An integrated and semi-automated micro-scaled approach to profile cis-regulatory elements by histone modification ChIP-Seq for large-scale epigenetic studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is the preferred approach to map histone modifications and identify cis-regulatory DNA elements throughout the genome. Multiple methods have been described to increase the efficiency of library preparation and to reduce hands-on time as well as costs. This review describes detailed steps to perform cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation for a batch of 48-96 samples with small cell numbers. The protocol implements a semiautomated platform to reduce technical variability and improve signal-to-noise ratio as well as reduce hands-on time, thus allowing large-scale epigenetic studies of clinical samples with limited cell numbers.</p

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression

Lab-on-a-chip for rapid electrochemical detection of nerve agent Sarin

This paper reports a lab-on-a-chip for the detection of Sarin nerve agent based on rapid electrochemical detection. The chemical warfare agent Sarin (C4H10FO2P, O-isopropyl methylphosphonofluoridate) is a highly toxic organophosphate that induces rapid respiratory depression, seizures and death within minutes of inhalation. As purified Sarin is colourless, odourless, water soluble and a easily disseminated nerve agent, it has been used as a weapon in terrorist or military attacks. To ascertain whether potable water supplies have been adulterated with this extremely potent poison, an inexpensive, sensitive and easy to use portable test kit would be of interest to first responders investigating such attacks. We report here an amperometric-based approach for detecting trace amounts of Sarin in water samples using a screen-printed electrode (SPE) integrated in a microfluidic chip. Enzymatic inhibition was obtained by exposing the immobilised biosensor in the microfluidic platform to Sarin in water samples. With the aid of cobalt phthalocyanine modified SPE, the device could detect Sarin at part-per-billion levels with concentration as low as 1 nM. The detection method reported here represents a significant improvement over the authors’previous optical-based detection method.ASTAR (Agency for Sci., Tech. and Research, S’pore

西安事件と中国共産党：政策決定過程に於けるいくつかの問題

Chromatin immunoprecipitation coupled with high-throughput, next-generation DNA sequencing (ChIP-seq) has enabled researchers to establish the genome-wide patterns of chromatin modifications and binding of chromatin-associated proteins. Well-established protocols produce robust ChIP-seq data for many proteins by sequencing the DNA obtained following immunoprecipitation of fragmented chromatin using a wide range of specific antibodies. In general, the quality of these data mainly depends on the specificity and avidity of the antibody used. However, even using optimal antibodies, ChIP-seq can become more challenging when the protein associates with chromatin via protein-protein interactions rather than directly binding DNA. An example of such a protein is the alpha-thalassaemia mental retardation X-linked (ATRX) protein; a chromatin remodeler that associates with the histone chaperone DAXX, in the deposition of the replication-independent histone variant H3.3 and plays an important role in maintaining chromatin integrity. Inherited mutations of ATRX cause syndromal mental retardation (ATR-X Syndrome) whereas acquired mutations are associated with myelodysplasia, acute myeloid leukemia (ATMDS syndrome), and a range of solid tumors. Therefore, high quality ChIP-seq data have been needed to analyze the genome-wide distribution of ATRX, to advance our understanding of its normal role and to comprehend how mutations contribute to human disease. Here, we describe an optimized ChIP-seq protocol for ATRX which can also be used to produce high quality data sets for other challenging proteins which are indirectly associated with DNA and complement the ChIP-seq toolkit for genome-wide analyses of histone chaperon complexes and associated chromatin remodelers. Although not a focus of this chapter, we will also provide some insight for the analysis of the large dataset generated by ChIP-seq. Even though this protocol has been fully optimized for ATRX, it should also provide guidance for efficient ChIP-seq analysis, using the appropriate antibodies, for other proteins interacting indirectly with DNA

Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex

Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1–H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex