Name: expression analysis of mammalian linker-histone subtypes
Uploaded: 2012-03-19T12:00:00
Description: Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

This work is supported by NIH grant GM085261 and a Georgia Cancer Coalition Distinguished Scholar Award (to Y.F.).

1. Sample Preparation and RNA Extraction
<ol>
 <li>Before RNA extraction, all working surfaces and pipettes should be wiped with 70% ethanol and treated with RNase decontamination solution, such as RNase Zap. This practice reduces the chances of RNase contamination and RNA degradation. Wear gloves for all procedures.</li>
 <li>To extract RNA from mouse tissue, dissect the organ of interest from euthanized mouse, and wash the tissue in ice cold Phosphate Buffered Saline (PBS: 0.13 M NaCl, 5 mM Sodium phosphate dibasic h...

<ol>
	<li>Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+linker+histones+are+essential+for+mouse+development+and+affect+nucleosome+spacing+in+vivo.">H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.</a> Mol. Cell Biol. 23, 4559-4572 (2003).</li><li>Woodcock, C. L., Skoultchi, A. I., Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+linker+histone+in+chromatin+structure+and+function:+H1+stoichiometry+and+nucleosome+repeat+length.">Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length.</a> Chromosome Res. 14, 17-25 (2006).</li><li>Shen, X., Gorovsky, M. 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U.S.A. 100, 5920-5925 (2003).</li><li>Happel, N., Doenecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+and+its+isoforms:+contribution+to+chromatin+structure+and+function.">Histone H1 and its isoforms: contribution to chromatin structure and function.</a> Gene. 431, 1-12 (2009).</li><li>Clausell, J., Happel, N., Hale, T. K., Doenecke, D., Beato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+subtypes+differentially+modulate+chromatin+condensation+without+preventing+ATP-dependent+remodeling+by+SWI&#47;SNF+or+NURF.">Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI&#47;SNF or NURF.</a> PLoS One. 4, 0007243-0007243 (2009).</li><li>Khadake, J. R., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-+chromatin-condensing+properties+of+rat+testes+H1a+and+H1t+compared+to+those+of+rat+liver+H1bdec;+H1t+is+a+poor+condenser+of+chromatin.">DNA- chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin.</a> Biochemistry. 34, 15792-15801 (1995).</li><li>Orrego, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+affinity+of+mammalian+histone+H1+somatic+subtypes+for+DNA+and+chromatin.">Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin.</a> BMC Biol. 5, 22-22 (2007).</li><li>Th'ng, J. P., Sung, R., Ye, M., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+family+histones+in+the+nucleus.+Control+of+binding+and+localization+by+the+C-terminal+domain.">H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain.</a> J. Biol. Chem. 280, 27809-27814 (2005).</li><li>Wang, Z. F., Sirotkin, A. M., Buchold, G. M., Skoultchi, A. I., Marzluff, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+histone+H1+genes:+gene+organization+and+differential+regulation.">The mouse histone H1 genes: gene organization and differential regulation.</a> J. Mol. Biol. 271, 124-138 (1997).</li><li>Brown, D. T., Sittman, D. 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The set of assays presented here enable comprehensive analysis of the expression levels of mammalian linker histone subtypes. Properly designed qRT-PCR assays provide highly sensitive and accurate measurements of RNA messages from any mammalian histone H1 genes. The critical part of qRT-PCR assays for linker histone subtype genes is the preparation of cDNA using random primer based reverse transcription. mRNA of most histone genes, including most H1 genes, do not contain a long poly-A tail presented in other cell...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>RNase Zap</td>
 <td>Applied Biosystems</td>
 <td>AM9780</td>
 </tr>
 <tr>
 <td>Trizol Reagent</td>
 <td>Invitrogen</td>
 <td>15596-018</td>
 </tr>
 <tr>
 <td>SuperScriptIII</td>
 <td>Invitrogen</td>
 <td>18080-051</td>
 </tr>
 <tr>
 <td>Absolute Ethanol</td>
 <td>Fisher Scientific</td>
 <td>BP2818-4</td>
 </tr>
 <tr>
 <td>IQ SYBR Green</td>
 <td>Bio-Rad</td>
 <td>170-8880</td>
 </tr>
 <tr>
 <td>RNeasy Mini Kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>Deoxyribonuclease I</td>
 <td>Sigma</td>
 <td>AMP-D1</td>
 </tr>
 <tr>
 <td>Microseal 96-well PCR plate</td>
 <td>Bio-Rad</td>
 <td>MSP-9605</td>
 </tr>
 <tr>
 <td>Microseal 'B' Adhesive Seals</td>
 <td>Bio-Rad</td>
 <td>MSB-1001</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Agros Organics</td>
 <td>AC40594</td>
 </tr>
 <tr>
 <td>Sodium phosphate dibasic heptahydrate (Na2HPO4&middot;7H2O)</td>
 <td>Fisher Scientific</td>
 <td>BP332</td>
 </tr>
 <tr>
 <td>Sodium chloride (NaCl)</td>
 <td>American Bioanalytical</td>
 <td>AB01915</td>
 </tr>
 <tr>
 <td>Sodium dihydrogen phosphate heptahydrate (NaH2PO4&middot;7H2O)</td>
 <td>Fisher Scientific</td>
 <td>BP-330</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Fisher Scientific</td>
 <td>BP310</td>
 </tr>
 <tr>
 <td>Complete Mini proteinase inhibitor cocktail tablet</td>
 <td>Roche Applied Science</td>
 <td>11836153001</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma</td>
 <td>E-5134</td>
 </tr>
 <tr>
 <td>Phenylmethanesulfonyl fluoride (PMSF)</td>
 <td>American Bioanalytical</td>
 <td>AB01620</td>
 </tr>
 <tr>
 <td>Nonidet-40 (NP-40)</td>
 <td>American Bioanalytical</td>
 <td>AB01425</td>
 </tr>
 <tr>
 <td>Potassium chloride (KCl)</td>
 <td>Fisher Scientific</td>
 <td>BP366</td>
 </tr>
 <tr>
 <td>Tris [hydroxymethyl aminomethane]</td>
 <td>American Bioanalytical</td>
 <td>AB02000</td>
 </tr>
 <tr>
 <td>Magnesium chloride (MgCl2)</td>
 <td>Fisher Scientific</td>
 <td>BP214</td>
 </tr>
 <tr>
 <td>Sulfuric acid (H2SO4)</td>
 <td>VWR</td>
 <td>VW3648-3</td>
 </tr>
 <tr>
 <td>Ammonium hydroxide (NH4OH)</td>
 <td>Agros Organics</td>
 <td>AC42330</td>
 </tr>
 <tr>
 <td>Bradford Protein Assay</td>
 <td>Bio-Rad</td>
 <td>500-0001</td>
 </tr>
 <tr>
 <td>Acetonitrile</td>
 <td>EMD</td>
 <td>AX0145-1</td>
 </tr>
 <tr>
 <td>Trifluoroacetic acid (TFA)</td>
 <td>J.T.Baker</td>
 <td>9470-01</td>
 </tr>
 </tbody>
</table>

expression analysis of mammalian linker-histone subtypes

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.

Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

Expression Analysis of Mammalian Linker-histone Subtypes

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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