Global analysis of expression, maturation and subcellular localization of mouse liver transcriptome identifies novel sex-biased and TCPOBOP-responsive long non-coding RNAs

- Transcript maturation was similar for lncRNAs in the chromatin fraction, but was significantly lower in the nucleoplasm and cytoplasm.
- significantly enriched in the nucleus than cytoplasm, and 100-fold more likely to be significantly chromatin-bound than nucleoplasmic.
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- In the liver, lncRNAs have been linked to liver fibrosis through their effects on glucose metabolism (LincIRS2) [13] and hep- atic stellate cell regulation (H19, Meg3, HOTTIP) [14–.
- Many lncRNAs are preferentially localized in the nu- cleus, where they can be visualized by single molecule RNA fluorescence in situ hybridization (smFISH) [17].
- furthermore, even nuclear lncRNAs may be present in the cytoplasm at significant levels [21, 25].
- However, by fractionating nuclei using a high urea buffer contain- ing salts and detergent, RNAs that are tightly bound to chromatin can be separated from RNAs that are soluble in the nucleoplasm, enabling the characterization of several thousand lncRNAs enriched in the insoluble chromatin fraction, as im- plemented in human cell lines and mouse macro- phages [30, 31]..
- A few hundred liver-expressed lncRNA genes were shown to respond to pituitary growth hor- mone secretory patterns [23], a key factor regulating sex-biased gene expression in the liver [32–34].
- expressed lncRNAs, which complicates efforts to deter- mine whether they have regulatory or other cellular functions in the cytoplasm, nucleoplasm or when bound to chromatin..
- We find a strong enrichment of thousands of liver-expressed lncRNAs in the chromatin fraction, including lncRNAs that respond to endogenous hormonal factors or exter- nal chemical exposures, many expressed at too low a level for discovery by traditional RNA-seq analysis of whole liver tissue or even in purified liver nuclei.
- In un- treated liver, Elovl3 showed strong, male-biased expres- sion in the cytoplasmic, nuclear and nucleoplasmic fractions (Fig.
- TCPOBOP induced Elovl3 expression in the chromatin-bound RNA fraction in male liver, and in all four fractions in female liver, which largely abol- ished its sex-dependent expression.
- transcript, pre-Elovl3 RNA was highest in the chromatin-bound fraction and was induced >.
- The lncRNA Neat1 (lnc14746) was exclusively found in the nuclear and chromatin-bound fractions (Fig.
- Xist (lnc15394), which is only expressed in female cells, was found at similar levels in the nuclear, nucleoplasmic and chromatin-bound fractions and was absent from cytoplasm (Fig.
- Median IO/EC ratios were highest in the chromatin- bound fractions and did not differ between PCGs and.
- Thus, the chromatin-bound fraction contains many more unspliced or partially spliced tran- scripts, in particular in the non-polyA selected fraction (Fig.
- b PreElovl3, assayed using qPCR primers that span an intron/exon boundary to amplify unspliced transcripts, which were significantly enriched in the chromatin-bound fraction after TCPOBOP exposure in both sexes.
- c Cyp2b10, validating TCPOBOP induction response, and also female-biased expression in the basal state.
- Black horizontal lines compare distributions of IO/EC ratios for lncRNAs vs PCGs in the cytoplasmic and nucleoplasmic fractions (other comparisons were not performed).
- Extensive reads seen across the gene body in the chromatin bound fraction are substantially depleted after polyA-selection (top vs second reads track).
- 0.001) in cytoplas- mic vs nuclear fractions, and separately, in nucleoplasmic vs chromatin-bound fractions, and in the chromatin-bound fractions with vs without polyA selection (Tables S2A-S2C)..
- Thus, PCG transcripts enriched in the cytoplasm are on average more mature (lower median IO/EC ratio) than the corresponding fraction-unbiased and nuclear- enriched transcripts (Fig.
- In the nucleus, transcript maturity was similar, or even higher, for nuclear-biased PCGs and lncRNAs as com- pared to non-compartment-biased PCGs and lncRNAs (Fig.
