Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development

The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

PARP inhibitors (PARPi) cause synthetic lethality in BRCA-deficient tumors. Whether specific vulnerabilities to PARPi exist beyond BRCA mutations and related defects in homology-directed repair (HDR) is not well understood. Here, we identify the ubiquitin E3 ligase TRIP12 as negative regulator of PARPi sensitivity. We show that TRIP12 controls steady-state PARP1 levels and limits PARPi-induced cytotoxic PARP1 trapping. Upon loss of TRIP12, elevated PARPi-induced PARP1 trapping causes increased DNA replication stress, DNA damage, cell cycle arrest, and cell death. Mechanistically, we demonstrate that TRIP12 binds PARP1 via a central PAR-binding WWE domain and, using its carboxy-terminal HECT domain, catalyzes polyubiquitylation of PARP1, triggering proteasomal degradation and preventing supra-physiological PARP1 accumulation. Further, in cohorts of breast and ovarian cancer patients, PARP1 abundance is negatively correlated with TRIP12 expression. We thus propose TRIP12 as regulator of PARP1 stability and PARPi-induced PARP trapping, with potential implications for PARPi sensitivity and resistance

Genotype-phenotype correlation in pseudoxanthoma elasticum

Background and aims: Pseudoxanthoma elasticum (PXE) is caused by variants in the ABCC6 gene. It results in calcification in the skin, peripheral arteries and the eyes, but has considerable phenotypic variability. We investigated the association between the ABCC6 genotype and calcification and clinical phenotypes in these different organs. Methods: ABCC6 sequencing was performed in 289 PXE patients. Genotypes were grouped as two truncating, mixed, or two non-truncating variants. Arterial calcification mass was quantified on whole body, low dose CT scans; and peripheral arterial disease was measured with the ankle brachial index after treadmill test. The presence of pseudoxanthoma in the skin was systematically scored. Ophthalmological phenotypes were the length of angioid streaks as a measure of Bruchs membrane calcification, the presence of choroidal neovascularizations, severity of macular atrophy and visual acuity. Regression models were built to test the age and sex adjusted genotype-phenotype association. Results: 158 patients (median age 51 years) had two truncating variants, 96 (median age 54 years) a mixed genotype, 18 (median age 47 years) had two non-truncating variants. The mixed genotype was associated with lower peripheral (13: 0.39, 95%CI:-0.62;-0.17) and total (13: 0.28, 95%CI:-0.47;-0.10) arterial calcification mass scores, and lower prevalence of choroidal neovascularizations (OR: 0.41 95%CI:0.20; 0.83) compared to two truncating variants. No association with pseudoxanthomas was found. Conclusions: PXE patients with a mixed genotype have less severe arterial and ophthalmological phenotypes than patients with two truncating variants in the ABCC6 gene. Research into environmental and genetic modifiers might provide further insights into the unexplained phenotypic variability

Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

Replication-coupled dilution of H4K20me2 guides 53BP1 to pre-replicative chromatin

The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1's target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR

Analysis of PARP inhibitor toxicity by multidimensional fluorescence microscopy reveals mechanisms of sensitivity and resistance

Exploiting the full potential of anti-cancer drugs necessitates a detailed understanding of their cytotoxic effects. While standard omics approaches are limited to cell population averages, emerging single cell techniques currently lack throughput and are not applicable for compound screens. Here, we employed a versatile and sensitive high-content microscopy-based approach to overcome these limitations and quantify multiple parameters of cytotoxicity at the single cell level and in a cell cycle resolved manner. Applied to PARP inhibitors (PARPi) this approach revealed an S-phase-specific DNA damage response after only 15 min, quantitatively differentiated responses to several clinically important PARPi, allowed for cell cycle resolved analyses of PARP trapping, and predicted conditions of PARPi hypersensitivity and resistance. The approach illuminates cellular mechanisms of drug synergism and, through a targeted multivariate screen, could identify a functional interaction between PARPi olaparib and NEDD8/SCF inhibition, which we show is dependent on PARP1 and linked to PARP1 trapping

RPA shields inherited DNA lesions for post-mitotic DNA synthesis

The paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity