Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy

Abstract

As host to the genome, the nucleus plays a critical role as modulator of cellular phenotype. To understand the totality of proteins that regulate this organelle, we used proteomics to characterize the components of the cardiac nucleus. Following purification, cardiac nuclei were fractionated into biologically relevant fractions including acid-soluble proteins, chromatin-bound molecules and nucleoplasmic proteins. These distinct subproteomes were characterized by liquid chromatography-tandem MS. We report a cardiac nuclear proteome of 1048 proteins-only 146 of which are shared between the distinct subcompartments of this organelle. Analysis of genomic loci encoding these molecules gives insights into local hotspots for nuclear protein regulation. High mass accuracy and complementary analytical techniques allowed the discrimination of distinct protein isoforms, including 54 total histone variants, 17 of which were distinguished by unique peptide sequences and four of which have never been detected at the protein level. These studies are the first unbiased analysis of cardiac nuclear subcompartments and provide a foundation for exploration of this organelle's proteomes during disease. Molecular & Cellular

Quantitative Analysis of the Chromatin Proteome in Disease Reveals Remodeling Principles and Identifies High Mobility Group Protein B2 as a Regulator of Hypertrophic Growth

“…Whole heart lysate (whole heart lysate) was made by homogenizing the entire heart in 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, followed by sonication and centrifugation at 13,000 rpm to remove insoluble material. We consistently achieve Ն80% enrichment of nuclei with this method as observed by electron microscopy and Western blotting analysis (22).…”

“…Purification of Cardiac Chromatin and Nucleoplasm and Acid Extraction of Nuclear Proteins-Following isolation of nuclei, further fractionation was carried out to separate nucleoplasm from chromatin as previously described (22). Briefly, isolated nuclei were resuspended in buffer (20 mM HEPES, pH 7.6, 7.5 mM MgCl 2 , 30 mM NaCl, 1 M urea, 1% Nonidet P-40) to solubilize the nuclear membrane and extract soluble proteins in the nucleoplasm.…”

“…Cardiac nuclei were isolated as previously described (22). Briefly, hearts were excised, washed in PBS, minced with scissors, and processed in a glass Dounce homogenizer in buffer containing 10 mM Tris (pH 7.4), 250 mM sucrose, 1 mM EDTA, 0.15% Nonidet P-40.…”

“…As with any fractionation study of a complex tissue like the heart, complete purification of individual fractions is not possible when using a nonbiased technique like mass spectrometry for detection. Our previous work demonstrates that this method significantly enriches for histone and other chromatin-bound proteins (22), although additional studies will be required to determine what role each individual proteins have in the chromatin fraction (i.e. to discriminate between true residents and contaminants).…”

Casz1 is required for cardiomyocyte G1-to-S phase progression during mammalian cardiac development

“…Ventricles were placed in liquid nitrogen and stored at −80°C. Similar genotypes were pooled (n=4 wild type, n=4 cardiac nulls) and homogenized in liquid nitrogen following a modified cardiac nuclei extraction protocol (Franklin et al, 2011). Briefly, ventricle powder was resuspended in homogenization buffer [10 mM Tris pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.1% NP-40, 10 mM sodium butyrate, 0.1 mM PMSF, 1× protease inhibitors (Roche)] and subjected to ten strokes with a Dounce homogenizer on ice.…”