- This suggests that other factors, such as chro- matin binding, examined below, contribute to transcript enrichment in the nucleus..
- significantly enriched in the chromatin fraction (3028 vs..
- Preferential enrichment in the chromatin fraction was also seen for 92% of more than 7000 other lncRNAs that did not meet our strin- gent criteria (adjusted p <.
- In contrast, PGCs were more likely to be significantly enriched in the nucleoplasm than in chromatin (Fig.
- Transcript maturity was significantly higher for all classes of PCGs, but not for lncRNAs, in the nucleoplasm than in the chromatin fraction, consist- ent with this model (Figure S4E).
- In contrast, PCGs were more com- monly enriched in the polyA-selected fraction (n = 3183 vs n = 2130) (Fig.
- Further, transcript maturity was significantly higher for the chromatin- bound PCG transcripts enriched in the polyA-selected compared to those enriched in the non-polyA-selected fraction (Figure S5E), consistent with the association of poly-adenylation with transcript maturation [43].
- In con- trast, the tendency for lncRNAs to be enriched in the non-polyA-selected fraction is consistent with splicing being delayed or incomplete for lncRNAs [44]..
- Chromatin-bound PCG transcripts enriched in the non- polyA-selected fraction had a significantly longer mean gene length and intron length as compared to PCGs transcripts enriched in the polyA-selected fraction (Fig..
- Finally, top enriched terms for the genes most highly enriched in the polyA-selected chromatin-bound fraction include ribosomal protein, oxidative phosphor- ylation/mitochondria, non-alcoholic fatty liver disease, and mRNA-splicing (Table S2F).
- 64-fold higher relative levels in the non-polyA-selected than in the polyA-selected fraction (Fig.
- All of these lncRNAs show their highest expression in the chromatin-bound, non-polyA-selected fraction across all four treatment groups (Fig.
- Similarly, 23 of the 26 PCGs were most highly expressed in the non-polyA-selected fraction (Fig..
- Comparison of lncRNA levels across fractions revealed more than a 10-fold increase in the number of lncRNAs expressed (fragments per kilobase length per million mapped sequence reads (FPKM) >.
- of the lncRNAs showed sex-biased expression in one or both chromatin-bound fractions, whereas only 18% showed sex-biased expression in the cytosol or nucleoplasm (Table S3A, Fig.
- 0.001 in at least one of the four biological conditions assayed.
- d Distributions of transcript maturity values (normalized IO/EC read density ratios, from Table S1F) in the cytoplasmic and nuclear fractions.
- In d, statistical analysis was used to compare Cyto vs UB, and UB vs Nuc, for lncRNAs and PCGs, based on expression data in the cytoplasm or in the nucleus.
- Figure S6A, S6B), consistent with its strong, male-bias expres- sion seen in the nuclear fractions by RNA-seq (Table S3A).
- Based on our RNA-seq data, lnc7423 is 4–6-fold enriched in the chromatin fraction in both sexes, whereas the female-biased lnc14770 only showed a significant nuclear bias in female liver (22-fold.
- In un- treated male liver, Cyp2b10 expression was very low, with a few RNA molecules detected in the cytoplasm, whereas expression of lnc5998 was essentially undetect- able.
- Integration with prior liver lncRNA expression datasets We integrated the above sets of regulated lncRNAs with prior, published datasets to help identify lncRNAs that are strong candidates for regulatory roles in the liver..
- Figure 7a presents expression data in both sexes across subcellular fractions for eight of these lncRNAs, and highlights the large increases in relative lncRNA levels, and hence the increased sensitivity for detection, in the chromatin-bound fractions.
- 0.001 in at least one of the four biological conditions assayed (Table S2B and Table S2C, columns D and E).
- Seventeen of the 506 lncRNAs show sex-biased expression (Table S3A), and 19 show TCPOBOP-responsiveness (Table S3B) in at least one fraction.
- 1 in at least one of the 5 subcellular fractions.
