Histone H1 Favors Folding and Parallel Fibrillar Aggregation of the 1–42 Amyloid‑β Peptide

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases of the central nervous system. The aggregation of the amyloid-β peptide, Aβ(1–42), is believed to play an important role in the pathogenesis of AD. Histone H1 is found in the cytoplasm of neurons in AD, and it has been shown to interact with aggregated amyloid-β peptides and with amyloid fibrils. We have used Thioflavin T (ThT) fluorescence enhancement, circular dichroism spectroscopy (CD), coprecipitation, and transmission electron microscopy (TEM) to study the interaction of histone H1 with Aβ(1–42). Both freshly prepared (monomeric) Aβ(1–42) and histone H1 solutions showed negative CD bands typical of the random coil. Mixing Aβ(1–42) and histone H1 led to the loss of the random coil, which was replaced mostly by β-structure. Therefore, both Aβ(1–42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Mutual structure induction demonstrates the interaction of Aβ(1–42) and histone H1. The interaction was confirmed by coprecipitation followed by SDS-PAGE. Mutual structure induction was also observed with the H1 terminal domains. Incubation of Aβ(1–42) for 1 week in the presence of histone H1 led to the formation of laminar aggregates and thick bundles, characterized by the parallel association of large numbers of fibrils. The aggregates were particularly large and ordered with the H1 subtype H1.2. Further aging of the complexes led to tight compaction of fibril bundles and to fiber growth. Stabilization of fibril–fibril interactions appeared to be determined by the C-terminal domain of histone H1. In summary, these observations indicate that histone H1 has at least two effects: it helps the folding of Aβ monomers and stabilizes the parallel association of fibrils

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin-1

<p><b>Copyright information:</b></p><p>Taken from "Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin"</p><p>http://www.biomedcentral.com/1741-7007/5/22</p><p>BMC Biology 2007;5():22-22.</p><p>Published online 11 May 2007</p><p>PMCID:PMC1890542.</p><p></p>. Displaced H1 formed an insoluble complex with the SAR that was separated by centrifugation. The inset shows the H1 remaining on the chromatin. The experimental points were fitted to a hyperbolic function. A plot of [H1Chr]/[Chr]on the ordinate and [H1SAR]/[SAR]on the abscissa gives a straight line. The slope of the line corresponds to /. The points are the average of three experiments. The bars show the interval spanned by the extreme values

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin-2

<p><b>Copyright information:</b></p><p>Taken from "Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin"</p><p>http://www.biomedcentral.com/1741-7007/5/22</p><p>BMC Biology 2007;5():22-22.</p><p>Published online 11 May 2007</p><p>PMCID:PMC1890542.</p><p></p>8 μg of SAR DNA and ~3 μg of one of the subtypes H1a-e and H1°. This amount of added H1 corresponds to ~0.4 molecules per nucleosome. The insoluble complex of SAR and H1 (p) was separated by centrifugation from chromatin (s) with its H1 complement redefined. Perturbation experiments with H1a, H1c and H1° were analyzed only by SDS gel electrophoresis. Experiments with H1b, H1d and H1e were analyzed by SDS and urea/acetic acid gel electrophoresis. Chromatin inputs analyzed by SDS and AU gel electrophoresis are shown (i). m, mixture of H1 subtypes separated by AU gel electrophoresis. The subtypes H1a-e and H1° are indicated (a-e, 0). CH, core histones

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin-0

<p><b>Copyright information:</b></p><p>Taken from "Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin"</p><p>http://www.biomedcentral.com/1741-7007/5/22</p><p>BMC Biology 2007;5():22-22.</p><p>Published online 11 May 2007</p><p>PMCID:PMC1890542.</p><p></p>. This weight ratio is equivalent to ~0.09 H1 molecules per base pair or ~3 H1 molecules per DNA binding site (assuming a DNA binding site of 33 base pairs [42]). The whole pellet and 1/3 of the supernatant were run on the gel. The subtypes H1a-e and H1° are indicated on the left (a-e, 0). SAR, scaffold attachment region from the histone cluster (657 bp); pUC19, HaeIII/HaeIII fragment from pUC19 (587 bp); i, input mixture of subtypes; p, pellet; s, supernatant. Competition between three subtypes. Competition with a protein/DNA ratio of 10:1 (w/w), equivalent to ~10 H1 molecules per DNA binding site. The whole pellet and supernatant were run on the gel

SETD7 interacting proteins identified by mass spectrometry.

