X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention

Mid-gestational gene expression profile in placenta and link to pregnancy complications.

Despite the importance of placenta in mediating rapid physiological changes in pregnancy, data on temporal dynamics of placental gene expression are limited. We completed the first transcriptome profiling of human placental gene expression dynamics (GeneChips, Affymetrix®; ~47,000 transcripts) from early to mid-gestation (n = 10; gestational weeks 5-18) and report 154 genes with significant transcriptional changes (ANOVA, FDR P<0.1). TaqMan RT-qPCR analysis (n = 43; gestational weeks 5-41) confirmed a significant (ANOVA and t-test, FDR P<0.05) mid-gestational peak of placental gene expression for BMP5, CCNG2, CDH11, FST, GATM, GPR183, ITGBL1, PLAGL1, SLC16A10 and STC1, followed by sharp decrease in mRNA levels at term (t-test, FDR P<0.05). We hypothesized that normal course of late pregnancy may be affected when genes characteristic to mid-gestation placenta remain highly expressed until term, and analyzed their expression in term placentas from normal and complicated pregnancies [preeclampsia (PE), n = 12; gestational diabetes mellitus (GDM), n = 12; small- and large-for-gestational-age newborns (SGA, LGA), n = 12+12]. STC1 (stanniocalcin 1) exhibited increased mRNA levels in all studied complications, with the most significant effect in PE- and SGA-groups (t-test, FDR P<0.05). In post-partum maternal plasma, the highest STC1 hormone levels (ELISA, n = 129) were found in women who had developed PE and delivered a SGA newborn (median 731 vs 418 pg/ml in controls; ANCOVA, P = 0.00048). Significantly higher expression (t-test, FDR P<0.05) of CCNG2 and LYPD6 accompanied with enhanced immunostaining of the protein was detected in placental sections of PE and GDM cases (n = 15). Our study demonstrates the importance of temporal dynamics of placental transcriptional regulation across three trimesters of gestation. Interestingly, many genes with high expression in mid-gestation placenta have also been implicated in adult complex disease, promoting the discussion on the role of placenta in developmental programming. The discovery of elevated maternal plasma STC1 in pregnancy complications warrants further investigations of its potential as a biomarker

Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders

Abstract Background Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta (TOP2B) facilitates rapid gene expression and functions at the later stages of development and differentiation. To gain new insight into the genome biology of TOP2B, we used proteomics (BioID), chromatin immunoprecipitation, and high-throughput chromosome conformation capture (Hi-C) to identify novel proximal TOP2B protein interactions and characterize the genomic landscape of TOP2B binding at base pair resolution. Results Our human TOP2B proximal protein interaction network included members of the cohesin complex and nucleolar proteins associated with rDNA biology. TOP2B associates with DNase I hypersensitivity sites, allele-specific transcription factor (TF) binding, and evolutionarily conserved TF binding sites on the mouse genome. Approximately half of all CTCF/cohesion-bound regions coincided with TOP2B binding. Base pair resolution ChIP-exo mapping of TOP2B, CTCF, and cohesin sites revealed a striking structural ordering of these proteins along the genome relative to the CTCF motif. These ordered TOP2B-CTCF-cohesin sites flank the boundaries of topologically associating domains (TADs) with TOP2B positioned externally and cohesin internally to the domain loop. Conclusions TOP2B is positioned to solve topological problems at diverse cis-regulatory elements and its occupancy is a highly ordered and prevalent feature of CTCF/cohesin binding sites that flank TADs

Glycoprotein hormone STC1 protein levels in maternal blood plasma.

<p>STC1 protein levels in maternal plasma from uncomplicated pregnancies (defined as controls) compared to (A) pregnancies resulting in the birth of small-for-gestational-age (SGA) and large-for-gestational-age (LGA) newborns, as well as pregnancies complicated with preeclampsa (PE) or gestational diabetes mellitus (GDM); (B) cases with PE and (C) GDM grouped by newborn birth weight. Median values are indicated by horizontal bars. Plotted values are represented with no adjustment for confounding effects. Statistical differences between controls and each of the patient groups were assessed by accounting for confounding factors with ANCOVA. Statistical tests were adjusted for newborn birth-weight (initial analyses of PE and GDM cases), gestational age (SGA, LGA, PE), mode of delivery and mother’s weight (all groups), height (PE, except when grouped by newborns birth weight) and age (GDM). The adjusted <i>P</i>-values are given above the data point of the respective study group.</p

Molecular functions of identified mid-gestation marker genes in placenta and their involvement in clinical conditions.

a<p>References are listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049248#pone.0049248.s018" target="_blank">Table S9</a></b>.</p

The immunostaining of CCNG2 and LYPD6 proteins was assessed in placental sections from term pregnancies with no complications (controls), with preeclampsia (PE) or with gestational diabetes mellitus (GDM).

