Additional file 1: Table S1. of CSF complement 3 and factor H are staging biomarkers in Alzheimer’s disease

Demographic and biomarker information for cognitive normal subjects and MCI patients with CSF t-Tau/Aβ42 < 0.39 in the ADNI and Emory cohorts. Table S2. Main effects from mixed linear modeling of CSF FH and C3 levels in ADNI. In each model, FH or C3 was entered as the dependent variable; age, gender, presence of APOE ε4 allele, diagnosis, Aβ42, t-Tau, p-Tau181, gender X age, presence of APOE ε4 allelle X age, and diagnosis X age were entered as fixed factors; and age was also entered as a random factor. Factors with main effect p > 0.10 were removed in a step-wise fashion to arrive at final model. See text and Fig. 2 for effects from different diagnostic categories. Table S3. Mixed linear modeling of PAD-based diagnostic classification and time (in months) on longitudinal memory and executive functions in the Emory validation cohort. A) Among patients initially classified as MCI with longitudinal follow-up (n=44), reclassification using PAD showed differences in absolute executive Z-scores between those reclassified as likely MCI vs. likely mild AD, but no difference in rates of executive function decline or memory functions. B) Among patients initially classified as mild AD with longitudinal follow-up (n=10), only time was associated with longitudinal memory and executive function decline in this underpowered subgroup. (DOCX 21 kb

High-Resolution Mapping of H1 Linker Histone Variants in Embryonic Stem Cells

<div><p>H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts <i>in vivo</i>. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1⁰ displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.</p> </div

H1 depletion leads to chromocenter clustering.

<p>(A) Typical images of WT (top), H1 TKO (middle), and RES ESCs (bottom) of FISH with a major satellite probe (left), DNA stain DAPI (middle), and merged images (right). Scale bar: 10 µm. (B) Box plots of chromocenter numbers in the nuclei of WT, H1 TKO, and RES ESCs. The line in the box indicates the median, while the bottom and top of the boxes are the 25^th and 75^th percentiles, respectively. ****: P<0.000001.</p

Increased nucleosome repeat length at major satellite repeats in ESCs.

<p>(A) Nucleosome repeat length analyses of bulk chromatin (left), major satellite sequences (middle) and minor satellite sequences (right) in WT ESCs. DNA isolated from ESC nuclei digested with MNase at different time points were analyzed by ethidium bromide (EB) –stained gel (left), transferred to membrane which was sequentially probed with major satellites (middle) and minor satellites (right) using Southern blotting. The positions of di-nucleosomes with 10-minute MNase digestion are marked by *. The dashed line indicates di-nucleosome position of major satellites, which is higher than that of bulk chromatin and minor satellites. (B) The NRLs were calculated from the images presented in (A) by extrapolating the corresponding curves to time “0” as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003417#pgen.1003417-Gilbert2" target="_blank">[72]</a>.</p

Generation of H1d^FLAG knock-in ESCs.

<p>(A) Schematic representation of the H1d^FLAG targeting construct and the knock-in strategy for insertion of the FLAG tag at N-terminus of the endogenous H1d gene. (B) Identification of ESC clones containing the modified FLAG-H1d allele. DNA isolated from Blasticidin resistant ESC clones were analyzed by Southern blotting. <i>Cis vs. trans</i> configurations of the homologous recombination events are schematically illustrated in the diagram above the Southern blotting image. (C) Reverse phase HPLC profiles of histone extracts from ce^het (left panel) and <i>cis</i>-targeted H1d^FLAG ESCs (right panel). mU, milliunits of absorbency at 214 nm. (D) Coomassie staining and Western blotting analysis of individual H1 fractions eluted from HPLC of histone extracts of ce^het (1) and H1d^FLAG (2) ESCs. (E) Calculated ratio of each H1 variant (and total H1) to nucleosome of ce^het and H1d^FLAG ESCs.</p

H1 depletion leads to increased expression of major satellite repeats independent of multiple epigenetic marks.

<p>(A) Analyses of expression of selected repeats in WT, H1 TKO, and RES ESCs by qRT-PCR. Data are represented as mean +/− S. D.. *: P<0.05; **: P<0.01. (B) qChIP analysis of three repressive histone marks and one active histone mark at selected repetitive sequences in WT and H1 TKO ESCs. Dashed lines indicate the highest level of signals detected by qChIP with IgG antibody. (C) Bisulfite sequencing analysis (i) and percent of methylated CpG (ii) of major, minor satellite sequences. The positions of CpG sites analyzed are marked as vertical ticks on the line.</p

H1d and H1c are enriched at the major satellite sequences.

<p>(A) Representative profiles of top H1d and H1c enriched regions (mapped to mm9). Repeat element tracks were obtained from UCSC genome browser. Dashed lines indicate the localization of these H1 peaks at major satellite sequences. (B) Fold enrichment of percent mappable repeats (mapped to RepBase) from H1d, H1c, and histone marks ChIP-seq libraries over that from corresponding chromatin input-seq library on all repeats (left), six most abundant repetitive sequences and the remaining other repeats (right). The dashed lines indicate the level of normalized input signal. P values calculated with Fisher's exact test comparing ChIP-seq with input-seq libraries are less than 2.5×10⁻⁵ for all repeat classes shown. Error bars represent the differences between replicates. Data are presented as average ± S.E.M.</p