Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.
16 Related JoVE Articles!
Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Institutions: Stowers Institute for Medical Research, Kansas University Medical Center.
Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.
Biochemistry, Issue 92, chromatin remodeling, INO80, SNF2 family ATPase, biochemical assays, ATPase, nucleosome remodeling, nucleosome binding
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
Detection of Histone Modifications in Plant Leaves
Institutions: RWTH Aachen University, RWTH Aachen University, Leibniz University.
Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2
. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2
. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9
, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10
, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4
photosynthesis in maize5,11
and systemic immunity in Arabidopsis3
Molecular Biology, Issue 55, chromatin, chromatin immunoprecipitation, ChIP, histone modifications, PCR, plant molecular biology, plant promoter control, gene regulation
Expression Analysis of Mammalian Linker-histone Subtypes
Institutions: Georgia Institute of Technology .
Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1
and regulates specific gene expression in vivo2-4
. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10
, 4 germ cell specific H1 subtypes, and H1x5
. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9
provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1
). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10
. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1
) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization.
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13
. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.
Genetics, Issue 61, H1 linker histones, histone H1 subtypes, chromatin, RT-PCR, HPLC, gene expression
Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells
Institutions: University of Virginia.
In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1
. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2
and autoimmune diseases3
. It is well established that transcription is regulated by alterations to the chromatin template4,5
. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6
. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.
Cellular Biology, Issue 41, chromatin, histone modification, transcription, antibody, cell culture, epigenetics, transcription factor, nucleosome
Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity
Institutions: University of Arkansas for Medical Sciences .
Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3
. As a result, these enzymes are prime targets for small molecule studies and drug development 4
. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5
. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5
Molecular Biology, Issue 61, LSD1, lysine demethylase, mass spectrometry, reductive methylation, demethylase quantification
Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation
Institutions: The Alfred Medical Research and Education Precinct, The Alfred Medical Research and Education Precinct, University of Melbourne.
An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2
. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3
. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3
. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7
. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8
. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9
. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.
Cellular Biology, Issue 42, H2AX, radiation, euchromatin, heterochromatin, immunofluorescence, 3D-modeling
An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis
Institutions: University of Zürich, Université de Montpellier II.
In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis
ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ
hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.
Plant Biology, Issue 88, Arabidopsis thaliana, ovule, chromatin modification, nuclear architecture, immunostaining, Fluorescence in situ Hybridization, FISH, DNA staining, Heterochromatin
Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA
Institutions: Stanford University , University of Connecticut, University of Connecticut.
The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41
. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3
. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.
Cellular Biology, Issue 50, Chromatin, Nucleosome, Epigenetics, ELISA, Histone, Modification, Methylation, Acetylation
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays
Institutions: Stuttgart University.
Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.
Biochemistry, Issue 93, Peptide arrays, solid phase peptide synthesis, SPOT synthesis, protein lysine methyltransferases, substrate specificity profile analysis, lysine methylation
Quantitative Analysis of Chromatin Proteomes in Disease
Institutions: David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah.
In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1
the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2
Transcriptional regulation during development and disease have been well studied in this organ,3-5
but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6
In the developed world, heart disease is the number one cause of mortality for both men and women.7
Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13
there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15
In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16
Additionally, cardiomyocytes are 40% mitochondria by volume17
which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo
experimentation in various animal models and organ systems where metabolic labeling is not feasible.
Medicine, Issue 70, Molecular Biology, Immunology, Genetics, Genomics, Physiology, Protein, DNA, Chromatin, cardiovascular disease, proteomics, mass spectrometry
Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes
Institutions: Stowers Institute for Medical Research, Kansas University Medical Center.
INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex's chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.
Biochemistry, Issue 92, chromatin remodeling, INO80, SNF2 family ATPase, structure-function, enzyme purification
Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Institutions: RWTH Aachen University.
-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for S
of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGA
T-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for m
ransfer of A
roups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.
Biochemistry, Issue 93, S-adenosyl-l-methionine, AdoMet, SAM, aziridine cofactor, double activated cofactor, methyltransferase, DNA methylation, protein methylation, biotin labeling, fluorescence labeling, SMILing, mTAG
Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment
Institutions: Philipps-Universität Marburg, Philipps-Universität Marburg.
During spermatogenesis in mammals and in Drosophila melanogaster,
male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin—the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster
offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo
in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.
Developmental Biology, Issue 91,
Ex vivo culture, testis, male germ-line cells, Drosophila, imaging, pharmacological assay
A Rapid Technique for the Visualization of Live Immobilized Yeast Cells
Institutions: Princeton University.
We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also Xu et al., Mol. Cell 2006.
Microbiology, Issue 1, yeast, HML, HMR, epigenetic, loci, silencing, cerevisiae