I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. Owing to the tremendous progress in next-generation sequencing technology, ChIP-seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP-chip. With the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this Review, I describe the benefits and challenges in harnessing this technique with an emphasis on issues related to experimental design and data analysis. ChIP-seq experiments generate large quantities of data, and effective computational analysis will be crucial for uncovering biological mechanisms.

/pdf/chip-seq-advantages-and-challenges-of-a-maturing-technology-57od1mnvrx.pdf

ChIP-Seq: advantages and challenges of a maturing technology

Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of underacetylated histones. Recently, these two epigenetic processes have been dynamically linked. The methyl-CpG-binding protein MeCP2 appears to reside in a complex with histone deacetylase activity. MeCP2 can mediate formation of transcriptionally repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase. Little is known, however, about the relative roles of methylation and histone deacetylase activity in the stable inhibition of transcription on densely methylated endogenous promoters, such as those for silenced alleles of imprinted genes, genes on the female inactive X chromosome and tumour-suppressor genes inactivated in cancer cells. We show here that the hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA alone in tumour cells in which we have shown that TSA alone can upregulate the expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2'deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each gene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylated promoters. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.

/pdf/synergy-of-demethylation-and-histone-deacetylase-inhibition-21alpb6eym.pdf

Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.

Developmental progression is driven by specific spatiotemporal domains of gene expression, which give rise to stereotypically patterned embryos even in the presence of environmental and genetic variation. Views of how transcription factors regulate gene expression are changing owing to recent genome-wide studies of transcription factor binding and RNA expression. Such studies reveal patterns that, at first glance, seem to contrast with the robustness of the developmental processes they encode. Here, we review our current knowledge of transcription factor function from genomic and genetic studies and discuss how different strategies, including extensive cooperative regulation (both direct and indirect), progressive priming of regulatory elements, and the integration of activities from multiple enhancers, confer specificity and robustness to transcriptional regulation during development.

http://materiais.dbio.uevora.pt/BD/Crescimento/TFs_2012.pdf

Transcription factors: from enhancer binding to developmental control.

We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.

A high-resolution atlas of nucleosome occupancy in yeast.

Genome-wide transcription factor binding: Beyond direct target regulation

Electron paramagnetic resonance studies of nitrosyl ferrous heme complexes. Determination of an equilibrium between two conformations.

The packaging of eukaryotic DNA into chromatin has profound consequences for gene regulation, as well as for other DNA transactions such as recombination, replication and repair. Understanding how this packaging is determined is consequently a pressing problem in molecular genetics. DNA sequence, chromatin remodelers and transcription factors affect chromatin structure, but the scope of these influences on genome-wide nucleosome occupancy patterns remains uncertain. Here, we use high resolution tiling arrays to examine the contributions of two general regulatory factors, Abf1 and Rap1, to nucleosome occupancy in Saccharomyces cerevisiae. These factors have each been shown to bind to a few hundred promoters, but we find here that thousands of loci show localized regions of altered nucleosome occupancy within 1h of loss of Abf1 or Rap1 binding, and that altered chromatin structure can occur via binding sites having a wide range of affinities. These results indicate that DNA-binding transcription factors affect chromatin structure, and probably dynamics, throughout the genome to a much greater extent than previously appreciated.

/pdf/extensive-role-of-the-general-regulatory-factors-abf1-and-2jofyj3611.pdf

Extensive role of the general regulatory factors, Abf1 and Rap1, in determining genome-wide chromatin structure in budding yeast

Oxidized cytochrome c oxidase is shown to exist in three conformations in addition to the transient "g5" conformation previously reported [Shaw, R. W., Hansen, R. E., & Beinert, H. (1978) J. Biol. Chem. 253, 6637-6640]. The "resting" and "g12" conformations are distinguished by an NO-induced cytochrome a3 electron paramagnetic resonance (EPR) signal and an EPR signal at g' = 12, respectively. The "oxygenated" conformatin exhibits an unusual EPR signal in the presence of fluoride and is identical with the "oxygenated" state first discovered by Okunuki et al. [Okunuki, K., Hagihora, B., Sekuzu, I., & Horio, T. (1958) Proc. Int. Symp. Enzyme Chem., Tokyo, Kyoto, 264]. It is proposed that when the reduced enzyme is reoxidized by dioxygen, the oxidized enzyme first relaxes from the "g5" into the "oxygenated" conformation after which a percentage of the molecules slowly relax into the "g12" conformation. The "resting" conformation is not formed when the enzyme is reoxidized. On the basis of the EPR observations, it is proposed that these various conformations of the oxidized enzyme differ in the structure of the cytochrome a3--Cua3 site. Structures for the cytochrome a3--Cua3 site are proposed for each conformation, and a mechanism by which these conformations undergo interconversion among themselves is described.

Randall H. Morse

Papers

A high-resolution atlas of nucleosome occupancy in yeast.

Genome-wide transcription factor binding: Beyond direct target regulation

Electron paramagnetic resonance studies of nitrosyl ferrous heme complexes. Determination of an equilibrium between two conformations.

Extensive role of the general regulatory factors, Abf1 and Rap1, in determining genome-wide chromatin structure in budding yeast

Conformations of oxidized cytochrome c oxidase.