Nuclear transcriptome profiling of induced pluripotent stem cells and embryonic stem cells identify non-coding loci resistant to reprogramming

<p>Identification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified super-enhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific super-enhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous <i>cis</i>-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications.</p

Late-Onset of Spinal Neurodegeneration in Knock-In Mice Expressing a Mutant BiP

<div><p>Most human neurodegenerative diseases are sporadic, and appear later in life. While the underlying mechanisms of the progression of those diseases are still unclear, investigations into the familial forms of comparable diseases suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis. Binding immunoglobulin protein (BiP) is an ER chaperone that is central to ER function. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence in order to evaluate the effect of a functional defect in an ER chaperone in multi-cellular organisms. Here we report that heterozygous mutant BiP mice revealed motor disabilities in aging. We found a degeneration of some motoneurons in the spinal cord accompanied by accumulations of ubiquitinated proteins. The defect in retrieval of BiP by the KDEL receptor leads to impaired activities in quality control and autophagy, suggesting that functional defects in the ER chaperones may contribute to the late onset of neurodegenerative diseases.</p></div

Motoneurons at the anterior horn of spinal cords of aged mutant BiP mice suffer from ER stress.

<p>(A) Motoneurons stained by an anti-choline acetyltransferase antibody (red) at the anterior horn in the spinal cord of both a 6 month-old wild type (+/+, 6 m) and a 6 month-old mutant BiP mouse (Bm/+, 6 m) express ER chaperones as well (green). Scale bars, 20 um. (B) The immunoreactivity with an anti-choline acetyltransferase antibody at the anterior horn is reduced in the aged 29 month-old mutant spinal cord (Bm/+, 29 m). Scale bars, 20 um. (C) Large cells at the anterior horn of the aged 29 month-old mutant spinal cord (Bm/+, 29 m) express ER chaperones as well as CHOP. Scale bars, 10 um. The nuclei were stained with Hoechst 33258 (blue, A and C).</p

Some motoneurons in the spinal cord revealed a degeneration accompanied by accumulations of ubiquitinated proteins.

<p>(A) TUNEL staining revealed some apoptotic cells at the anterior horn in the spinal cord of a 29 month-old mutant BiP mouse (Bm/+, 29 m). Scale bars, 10 um. (B) The immunoreactivity with an anti-GFAP antibody at the anterior horn is increased in a 17 month-old mutant spinal cord (Bm/+, 17 m). Scale bars, 20 um. GFAP positive cells are counted (five fields in each mouse, GFAP positive cells/the number of nucleus). +/+, 16 m; 33/199, 35/174, 34/192, 40/181, 42/207, Bm/+, 17 m; 42/132, 50/140, 59/147, 39/154, 58/143 +/+, 6 m; 5/143, 4/131, 4/141, 0/95, 0/107. The ratio of GFAP positive cells is significantly higher in the mutant BiP spinal cord (Bm/+, 17 m) compared to that in the wild type (+/+, 16 m) by t test (p value is 0.0009). (C) The aggregations were stained by an anti-ubiquitin antibody in the large cells at the anterior horn of the 29 month-old mutant spinal cord (Bm/+, 29 m, arrowheads). Scale bars, 10 um.</p

The expressions of chaperones in the mutant BiP mice.

<p>The heterozygous mutant BiP mice and the litter mate wild type mice were anesthetized by pentobarbital, and the brains and spinal cords were removed. They were subjected to Western blot analysis with an anti-KDEL mouse mAb for BiP and GRP94, an anti-HA mouse mAb for mutant BiP, an anti-CHOP rabbit antiserum, and an anti-SOD1 rabbit antiserum.</p

Aggregations were obvious in the mutant BiP MEF.

<p>The aggregations by transient expressions of SOD1-GFP were evaluated by immunofluorescence microscopy with labeling by using a rabbit anti-Derlin1 antibody for the ER staining (red) and SOD1-GFP (green) in wild type (+/+) and the homozygous mutant (Bm/Bm) MEF with Hoechst 33342 for nuclear staining. Scale bars, 10 um. Aggregations of SOD1were observed in the mutant BiP MEF as well as in the wild type MEF treated with a proteasome inhibitor, ALLN (10 ug/ml), at 37°C for 12 h.</p

