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Proteins that possess a chromo domain are well‐known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy‐terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi‐directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates.

A model for integrating course-based research with community genome annotation efforts at model organism databases is presented. Disseminating gene function discoveries directly to an interested audience increased student motivation to more deeply engage all aspects of an authentic research experience., Use of inquiry-based research modules in the classroom has soared over recent years, largely in response to national calls for teaching that provides experience with scientific processes and methodologies. To increase the visibility of in-class studies among interested researchers and to strengthen their impact on student learning, we have extended the typical model of inquiry-based labs to include a means for targeted dissemination of student-generated discoveries. This initiative required: 1) creating a set of research-based lab activities with the potential to yield results that a particular scientific community would find useful and 2) developing a means for immediate sharing of student-generated results. Working toward these goals, we designed guides for course-based research aimed to fulfill the need for functional annotation of the Tetrahymena thermophila genome, and developed an interactive Web database that links directly to the official Tetrahymena Genome Database for immediate, targeted dissemination of student discoveries. This combination of research via the course modules and the opportunity for students to immediately “publish” their novel results on a Web database actively used by outside scientists culminated in a motivational tool that enhanced students’ efforts to engage the scientific process and pursue additional research opportunities beyond the course.

Class I histone deacetylases (HDACs) regulate DNA-templated processes such as transcription. They act both at specific loci and more generally across global chromatin, contributing to acetylation patterns that may underlie large-scale chromatin dynamics. Although hypoacetylation is correlated with highly condensed chromatin, little is known about the contribution of individual HDACs to chromatin condensation mechanisms. Using the ciliated protozoan Tetrahymena thermophila , we investigated the role of a specific class I HDAC, Τhd1p, in the reversible condensation of global chromatin. In this system, the normal physiological response to cell starvation includes the widespread condensation of the macronuclear chromatin and general repression of gene transcription. We show that the chromatin in Thd1p-deficient cells failed to condense during starvation. The condensation failure correlated with aberrant hyperphosphorylation of histone H1 and the overexpression of CDC2 , encoding the major histone H1 kinase. Changes in the rate of acetate turnover on core histones and in the distribution of acetylated lysines 9 and 23/27 on histone H3 isoforms that were found to correlate with normal chromatin condensation were absent from Thd1p mutant cells. These results point to a role for a class I HDAC in the formation of reversible higher-order chromatin structures and global genome compaction through mechanisms involving the regulation of H1 phosphorylation and core histone acetylation/deacetylation kinetics.

Class I histone deacetylases (HDACs) participate in the regulation of DNA-templated processes such as transcription and replication. Members of this class can act locally at specific sites, or they can act more globally, contributing to a baseline acetylation state, both of which actions may be important for genome maintenance and organization. We previously identified a macronuclear-specific class I HDAC in Tetrahymena thermophila called Thd1p, which is expressed early in the development of the macronucleus when it initially becomes transcriptionally active. To test the idea that Thd1p is important for global chromatin integrity in an active macronucleus, Tetrahymena cells reduced in expression of Thd1p were generated. We observed phenotypes that indicated loss of chromatin integrity in the mutant cells, including DNA fragmentation and extrusion of chromatin from the macronucleus, variable macronuclear size and shape, enlarged nucleoli, and reduced phosphorylation of histone H1 from bulk chromatin. Macronuclei in mutant cells also contained more DNA. This observation suggests a role for Thd1p in the control of nuclear DNA content, a previously undescribed role for class I HDACs. Together, these phenotypes implicate Thd1p in the maintenance of macronuclear integrity in multiple ways, probably through site-specific changes in histone acetylation since no change in the acetylation levels of bulk histones was detected in mutant cells.

