Alternative Linker Histone Permits Fast Paced Nuclear Divisions in Early Drosophila Embryo

In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis

Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA

The tumour suppressor brain tumour (Brat) regulates linker histone dBigH1 expression in the Drosophila female germline and the early embryo

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too

Terminin components co-purify as discrete sub-complexes.

<p>(A) PAGE of protein fractions eluted by increasing salt gradient from a heparin-sepharose column onto which a cell lysate containing co-expressed HOAP, Ver, DTL/Moi and HP1 proteins was loaded. The tricine SDS-gel was stained by Coomassie. Ver and DTL/Moi co-eluted in the first 4 fractions, whereas HOAP and HP1 co-eluted at higher salt concentrations. HP1 elutes in two peaks: in fractions 4–6, and fractions 8–12, the latter being observed only if HOAP is present. Protein identities were confirmed by either western blot or mass spectrometry (Panel B). On panel A different parts of the same gel are shown. The bands marked were subjected to mass spectrometry. (B) Results of mass spectrometry identification of heterologously expressed terminin proteins. The peptide regions identified by mass spectrometry are underlined in the amino acid sequences of expressed proteins. (C) Fractions of gel filtrations obtained after heparin-sepharose column purification: fractions containing HOAP with HP1 and Ver with DTL/Moi were re-mixed at low salt concentration and gel filtrated. Molecular weight marker ladder (L), input (Inp), flow-through (FT) and fraction numbers and the position of the respected proteins are indicated.</p

Co-expression of interacting partners increases the solubility of terminin proteins.

<p>(A) Presumed interactions among terminin proteins based on GST pull-down assays [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142771#pone.0142771.ref017" target="_blank">17</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142771#pone.0142771.ref019" target="_blank">19</a>]. (B) Solubility of terminin components expressed individually in Arctic Express cells. Arrowheads point to bands corresponding to specific terminin proteins. HipHop expression cannot be observed on Coomassie-stained gel. On panel B images of different parts of the same gel are shown. (C) Co-expression of terminin components from polycistronic constructs. On panel C different parts of the same gel are shown except for the part with DTL/Moi-Ver data. L: molecular weight marker ladder, W: whole cell extract, S: supernatant, P: pellet, NC: control supernatant without any heterologous protein.</p

Sequence alignments reveal discrete parts of terminin proteins being subject to accelerated evolution.

<p>Homology plots show different conservation rates in distinct parts of terminin proteins. Each column represents the percentage of the consensus amino acid at the given position. Blue columns indicate regions where a gap occurs in some of the compared sequences as a result of deletion or insertion. Arrowheads and numbers represent domain boundaries considered during pN/pS calculations.</p

<i>D</i>. <i>yakuba</i> Ver and <i>D</i>. <i>melanogaster</i> DTL/Moi form co-purifying hybrid complex.

<p>(A) Cell lysate containing co-expressed <i>D</i>. <i>yakuba</i> Ver and <i>D</i>. <i>melanogaster</i> DTL/Moi was loaded onto heparin-sepharose column and the proteins were eluted by an increasing NaCl gradient. The protein content of the fractions was analysed on tricine SDS-PAGE. Different parts of the same gel are shown. (B) Fractions of the purification shown above containing <i>D</i>. <i>yakuba</i> Ver and <i>D</i>. <i>melanogaster</i> DTL/Moi (fractions:1–6) were combined and gel-filtrated on Superdex 200 10/300 GL column. The two proteins eluted in the same fractions in 1:1 ratio at 45kDa suggesting the formation of a hybrid complex. Molecular weight marker (L), input (Inp), flow through (FT) and fraction numbers, and the position of the respected proteins are indicated.</p

Ver and DTL/Moi form a heterodimer that could be purified independently from other terminin proteins.

<p>(A) Heparin-sepharose chromatography fractions of co-expressed Ver and DTL/Moi. Different parts of the same gel are shown. (B) The first 5 fractions of the purification shown on panel A containing Ver and DTL/Moi proteins were combined and gel-filtrated on Superdex 200 10/300 GL column. Molecular weight marker (L), input (Inp), flow through (FT) and fraction numbers and the position of the respected proteins are indicated.</p

mRNA levels of related Abcb genes change opposite to each other upon histone deacetylase inhibition in drug-resistant rat hepatoma cells.

The multidrug-resistant phenotype of tumor cells is acquired via an increased capability of drug efflux by ABC transporters and causes serious problems in cancer treatment. With the aim to uncover whether changes induced by epigenetic mechanisms in the expression level of drug transporter genes correlates with changes in the drug resistance phenotypes of resistant cells, we studied the expression of drug transporters in rat hepatoma cell lines. We found that of the three major rat ABC transporter genes Abcb1a, Abcb1b and Abcc1 the activity of only Abcb1b increased significantly in colchicine-selected, drug-resistant cells. Increased transporter expression in drug-resistant cells results primarily from transcriptional activation. A change in histone modification at the regulatory regions of the chromosomally adjacent Abcb1a and Abcb1b genes differentially affects the levels of corresponding mRNAs. Transcriptional up- and down-regulation accompany an increase in acetylation levels of histone H3 lysine 9 at the promoter regions of Abcb1b and Abcb1a, respectively. Drug efflux activity, however, does not follow tightly the transcriptional activity of drug transporter genes in hepatoma cells. Our results point out the need for careful analysis of cause-and-effect relationships between changes in histone modification, drug transporter expression and drug resistance phenotypes