Hematological results from primary blood samples (n = 10) that were used to derive blood preparations.

<p>Hematological results from primary blood samples (n = 10) that were used to derive blood preparations.</p

Hematological results from primary blood samples (n = 10) that were used to derive blood preparations.

<p>Hematological results from primary blood samples (n = 10) that were used to derive blood preparations.</p

Scatter plot of the change of mtDNAcn(WB) per unit increase of the platelets/leukocyte ratio.

<p>This scatter plot shows the change of mtDNAcn(WB) per unit increase of the platelets/leukocyte ratio. Data from 46 preparations are shown. Individual mtDNAcn(WB) for each preparation are plotted as white circles. The line represents the fit of a linear model (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163770#pone.0163770.t003" target="_blank">Table 3</a>, Model 6). mtDNAcn(WB): mitochondrial DNA copy number measured in whole blood.</p

Coefficients of multivariate regression models for mtDNAcn(WB) measured in blood preparations (n = 46).

<p>Coefficients of multivariate regression models for mtDNAcn(WB) measured in blood preparations (n = 46).</p

Platelet count, Leukocyte count and mtDNAcn(WB) distributions in all subjects from the AWHS cohort study (n = 3389).

<p>Platelet count, Leukocyte count and mtDNAcn(WB) distributions in all subjects from the AWHS cohort study (n = 3389).</p

Transcriptional changes in <i>Ctcf</i> mutant hearts.

<p>(A) Iris plot showing enriched GO terms in differentially expressed genes between control and mutant E10.5 hearts (outside circle), together with changes in expression of individual genes (inner circle; red, upregulated in mutants; blue, downregulated). (B) Genes that are up- or downregulated in <i>Ctcf</i> mutant hearts are more likely to have a CTCF binding site in their vicinity (10 or 20 kb surrounding the transcriptional start site) than genes that are expressed in the embryonic heart but do not change. However, only downregulated genes are located closer to heart-specific enhancers. Frequency is expressed relative to that of expressed but unchanged genes. *, p<0.01; **, p<0.0005; Mann-Whitney test. (C) Mean-plots of the distribution of CTCF binding, as determined by ChIP-seq [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006985#pgen.1006985.ref010" target="_blank">10</a>], relative to the TSS (0 on the x-axis) of different groups of genes (legend on the right). Note that the peak of the distributions is located slightly upstream of the TSS, indicative of binding to proximal promoter sequences.</p

Morphological defects in <i>Ctcf</i> mutant embryonic hearts.

<p>Whole mount control and mutant (<i>Ctcf</i>^{<i>fl</i>/<i>fl</i>};<i>Nkx2-5-Cre</i>) embryos at E10.5 (A, B), E11.5 (C, D) and E12.5 (E, F). Arrows in (E) point to the pericardial edema present in mutant embryos. Higher magnification of the heart show morphological defects in <i>Ctcf</i> mutant hearts becoming manifest at E12.5 (F). (G-L) Sections from control and mutant embryos stained with hematoxylin and eosin; higher magnifications (black dashed boxes) for each are shown on the right of each section. Disorganization of the interventricular septum and thinning of the myocardial wall is apparent in <i>Ctcf</i> mutant hearts at E11.5. A, atria; V, ventricle; RA, right atria; LA, left atria; RV, right ventricle; LV, left ventricle; AVC, atrioventricular canal; IVS, interventricular septum. Scale bars, 750 μm (A), 200 μm (B, D), 1 mm (C), 2 mm (E), 800 μm (F), 100 μm (G, H), 200 μm (I-L), and 100 μm in all higher magnifications.</p

The cardiac developmental program is abrogated in <i>Ctcf</i> mutant hearts.

<p>(A) Deletion of <i>Ctcf</i> leads to downregulation of genes encoding key cardiac developmental transcription factors and signaling-pathway components. The heatmap shows normalized gene expression values from the RNA-seq data for controls (<i>Ctcf</i>^<i>fl/+</i>), heterozygotes (<i>Ctcf</i>^<i>fl/+</i>;<i>Nkx2</i>.<i>5-Cre</i>) and homozygotes (<i>Ctcf</i>^<i>fl/fl</i>;<i>Nkx2</i>.<i>5-Cre</i>) across three biological replicates each. (B-E) <i>In situ</i> hybridization in sections of E10.5 control and mutant (<i>Ctcf</i>^{<i>fl</i>/<i>fl</i>};<i>Nkx2-5-Cre</i>) hearts for <i>Nkx2</i>.5 (B), <i>Hopx</i> (C), which are strongly downregulated; and <i>Nppa</i> (D) and <i>Pitx2</i> (E), which lose expression in the left ventricle (brackets and asterisks in D) or right ventricle (arrow in E), respectively. (F, G) Genomic region containing <i>Tnnt1</i> and <i>Tnni3</i> (F; mm9, chr7:4,433,080–4,493,651) or <i>Tnni2</i> and <i>Tnnt3</i> (G; mm9, chr7:149,623,608–149,703,181). Troponin genes are highlighted in black. (H-K) <i>In situ</i> hybridization in sections of E10.5 control and mutant hearts for <i>Tnnt1</i> (H), <i>Tnni3</i> (I), <i>Tnni2</i> (J) and <i>Tnnt3</i> (K). RA, right atria; LA, left atria; RV, right ventricle; LV, left ventricle; AVC, atrioventricular canal; IVS, interventricular septum. Scale bar, 200 μm.</p

<i>Ctcf</i> deletion causes global changes in the expression of the <i>IrxA</i> cluster in the developing heart.

<p>(A) Diagram of the extended <i>IrxA</i> cluster showing the location of <i>Irx1</i>, <i>Irx2</i>, <i>Irx4</i> and the neighboring genes <i>Ndufs6</i>, <i>Mrpl36</i> and <i>Lpcat1</i> (not to scale). (B-J) <i>In situ</i> hybridization in sections of control and mutant hearts for <i>Irx4</i> at E9.5 (B), E10.5 (C) and E11.5 (D); and <i>Irx1</i> (E), <i>Irx2</i> (F) and <i>Ndufs6</i> (G) at E11.5. (H-I) Higher magnifications (black dashed boxes) of <i>Irx1</i> (H), <i>Irx2</i> (I) and <i>Irx4</i> (J) expression in control and mutant hearts at E11.5. Arrows indicate extension in the expression territories of <i>Irx1</i> (H) and <i>Irx2</i> (I). A,atria; V, ventricle, RA; right atria; LA, left atria; RV, right ventricle; LV, left ventricle; AVC, atrioventricular canal; IVS, interventricular septum. Scale bars, 100 μm (B), 200 μm (C-J).</p

Defective mitochondrial biogenesis upon CTCF loss.

<p>Western blot of Cox I, Cox IV (A), Grp75, Tfam and Tom20 (B) of control and mutant (<i>Ctcf</i>^{<i>fl</i>/<i>fl</i>};<i>Nkx2-5-Cre</i>) hearts at E10.5 and E11.5. (C) Blue native gel of control and mutant hearts at E10.5 and E11.5 showing abundance and distribution of mitochondrial respiratory complexes (CI, CIV, CVm) and supercomplexes (CI+CIII). (D) Densitometry of blue native gel quantifying changes between E10.5 and E11.5 in complexes and supercomplexes, in control and mutant hearts. (E) Transmission Electron Microscopy at E10.5 and E11.5 in control and mutant cardiomyocytes. n, nucleus; sm, sarcomere. Arrowheads point to mitochondria. Scale bar, 1 μm top row, 0.5 μm bottom row.</p