter (p) was used as unfavorable control. Values are the mean??SEM of three independent experiments performed and were expressed as percentages of Input. Statistical significance: value ?0.05 (*). Right: qPCR analysis of the MeDIP assays performed in polymorphic fibroblasts (C57B/6 female??SD7 male) EPZ-6438 irreversible inhibition using allele-specific primers for the intragenic region (M-and P-promoter (p) was EPZ-6438 irreversible inhibition used as a negative control. The results shown represent one of two impartial experiments performed. Values were expressed as percentages??SEM of Input DNA for each sample analyzed in triplicate. 13072_2019_253_MOESM4_ESM.pdf (1.1M) GUID:?C334CAB3-CD7C-4342-83D6-4E6E59036DB0 Additional file 5. Verification of Kcnq1ot1 depletion in cells used for the ChIP assays reported in Fig.?6. C2.7 myoblasts were transfected with Kcnq1ot1 siRNAs as in Fig.?1a and analyzed by RT-qPCR for Kcnq1ot1 RNA levels in siCTR and siKcnq1ot1 samples. Values were normalized to Tbp RNA levels and expressed as percentages of the control. Results are the mean??SEM of three independent experiments. Statistical significance: value ?0.001 (***) 13072_2019_253_MOESM5_ESM.pdf (243K) GUID:?3AB667E0-61CD-4AD2-8442-BE5554CBEF9F Additional file 6. intragenic region (M-promoter (p) used as an invariant control and -Actin promoter (-Act p) as a negative control. Values obtained are expressed as percentages of Input chromatin and normalized to those of promoter, used as an additional invariant control. The results shown represent one of two impartial experiments and error bars represent the mean??SEM of each sample analyzed in triplicate. 13072_2019_253_MOESM6_ESM.pdf (803K) GUID:?6F95FB9E-4F56-4B36-8F83-65C63CB28D62 Additional file 7. H3K27me3 association to the p57 intragenic region decreases during differentiation. ChIP-qPCR analysis of H3K27me3 association to the intragenic region (promoter p) in undifferentiated (U) and differentiated (D) C2.7 muscle cells. promoter (p) was used as a negative control. Values obtained were expressed as percentages of Input chromatin and normalized to those of promoter, used as an invariant control. Lum The results are the mean??SEM of three independent experiments. Statistical significance: value ?0.05 (*). 13072_2019_253_MOESM7_ESM.pdf (921K) GUID:?B10D350C-7FC8-4820-9E7A-6DF1F33505F8 Additional file 8. Kcnq1ot1 and HOTAIR are differentially associated with LSD1. Cell extracts of differentiated C2.7 muscle cells were immunoprecipitated using anti-LSD1 antibody or control IgG. Immunopurified materials were subjected to RT-qPCR with specific primers for Kcnq1ot1 and HOTAIR transcripts. Values, relative to a representative EPZ-6438 irreversible inhibition experiment, were expressed as fold enrichment respect to IgG. 13072_2019_253_MOESM8_ESM.pdf (188K) GUID:?4D3A2F64-43D8-430D-A874-5AC51D521DC3 Additional file 9. Additional methods. 13072_2019_253_MOESM9_ESM.pdf (285K) GUID:?15755657-9FD7-4F70-8559-CDA77BAACB59 Data Availability StatementData sharing is not applicable to this article as no datasets were generated or analyzed during the study. Abstract Background The cell-cycle inhibitor p57kip2 plays a critical role in mammalian development by coordinating cell proliferation and differentiation in many cell types. p57kip2 expression is usually finely regulated by several epigenetic mechanisms, including paternal imprinting. Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the imprinting domain name, is expressed exclusively from the paternal allele and participates in the allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of expression in muscle cells. Results We found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel regulatory region, located intragenically and made up of two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion exhibited that this LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal.