Volume 32, Issue 2, Pages 232-246 (October 2008) Kcnq1ot1 Antisense Noncoding RNA Mediates Lineage-Specific Transcriptional Silencing through Chromatin-Level Regulation  Radha Raman Pandey, Tanmoy Mondal, Faizaan Mohammad, Stefan Enroth, Lisa Redrup, Jan Komorowski, Takashi Nagano, Debora Mancini-DiNardo, Chandrasekhar Kanduri  Molecular Cell  Volume 32, Issue 2, Pages 232-246 (October 2008) DOI: 10.1016/j.molcel.2008.08.022 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Kcnq1ot1 Is an RNAPII-Encoded 91 kb-Long Moderately Stable Nuclear Transcript (A) qPCR analysis of ChIP DNA, obtained from MEFs, using RNAPII antibody. (B) Physical map of Kcnq1 and Kcnq1ot1 transcription units and the RPA probes used in mapping the 3′ end of Kcnq1ot1. (C) Autoradiograms of strand-specific RPAs. (D) Autoradiogram showing that Kcnqot1 is exclusively localized in the nuclear compartment in MEFs. (E) qPCR assay for analyzing the stability of Kcnq1ot1 and c-Myc RNAs in control and Actinomycin-D-treated MEFs. Kcnq1ot1 and c-Myc RNA levels were normalized to β-actin, their levels in control were set as 100%, and the levels in Actinomycin-D-treated cells were presented relative to control. Kcnq1ot1 half-life was calculated by linear regression analysis. The qPCR values in (A) and (E) represent mean ± SD (standard deviation) of three independent biological replicates. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Kcnq1ot1 RNA Per Se Mediates Bidirectional Silencing through Interacting with Chromatin (A) Physical maps of PH19, PS7, and PS7fosUTR+/− episomes. (B) Analysis of the stability of Kcnq1ot1 in the PS7, PS7fosUTR−, and PS7fosUTR+ episomes by qPCR using linear regression analysis. The values presented were normalized to GAPDH and represent mean ± SD of three independent biological replicates. (C) RPA analysis shows that the activity of the Kcnq1ot1 promoter in the PS7fosUTR− and PS7fosUTR+ episomes remains unaffected. (D) Bar graph shows the percentage activity of the hygromycin gene, as determined by counting the hygromycin-resistant colonies after selection. The value for the control PH19 episome was set at 100%, and the values of other experimental constructs were shown relative to this. The data in (C) and (D) represent mean ± SD of three independent biological replicates. (E and F) Kcnq1ot1 interacts with chromatin in a lineage-specific manner. (E) qPCR analysis of Kcnq1ot1, Tsix, and β-actin transcripts. The data represent mean ± SD of three independent experiments. (F) Semiquantitative analysis of H3 antibody-purified chromatin material (ChIP) from the placenta and fetal liver. The upper panel verifies that the histone H3 antibody has pulled down the chromatin-associated Kcnq1ot1 DNA sequence equally from both tissues. The middle and lower panels show the RNA (ChRIP) analysis of placenta and fetal liver using primers specific for Kcnq1ot1 and the negative control β-actin, respectively. (G) qPCR analysis of ChOP-purified DNA from E14.5 placenta. Kcnq1P1 and Kcnq1P2 represent different primer positions. The enrichment with antisense oligo over scrambled oligo was plotted in logarithmic scale. Imprinted genes are marked with asterisks. The data represent mean ± SD of two independent experiments. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Kcnq1ot1 RNA Establishes Lineage-Specific Patterns of an Inactive Chromatin-Specific Modification, H3K27me3, along the Kcnq1 Domain (A) Physical map of the 1 Mb Kcnq1 domain. Genes imprinted only in placenta are depicted with blue boxes, while genes imprinted in both placenta and liver are shown in red. Biallelically expressed genes are shown in green boxes. Black lines represent primer positions used in qPCR validation experiments. Signal maps of H3K27me3 along the Kcnq1 domain are shown underneath the physical map. Peaks indicate the log2 signal ratio (ChIP/input enrichment) of each oligonucleotide on the array. Signal maps represent one of the two independent experiments. (B) Smoothed ChIP-chip log2 signal of H3K27me3 in placenta and liver of wild-type mice. Transcription start sites of genes in the region are indicated by vertical dotted lines. Transparent columns represent regions showing H3K27me3 enrichment in placenta as compared to fetal liver, and the numbers on top of the columns represent the P values, which were calculated using a normalized paired Z test on placenta-liver differences. (C) qPCR verification of H3K27me3 ChIP-on-chip data in wild-type mice using a select group of primers; data represent mean ± SD of three independent experiments. (D) Smoothed ChIP-chip log2 signal of H3K27me3 in placenta and liver of ΔKcnq1ot1 mice. (E) qPCR verification of H3K27me3 data in ΔKcnq1ot1 mice. qpCR data represent mean ± SD of three independent experiments. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 ChIP-on-chip Showing the Distribution of H3K9ac and H3K9me3 along the Kcnq1 Domain (A) Physical map of the Kcnq1 domain and the signal maps of H3K9me3 and H3K9ac in wild-type placenta and fetal liver. (B and D) Smoothed ChIP-chip log2 signal of H3K9me3 (B) and H3K9ac (D) in both liver and placenta. (C and E) qPCR verification of H3K9me3 (C) and H3K9ac (E) ChIP-on-chip data; the data represent mean ± SD of three independent experiments. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 High-Resolution Signal Maps Showing Histone Modification Profiles of Placentally Imprinted Genes ChIP-on-chip showing the H3K27me3, H3K9me3, and H3K9ac profiles (zoom in signal maps) over placental imprinted genes; Ascl2, Tspan32, Cd81, and Tssc4 in E14.5 placenta and liver. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 High-Resolution Signal Maps Showing Histone Modification Profiles of Genes Imprinted in Both Liver and Placenta ChIP-on-chip showing the H3K27me3, H3K9me3, and H3K9ac profiles (zoom in signal maps) of Kcnq1, Kcnq1ot1, Cdkn1c, and Slc22al8, imprinted in both embryo as well as placenta in E14.5 placenta and liver. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Kcnq1ot1 Interacts with G9a and PRC2 Complex Members in a Lineage-Specific Manner (A) Agarose gel pictures showing semiquantitative RT-PCR analysis of Kcnq1ot1 and β-actin RNA profiles in the immunoprecipitated RNA, obtained from nuclear extracts of fetal liver and placenta, incubated with antibodies against IgG, G9a, EZH2, and SUZ12. β-actin is used as a negative control for the analysis. (B) Immuno-DNA FISH analysis on placenta and fetal liver. DNA signals are detected in green; nucleolar marker, nucleophosmin detected in red; and DAPI staining in blue. Scale bar, 5 μm. Molecular Cell 2008 32, 232-246DOI: (10.1016/j.molcel.2008.08.022) Copyright © 2008 Elsevier Inc. Terms and Conditions