Methylation of the <i>A^vy</i> Allele following Paternal Transmission

<div><p>The methylation status of each CpG dinucleotide was determined by sequencing PCR clones of bisulfite-converted DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. Each line represents an individual clone, theoretically from one cell, and each circle an individual CpG. Open circles indicate an unmethylated CpG, and closed circles a methylated CpG. Each block of lines represents the clones derived from the sperm of one adult male mouse, one set of ten zygotes, ten two-cell embryos or one blastocyst. The percentage of methylation in each dataset is shown (calculated from the number of methylated CpGs divided by the total CpGs sequenced, multiplied by 100). The position of each circle is representative of the relative location along the length of the PCR product. Any clones with greater than 5% non-CpG methylation were excluded from the dataset, and these clones made up less than 5% of all clones sequenced. These clones tended to have very high levels of non-CpG methylation, an indication of incomplete bisulfite conversion. Zygotes, two-cell embryos, or blastocysts were collected from yellow or pseudoagouti <i>A^vy/a</i> sires mated to <i>a/a</i> dams, as indicated. Clones were only included in the zygote or blastocyst samples if they could be distinguished from others in the sample by CpG or low-level non-CpG methylation.</p><p>(A) The IAP LTR pseudoexon 1a junction shown in detail. The bisulfite sequencing primers are shown [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. The PCR product contains 11 CpG dinucleotides, depicted as circles, all of which are in the LTR.</p><p>(B) Data obtained from the sperm of four pseudoagouti males, and 10 × 10 zygotes, 10 × 10 two-cell embryos or 10 × 1 blastocysts collected from pseudoagouti <i>A^vy/a</i> sires mated to <i>a/a</i> dams.</p><p>(C) Data obtained from the sperm of four yellow male and 9 × 1 blastocysts collected from yellow <i>A^vy/a</i> sires mated to <i>a/a</i> dams. These data indicate that the <i>A^vy</i> allele is subject to rapid demethylation immediately postfertilisation following paternal transmission.</p></div

Methylation of the <i>A^vy</i> Allele in Mature Sperm

<p>(A) Expression of the <i>A^vy</i> allele is controlled by an IAP, inserted into pseudoexon 1a (grey box). A cryptic promoter within the 3′ LTR of the IAP (black arrows) directs transcription of the <i>agouti</i> coding exons. The BamHI (B) and MspI (M) sites are shown in the region of the unique 400-bp probe B. Tail and mature sperm from a yellow and a pseudoagouti male were collected. DNA was prepared and samples digested with BamHI followed by MspI or its isoschizomer HpaII, transferred and hybridised with the <i>agouti</i> probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b003" target="_blank">3</a>]. The <i>A^vy</i> allele produces a 9-kb BamHI band, while the <i>a</i> allele produces a 3.3-kb band. Membranes were stripped and rehybridised with a murine <i>α-globin</i> probe to check for equal digestion within the tissue samples (shown in [B]). These results represent experiments performed on sperm and tail DNA from seven yellow and five pseudoagouti males, a further one of each are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-sg001" target="_blank">Figure S1</a>. Mature sperm were isolated from both epididymes of the male (each sample contained in the order of 10⁶ to 10⁷ spermatocytes). Sperm samples were checked by light microscopy and found to be greater than 95% spermatocytes. The methylation state of the tissues is indicated by the ratio of the 9-kb BamHI band to the 7-kb band remaining after HpaII digestion. The 9-kb band is marked by an asterisk. The methylation state of the sperm reflects the phenotype of the father rather than the range of phenotypes seen in the offspring.</p

Methylation of the <i>A^vy</i> Allele in 12.5-dpc Embryos following Paternal Transmission

<p>The 12.5-dpc embryos produced from an <i>A^vy/a</i> sire mated with an <i>a/a</i> dam. Samples were digested and subjected to Southern transfer as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-g001" target="_blank">Figure 1</a>. A range of methylation states were observed, evidenced by the varying amounts of the 9-kb BamHI band remaining after HpaII digestion. This indicates that the methylation is likely to be reset by this stage of development.</p

ubH2A signal decreased when both USP21 variants are over-expressed.

<p>Hela cells were transfected either with EGFP-USP21SV (A–H, M–P) or EGFP-USP21LV (I–L, Q–T) for 24 hours. Anti-lamin antibody was used for immunofluorescence identification of the nucleus (A, E, I) and anti-tubulin antibody was used for immunofluorescence identification of the cytoplasm (M, Q). Anti ubH2A antibody was used to evaluate nuclear ubH2A (G, K, O, S). Control IgG was used as a negative control for immunofluorescence of ubH2A (C). The merged image illustrates the relationship of either EGFP-USP21SV (H, P) or EGFP-USP21LV (L, T) expression and ubH2A. Merging of Control and GFP showed features of the cell with GFP but without H2A ubiquitylation (D). Scale bar indicated 50 μm.</p