- Expression of each gene (row) is normalized to the highest expression (or strongest sex-bias) of the gene across all fractions.
- Many also responded to other chemicals that dysregulate gene expression in the liver, including phenobarbital (n = 45 lncRNAs), acetaminophen (n = 27) and agonists of PPARA (either WY14634 or fenofibrate) (n = 33).
- examined (phenobarbital, acetaminophen, WY14634 and fenofibrate), and 282 (84%) were maximally expressed in one of the chromatin-bound fractions, which may explain why they were not identified previously.
- All three lncRNAs show strongly female-biased expression (up to 66-fold) with FPKM values as high as 4.5 – 7.5 in the nucleoplasm or in chromatin (Tables S2A-S2C).
- Slc22a28 could be regulated by the one male-specific DHS found far upstream of Slc22a28 (342 kb away) but in the same inter-TAD re- gion.
- alternatively, the female-biased lncRNA(s) could act via a looping mechanism to silence Slc22a28 expres- sion in female liver, resulting in the observed male- biased expression..
- Of note, a majority of the genes in the TCPOBOP-repressed middle segment are more highly responsive to TCPOBOP in female liver, where 4 of 5 genes show female-biased expression in vehicle-treated liver.
- Solid lines indicate expression level in livers of the dominant sex, and dashed lines indicate expression in the opposite sex.
- Shown at the bottom are four tracks with normalized RNA-seq reads in the nucleoplasmic fraction for vehicle-treated male and female liver on the forward and reverse strands, as indicated.
- Socs2 has several isoforms, and the major transcript in the cytoplasmic, nuclear and nu- cleoplasmic fractions has its TSS within an intra-TAD structure together with the TSS of its divergent, sex- biased lncRNA partner.
- This genomic organization may insulate lncRNA-driven regulation of Socs2 in female liver from other genes elsewhere in the TAD that are not sex-biased.
- Hnf6, a major liver-enriched transcription factor impli- cated in the expression of many liver-specific genes, in- cluding sex-biased genes Fig.
- This repression of One- cut1/Hnf6 RNA was observed in the chromatin fraction, indicating a decrease in of Onecut1/Hnf6 transcription, but surprisingly, it did not result in a decrease in cyto- plasmic Onecut1/Hnf6 RNA (Table S3B).
- Transcripts enriched in the chromatin-bound fraction were the least mature, as indi- cated by a high fraction of sequence reads mapping to introns, while cytoplasmic transcripts were the most ma- ture.
- In contrast to PCGs, lncRNAs were highly enriched in the nucleus, and specifically in the chromatin-bound fraction, rather than the nucleoplasm.
- Using our approach, we found splicing was comparatively low in the chromatin-bound nuclear frac- tion for both lncRNAs and PCGs, independent of polya- denylation selection.
- Further, while PCG transcripts ap- parently became increasingly more mature in moving from chromatin to the nucleoplasm and then on to the cytoplasm, lncRNA transcripts showed less extensive splicing than PCG transcripts in the nucleoplasmic, nu- clear and cytoplasmic compartments, despite the pres- ence of multiple splice sites junctions in many lncRNA transcripts.
- Comparing relative transcript levels (relative transcript concentrations) between subcellular fractions, we found that many liver-expressed lncRNAs are highly enriched in the nuclear and chromatin-bound fractions and are substantially depleted from the cytoplasm and the nu- cleoplasm.
- Overall, 92% of liver lncRNAs showing sig- nificant differential enrichment between cytoplasm and nucleus were enriched in the nucleus, and 99% of lncRNAs differentially expressed between the chromatin fraction and nucleoplasm were chromatin-enriched.
- This strong apparent enrichment of lncRNAs for chromatin binding likely involve multiple mechanisms, ranging from enhanced lncRNA degradation in the nucleoplasm or cytoplasm to active lncRNA retention in the chroma- tin fraction.
- These genes fall within a sex-independent intra-TAD loop (black) with several female-biased DHS (pink) in the promoter regions of both genes.