Gene expression in eukaryotes relies upon the accessibility of the DNA template to a variety of components in the transcription apparatus, which is controlled in turn by a locus-specific chromatin structure. Chromatin of higher eukaryotes is often discussed in terms of two cytologically distinct domains: euchromatin and heterochromatin. Heterochromatin is classically distinguished from euchromatin by its paucity of genes, its highly condensed chromatin structure throughout the cell cycle, its late replication in S phase, and its enrichment in repetitive DNA sequences (25, 32). However, genes have been identified that reside within heterochromatic regions (21, 27), and the expression of some of these genes is dependent upon their placement in heterochromatin. Furthermore, recent evidence has shown that heterochromatin plays important roles in long-range intra- and interchromosomal interactions that affect nuclear organization (8, 10), chromosome segregation (9, 33, 46), and locus-controlled patterns of transcriptional activity (42). Heterochromatin and euchromatin represent two different structural environments, and each has profound effects on gene expression (55, 58). A large body of genetic evidence suggests that heterochromatin interferes with the expression of normally euchromatic genes. In yeast cells, for example, mitotically transmissible silencing of silent mating-type loci depends upon nearby sequences, as well as on histone and nonhistone chromosomal components (44). Sequences not related to mating type become transcriptionally repressed when placed at or near this specialized chromatin environment, and similar positional silencing occurs near telomeres (24). Likewise in Drosophila melanogaster, heterochromatic position-effect variegation (PEV) is characterized by mitotically transmissible silencing and position-dependent gene silencing (55, 57, 58). Silencing by pericentric heterochromatin has also been observed in transgenic mice (18). Thus, the underlying mechanism leading to positional silencing appears to exist in yeast, flies, and mammals. PEV in flies is influenced by modifiers (both enhancers and suppressors), and these gene products have been proven important in dissecting the composition of heterochromatin. One of the PEV modifier genes, Su(var)205, encodes HP1, which is perhaps the best-studied structural protein associated with heterochromatin (11, 12). A loss-of-function mutation in Su(var)205 leads to increased expression of euchromatic genes that suffer from position effect (i.e., White), while overproduction of HP1 results in decreased expression of these genes (12). Opposite results have been obtained for heterochromatin-positioned genes (i.e., Light), suggesting that genes localized in heterochromatin differ in their regulatory requirements (26). Conservation of HP1 throughout evolution (50) and the lethality of HP1 null alleles (13) suggest that HP1 is important for cell viability and development in flies. In support of this, Swi6p, a chromodomain-containing protein from fission yeast, while not essential, is required to maintain transcriptional repression at the silent mating-type loci and centromeres (2, 15, 38). Recent studies of Δswi6 cells have shown a reduced spore viability, along with altered expression of some meiotic genes, suggesting that proteins of this general family play an important yet poorly understood role in gene expression (37). Like other ciliates, Tetrahymena spp. exhibit nuclear dimorphism, wherein each cell contains both a germ line micronucleus and a somatic macronucleus (22, 23). Even though these nuclei coexist as neighbors in a common cytoplasm, they differ considerably in function and provide a useful model for studying the function of HP1-like proteins. Recently, we reported the identification of an HP1-like protein, Hhp1p, that is absent from transcriptionally silent micronuclei but is modestly enriched in the condensed chromatin regions (referred to as chromatin bodies) that punctuate transcriptionally active macronuclei (30). Here, we report the disruption of all somatic (macronuclear) copies of the endogenous HHP1 genes in Tetrahymena thermophila. Our results demonstrate that, unlike Drosophila HP1, Hhp1p is not essential. Although no phenotypic difference is observed in vegetative cells lacking Hhp1p, a clear phenotypic difference is observed when cells are starved for nutrients. During a shift to nongrowth (starvation) conditions, the survival rate of cells lacking Hhp1p (ΔHHP1) is reduced relative to wild-type cells, morphological changes in chromatin body size fail to occur, and the activation of two starvation-induced genes is markedly reduced. These results suggest a model wherein the loss of Hhp1p leads to aberrant assembly of the condensed chromatin in macronuclei during the transition from growth to nongrowth conditions. We suspect that failure to properly assemble condensed chromatin domains leads to the reduced expression of starvation-induced genes that reside in and utilize condensed chromatin in a positive fashion to bring about their expression.