<p>SETD7 interacting proteins identified by mass spectrometry.</p

SETD7 is expressed at very low levels in pluripotent human cells and induced during differentiation.

<p>(A) Average rank of the top 700 most differentially expressed genes between pluripotent (iPSCs or ESCs) and fibroblasts, including those upregulated in pluripotent cells (left panel) and upregulated in fibroblasts (right panel). (B) <i>SETD7</i> mRNA levels in human ESCs grown under self-renewal conditions (UndES[4]), <i>in vitro</i> differentiated human ESCs (DifES[4]), human fibroblasts (HFF), two lines of human keratinocytes (HEK1 and HEK2) and two lines of iPSCs generated from keratinocytes ([H]KiPS4F and KiPS4F1). Mean and standard deviation of three technical replicates is shown. Induction of SETD7 mRNA levels during ES[4] differentiation was confirmed in more than four independent differentiation experiments. (C) Western blot showing SETD7 protein levels in pluripotent and somatic cells. Loading control beta actin (ACTB) is also shown. (D) Western blot showing protein levels of SETD7, AFP, OCT4 and SOX2 in under self-renewing conditions and <i>in vitro</i> differentiated human ESCs. Loading control alpha tubulin (TUBA) is also shown. One representative experiment out of three is shown. (E) Genomic visualization of the levels of H3K72me3, H3K4me3, H3K4me2, H3K36me3 and RNA polymerase II (Pol II) in the human embryonic stem cell line H1 around the <i>SETD7</i> gene according to ENCODE. A non-methylated CpG island is depicted in green. (F) Levels of H3K4me2 and H3K27me3 at <i>SETD7</i> gene promoter region (27 bp upstream of the transcription start site) in pluripotent and somatic cells determined by chromatin immunoprecipitation (ChIP) and ploted relative to the input. IgGs wer used as negative control. Bars show the mean and standard deviation of three independent immunoprecipitations.</p

The SETD7 knock-down affects the cell cycle profile of differentiating cells.

<p>(A) Cell cycle profile of undifferentiated and after 15 days of <i>in vitro</i> differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7). (B) Mean of the percentage of cells in each phase of the cell cycle in three independent differentiation experiments. *Differences in the percentage of cells in S-phase between shSETD7 and shSCR in differentiated cells was found significant at a p-value<0.05.</p

Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.

<p>Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.</p

Analysis of SETD7 interacting proteins.

<p>(A) Strategy for the purification of interacting proteins. (B) Levels of endogenous and overexpressed SETD7 in infected HeLa cells infected with pWPI-FLAG (FLAG) or pWPI-FLAG:SETD7 (FLAG:SETD7) determined by western blot. (C) Coomassie blue staining of the immunoprecipitated proteins. Molecular weight markers, anti FLAG-immunoprecipitated proteins from cells transduced with empty vector and cells transduced with FLAG:SETD7 expressing vector are shown.</p

The SETD7 knock-down causes defects in the silencing of pluripotency genes.

<p>(A) Western blot showing the levels of OCT4 at different days during the in vitro differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7) (B) Immunolocalization of SOX2 (green) and OCT4 (red) expression at day 4 and day 15 of in vitro differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7) (C) Quantification of the percentage of embryoid bodies negative or positive for OCT4 staining at day 4 and day 7 of differentiation of cells treated with vehicle (DMSO), 1μM or 5μM PFI-2. (D) Immunolocalization of OCT4 (red) in embryoid bodies at day 4 and day 7 of in vitro differentiation of ES[4] treated with vehicle (DMSO) or 5μM PFI-2.</p