The genetic history of the Southern Arc: a bridge between West Asia and Europe

By sequencing 727 ancient individuals from the Southern Arc (Anatolia and its neighbors in Southeastern Europe and West Asia) over 10,000 years, we contextualize its Chalcolithic period and Bronze Age (about 5000 to 1000 BCE), when extensive gene flow entangled it with the Eurasian steppe. Two streams of migration transmitted Caucasus and Anatolian/Levantine ancestry northward, and the Yamnaya pastoralists, formed on the steppe, then spread southward into the Balkans and across the Caucasus into Armenia, where they left numerous patrilineal descendants. Anatolia was transformed by intra–West Asian gene flow, with negligible impact of the later Yamnaya migrations. This contrasts with all other regions where Indo-European languages were spoken, suggesting that the homeland of the Indo-Anatolian language family was in West Asia, with only secondary dispersals of non-Anatolian Indo-Europeans from the steppe

Analysis of protein dynamics at active, stalled, and collapsed replication forks

Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation

ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells.

Homologous recombination (HR) is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB). However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents

Inhibition of histone deacetylase 3 causes replication stress in cutaneous T cell lymphoma.

Given the fundamental roles of histone deacetylases (HDACs) in the regulation of DNA repair, replication, transcription and chromatin structure, it is fitting that therapies targeting HDAC activities are now being explored as anti-cancer agents. In fact, two histone deacetylase inhibitors (HDIs), SAHA and Depsipeptide, are FDA approved for single-agent treatment of refractory cutaneous T cell lymphoma (CTCL). An important target of these HDIs, histone deacetylase 3 (HDAC3), regulates processes such as DNA repair, metabolism, and tumorigenesis through the regulation of chromatin structure and gene expression. Here we show that HDAC3 inhibition using a first in class selective inhibitor, RGFP966, resulted in decreased cell growth in CTCL cell lines due to increased apoptosis that was associated with DNA damage and impaired S phase progression. Through isolation of proteins on nascent DNA (iPOND), we found that HDAC3 was associated with chromatin and is present at and around DNA replication forks. DNA fiber labeling analysis showed that inhibition of HDAC3 resulted in a significant reduction in DNA replication fork velocity within the first hour of drug treatment. These results suggest that selective inhibition of HDAC3 could be useful in treatment of CTCL by disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death

An HDAC3 selective inhibitor triggers apoptosis associated with increased DNA damage and cell cycle defects.

<p>(A) Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and apoptosis assessed by Annexin V staining and flow cytometry. Cells were also labeled with propidium iodide to assess DNA content. Untreated (UT) and DMSO treated cells were used as controls. Shown is a representative graph from an experiment performed in duplicate that is consistent with other biological replicates. (B) Western blot analysis of γH2aX levels in Hut78 cells treated with DMSO, 10 nM Depsi, 10 µM 233, or 10 µM 966 for 8 hrs. Untreated and DMSO treated cells were used as controls. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side by side comparison of DMSO and Depsipeptide. (C) Cell cycle status was analyzed using BrdU incorporation and propidium iodide to assess DNA content by flow cytometry. Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and pulsed for an hour and a half with BrdU prior to cell harvest and analysis. Shown are representative flow cytometry plots from an experiment performed in duplicate that is consistent with other biological replicates. (D) Graphical representation of BrdU incorporation from the experiment described in (C). (E) Graphical representation of the percent of S phase cells that did not incorporate BrdU (shown by box in panel (C)). Statistical analysis for both the Annexin V and BrdU experiments was performed using a two-tail T-test and comparing the HDI treated cells to the DMSO treated cells resulting in the following p-values: (A) Depsi: p = 0.0002, 233: p = 0.003, and 966: p = 0.0003. (D) Depsi: p = 0.003, 233: p = 0.01, and 966: p = 0.08. (E) Depsi: p = 0.003, 233: p = 0.003, and 966: p = 0.004.</p

HDIs show selective inhibition of HDACs in CTCL cell lines.

<p>(A)Western blot analysis of whole cell lysates from Wild-type (WT) and <i>Hdac3</i>-null livers. Histones H3 and H4 served as loading controls. (B) Upper Panel: Western blot analysis of NIH 3T3 cells following treatment with various HDIs (indicated above each lane). Anti-histone H3 was used as a loading control. Lower panel: Western blot analysis of NIH 3T3 cells treated with either Trichostatin A (TSA) (1 µM), sodium butyrate (NaB) (5 mM), or increasing concentrations of nicotinamide (mM). (C) Western blot analysis of whole cell lysates prepared from cells that were transfected with either non-targeting siRNAs (NT) or siRNAs directed to the indicated Hdacs. (D) Western blot analysis of H3K56ac using whole cell lysates prepared from cells treated with the indicated amounts of RGFP966 for 24 hr. (E & F) Western blot analysis of (E) HH or (F) Hut78 cell lines treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, 10 µM 136, or 10 µM 966. Cells were treated for 24 hr and then harvested for protein isolation. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side-by-side comparison of DMSO and Depsipeptide. Histones H3 and H4 were used as loading controls.</p

CTCL cell lines are sensitive to pan and selective HDIs.

<p>(A) Growth curves of HDI treated HH cells (left) or Hut78 cells (right). Cells were treated once with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 at hour 0. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (B) Dose curves of 966 treated HH cells (left) and Hut78 cells (right). The experiment was performed in the same manner as (A) except that the cells treated were treated once with 2 µM, 5 µM, or 10 µM of 966 at hour 0. For both (A) and (B), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 233: p = 0.004, and 966: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 233: p = 0.006, and 966: p = 0.006. (B) HH cells (left), Depsi: p = 0.0008, 966 2 µM: p = 0.02, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 966 2 µM: p = 0.03, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006.</p

iPOND analysis reveals HDAC3 association with replication forks in Hut78 CTCL cells.

<p>Hut78 cells were pulsed for 15 minutes with EdU followed by either no thymidine chase or a 60 minute thymidine chase. The protein-DNA complexes were then cross-linked, nascent DNA was conjugated to biotin using click chemistry, and then protein-DNA complexes were purified using Streptavidin beads. The eluted proteins were then analyzed using western blot analysis. A no click reaction sample (No Clk) that did not include biotin azide was used as a negative control. 0.1% input samples were included for positive controls of each protein analyzed. PCNA served as a positive control for a replication fork bound protein and H2B served as a loading control and positive control for a chromatin bound protein.</p