- Chromatin-bound lncRNAs may act in cis at sites in the genome close to their transcrip- tion [55], but some may transit through the nucleoplasm and be trans-acting [8].
- Indeed, many of the lncRNAs enriched in the chromatin fraction were also present in the nucleoplasm at significant concentrations, which could allow them access to multiple trans sites within the nucleus.
- Finally, we note that the subcellular fraction enrichments presented here, which are based on relative transcript concentrations, do not equate with absolute localizations, as they do not take into account differ- ences in the total amount of polyA RNA in each fraction [21].
- 60-fold greater abundance in the non-polyA-selected fraction (Fig.
- Moreover, PCGs enriched in the cytoplasm were more extensively spliced in both the cytoplasm and the nucleus than their nuclear-enriched PCG counterparts.
- their enrichment in the non-polyadenylated frac- tion can thus be explained by the longer times required for gene transcription and splicing as compared to shorter, lower intronic content PCGs.
- These events are thought to aid in the stress response by stockpiling mRNAs for rapid release [97] and may also minimize fluctuations in protein levels due to bursty transcription [98]..
- Many of these lncRNA gene regulatory responses were observed in the chromatin-bound RNA fractions, consistent with both processes being regulated at the transcriptional level.
- For many lncRNAs, the highest level of expression was often seen in the chromatin fraction, which increased the sen- sitivity for their detection and helps explain why a subset of these regulated lncRNAs were not identified in earlier whole liver or total nuclear RNA-seq analyses.
- These and other putative regulatory lncRNAs may be investigated using a variety of experi- mental and computational approaches in- cluding innovative knockout technologies [36] that may uncover their biological functions and gene targets in the liver..
- 20 °C in the cooling chamber of a Leica CM1950 cryo- stat.
- At the start of the next day, a water bath and an Advanced Cell Diagnostics HybEZ oven were set to 40 °C after placing Advanced Cell Diagnostics humidity paper soaked in distilled water in the oven’s humidity control tray.
- After 30 min, excess liquid was flicked off each slide and the slides were washed in 1x PBS twice by submerging the rack in the PBS wash 3–5 times.
- Four drops of the above described probe mixture was added to each slide and the rack was placed in the oven for 2 h at 40 °C.
- Due to the high auto-fluorescence of liver tissue in the green spectrum, we visualized the most highly expressed RNAs in Channel 1 and the less highly expressed RNAs in Channel 2.
- #8961, Cell Signaling Technology) was placed in the cen- ter of the slide.
- A coverslip was added and the slide stored in the dark at 4 °C..
- Nuclei were counted manually for each image due to the variation of DAPI staining in the nucleus caused by euchromatin and heterochromatin..
- The MultiOverlap op- tion of featureCounts was enabled by using the –O op- tion, so that reads that overlap two or more genes in a GTF file were included in the counts for each gene.
- The rela- tionship between Gene Body (GB) read counts and read counts mapping to Exon Collapsed (EC) regions plus those that map to Intronic Only (IO) regions is de- scribed in the legend to Table S1B-S1C and in data presented in those tables..
- Im- portantly, these analyses identified transcripts that show significant differential enrichment in the indicated sub- cellular fraction, but do not imply that a given transcript is exclusively present in that fraction.
- sex-biased or TCPOBOP-responsive if it met an edgeR- adjusted p-value cutoff of 0.05 for differential expression in at least one of the five fractions..
- Class 1 lncRNAs are those that, following hypophysectomy, show decreased expression in the sex where the lncRNA is more highly expressed in intact mouse liver.
- In con- trast, Class 2 lncRNAs increase in expression following hypophysectomy in the sex where they show lower expression in intact mice [26].
- RNA sequencing reveals age and species differences of constitutive Androstane receptor-targeted drug-processing genes in the liver.
- Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs.
- Structure and function of the type 3 deiodinase gene..
- Macro lncRNAs: a new layer of cis-regulatory information in the mammalian genome

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