<p>(A) Hematoxylin-eosin staining was used to describe histopathological findings in analyzed placental samples (100-fold microscope magnification). In term placentae the mature intermediate (IMV) and small terminal villi (STV) were seen. Characteristic to PE, villous agglutination and infarction (IN; intense eosinophilic staining) and increased number of syncytial knots (SK) were detected. GDM presented with degenerative placental lesions such as focal villous fibrinoid necrosis (FN). (B) Diffuse cytoplasmic staining of LYPD6 antibody was detected in syncytiotrophoblast (ST) cells in all villous types (IMV, STV). Additionally LYPD6 antibody strongly stained the cytoplasm and the nucleus of villous stroma Hoffbauer cells (H), fibroblasts (F) and endothelial cells (E) of villous vessels. No localization differences in LYPD6 antibody stain between the groups were found; however strong tendency to higher staining intensity was observed in PE and GDM placentas compared to normal term placenta. (C) CCNG2 antibody showed fine granular cytoplasmic staining of villous stromal Hoffbauer (H) and fibroblast (F) cells. In addition, weak cytoplasmic staining of syncytiotrophoblast (ST) and endothelial cells (E) of vessel wall was found. No localization differences in CCNG2 staining between the normal, PE and GDM groups were detected. Higher tendency to positivity was seen in PE placental sections. (D) Negative control (NC) staining was performed without primary antibody. Scale bar, 100 µm. Microscope magnifications ×100 and ×400 were used. Brown color indicates chromogen-labeled antibody and blue color indicates hematoxylin nuclear staining.</p

Genes with significant dynamic increase in expression during early and mid-gestation as experimentally confirmed by RT-qPCR.

<p>Relative mRNA expression levels in the extended sample set of first and second trimester placentas (<i>n</i> = 31; from gestational days 38 to 147) were determined by TaqMan assays. <i>P-</i>values were calculated by ANOVA and subjected to multiple testing correction (FDR). Genes below the significance threshold of <i>P</i>-value>0.02 are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049248#pone.0049248.s007" target="_blank">Figure S4</a></b>.</p

Expressional dynamics of identified mid-pregnancy specific genes in placenta from early to term gestation.

<p>Relative mRNA levels were determined by RT-qPCR TaqMan assays in placental tissues from early- (5–13 gestational weeks; <i>n</i> = 23), mid- (17–21 gestational weeks; <i>n</i> = 8) and term-gestation (36–41 gestational weeks; <i>n</i> = 12) samples of uncomplicated pregnancy cases. Boxplots show mid-gestation marker genes with (A) significantly increased mRNA expression compared to early- or late-gestation placental samples, (B) significantly increased expression levels compared to early gestation placental samples, and (C) gradual increase in expression during pregnancy. <i>P</i>-values were calculated by Student t-test and subjected to multiple testing correction (FDR) (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049248#pone.0049248.s016" target="_blank">Table S7</a></b>).</p

Placental genes with considerable expressional change in progression from early to mid-pregnancy.

<p>Ten placental tissue samples subjected to dynamic linear transcriptome expression profiling (Affymetrix HG-U133 plus 2.0 GeneChips) represented gestatational weeks of 5 (38 days), 8 (days 55, 56, 56), 11 (81 days), 13 (91 days), 17 (days 120, 121) and 18 (days 126, 132). The heatmap shows 154 genes (180 probe-sets) with significant gradual increase (red) or decrease (blue) in placental transcription in first and second trimester placentas (ANOVA, FDR corrected <i>P</i><0.1). Color intensity reflects mean log2 fold change in gene expression with one first trimester sample (week 5; 38 gestational days) as reference. For the placental samples collected at close gestational age, log2 fold change calculations represent the median values of 2–3 samples (55/56/56; 120/121; 126/132 gestational days). Hierarchical clustering with Euclidean distance, visualized by the dendrogram on the left, clearly separated genes with increased and decreased expression. Colorstrip on the right of the heatmap highlights genes with strongest statistical significance of differential expression. A subset 24 genes selected for further experiments are labeled on the vertical axis.</p