BackgroundThe NAD+-dependent histone deacetylases, known as "sirtuins", participate in a variety of processes critical for single- and multi-cellular life. Recent studies have elucidated the importance of sirtuin activity in development, aging, and disease; yet, underlying mechanistic pathways are not well understood. Specific sirtuins influence chromatin structure and gene expression, but differences in their pathways as they relate to distinct chromatin functions are just beginning to emerge. To further define the range of global chromatin changes dependent on sirtuins, unique biological features of the ciliated protozoanTetrahymena thermophilacan be exploited. This system offers clear spatial and temporal separation of multiple whole genome restructuring events critical for the life cycle.ResultsInhibition with nicotinamide revealed that sirtuin deacetylase activity inTetrahymenacells promotes chromatin condensation during meiotic prophase, differentiation of heterochromatin from euchromatin during development, and chromatin condensation/degradation during programmed nuclear death. We identified a class I sirtuin, called Thd14, that resides in mitochondria and nucleoli during vegetative growth, and forms a large sub-nuclear aggregate in response to prolonged cell starvation that may be peripherally associated with nucleoli. During sexual conjugation and development Thd14 selectively concentrates in the parental nucleus prior to its apoptotic-like degradation.ConclusionsSirtuin activity is important for several functionally distinct events requiring global chromatin condensation. Our findings suggest a novel role for sirtuins in promoting programmed pycnosis by acting on chromatin destined for degradation. The sirtuin Thd14, which displays physiological-dependent differential localization within the nucleus, is a candidate for a chromatin condensation enzyme that is coupled to nuclear degradation.

Foreword Preface Contributors List Conversion Factors Flow Chart: Application of Techniques Emily Wiley, Claremont Colleges Chapter 1 Volume/Weight Measurement Unit 1.1 Volume Measurement Andrew Zanella and Tom Davis, Claremont Colleges Unit 1.2 Weight Measurement Michael Guzy, Ohaus, Corp. Chapter 2 Concentration Measurement Unit 2.1 Spectrophotometry Rob Morris, Ocean Optics, Inc. Unit 2.2 Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy Sean R. Gallagher, UVP, LLC Chapter 3 Reagent Preparation Unit 3.1 Theoretical Discussion Deb Chakravarti, Keck Graduate Institute Unit 3.2 Practical Discussion Deb Chakravarti, Keck Graduate Institute Unit 3.3 Recipes In-House Chapter 4 Cell Culture Techniques Unit 4.1 Aseptic Technique Thomas Bykowski and Brian Stevenson, University of Kentucky College of Medicine Unit 4.2 Culture of Escherichia coli and Related Bacteria Sean P. Riley, Michael E. Woodman, and Brian Stevenson, University of Kentucky College of Medicine Chapter 5 Sample Preparation Unit 5.1 Centrifugation and Cell Fractionation In-House Unit 5.2 Purification and Concentration of Nucleic Acids Dennis H. Dowhan, University of Queensland Chapter 6 Chromatography Unit 6.1 Overview of Chromatography Buddhadeb Mallik, Bulbul Chakravarti, and Deb N. Chakravarti, Keck Graduate Institute of Applied Life Sciences Unit 6.2 Column Chromatography Deb Chakravarti, Keck Graduate Institute Chapter 7 Electrophoresis Unit 7.1 Overview of Electrophoresis Sean R. Gallagherr, UVP, LLC Unit 7.2 Agarose Gel Electrophoresis Jennifer A. Armstrong, The Claremont Colleges, and Joseph R. Schulz, Occidental College Unit 7.3 SDS-PAGE Sean Gallagher, UVP, LLC Unit 7.4 Staining of Gels Sean Gallagher, UVP, LLC Chapter 8 Blotting Unit 8.1 Overview of Blotting Maria Cristina Negritto, Pomona College, and Glenn M. Manthey, City of Hope, Beckman Research Institute Unit 8.2 Nucleic Acid Blotting: Southern and Northern Laura L. Mays Hoopes, Pomona College Unit 8.3 Protein Blotting: Immunoblotting Sean Gallagher, UVP, LLC Unit 8.4 Labeling DNA and Probe Preparation Karl A. Haushalter, Harvey Mudd College Chapter 9 Microscopy Unit 9.1 Conventional Light Microscopy Eric S. Cole, St. Olaf College Unit 9.2 Immunofluorescence Microscopy David J. Asai, Harvey Mudd College Chapter 10 Enzymatic Reactions Unit 10.1 Working with Enzymes David Skrincosky and Cindy Santangelo, Worthington Biochemical Corp. Unit 10.2 Overview of PCR Christine D. Kuslich, Molecular Profiling Institute, Buena Chui, GE Healthcare, and Carl T. Yamashiro, Molecular Profiling Institute Unit 10.3 Real-Time PCR Dean Fraga, College of Wooster, Tea Meulia, Ohio Agricultural Research and Development Center, Steven Fenster, Ashland University Chapter 11 Bioinformatics Unit 11.1 Using NCBI Blast Nicholas A. Stover, Bradley University, and Andre R.O. Cavalcanti, Pomona College

Newly synthesized histones are acetylated prior to their deposition into nucleosomes. Following nucleosome formation and positioning, they are rapidly deacetylated, an event that coincides with further maturation of the chromatin fiber. The histone deacetylases (HDACs) used for histone deposition and de novo chromatin formation are poorly understood. In the ciliate Tetrahymena thermophila , transcription-related deacetylation in the macronucleus is physically separated from deposition-related deacetylation in the micronucleus. This feature was utilized to identify an HDAC named Thd2, a class II HDAC that acts on newly synthesized histones to remove deposition-related acetyl moieties. The THD2 transcript is alternatively spliced, and the major form contains a putative inositol polyphosphate kinase (IPK) domain similar to Ipk2, an enzyme that promotes chromatin remodeling by SWI/SNF remodeling complexes. Cells lacking Thd2, which retain deposition-related acetyl moieties on new histones, exhibit chromatin and cytological phenotypes indicative of a role for Thd2 in chromatin maturation, including the proteolytic processing of histone H3.

The measurement of pH is one of the most basic and necessary skills in a life science laboratory. The function and physical characteristics of biological molecules are highly sensitive to the pH of the environment. Common biological buffers must be prepared with the appropriate pH, usually close to neutral for most biological applications. This unit includes a discussion of the different pH instrumentation, notably pH electrodes. It also relates basic pH measurement theory to critical parameters for the technique, and covers the correct use, handling, and storage of pH instruments. Curr. Protoc. Essential Lab. Tech. 3:3.2.1-3.2.16. © 2010 by John Wiley & Sons, Inc. Keywords: electrode; meter; calibration; Nernst; electrical potential

Publisher Summary Chromatin in vivo is intrinsically a dynamic structure, undergoing pronounced conformational changes in response to specific biological stimuli. Histones themselves are subject to multiple posttranslational modifications such as phosphorylation and acetylation. Histone acetylation is brought about and maintained by a balance of two classes of competing enzymes: the on reaction is catalyzed by enzymes called histone acetyltransferases (HATS); the opposing off reaction (that is, the removal of acetyl groups from histones) is catalyzed by deacetylase (HDACs) enzymes. This chapter discusses the techniques used for isolating and analyzing Tetrahymena histones modified in vivo. Histones are easily extracted from isolated nuclei by virtue of their acid solubility. Following extraction, individual histones can be isolated by reverse phase high-performance liquid chromatography (RP-HPLC) or be analyzed by gel electrophoresis. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) effectively resolves all Tetrahymena histones except H3 and H2A, better resolution is achieved on acid-urea (or triton-acid-urea) gels that separate proteins according to their size, charge, and, in the case of triton-containing acid-urea gels, detergent-binding properties.

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila . Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.