Journal: Nucleic Acids Research

Article Title: Interactome of intact chromatosome variants with site-specifically ubiquitylated and acetylated linker histone H1.2

doi: 10.1093/nar/gkad1113

Figure Lengend Snippet: Generation of chromatosome variants with site-specifically ubiquitylated and acetylated linker histone H1.2. ( A ) Schematic overview of affinity-tagged chromatosome assembly using site-specifically modified H1.2 variants from building blocks. Core histones were expressed in E. coli and assembled into the histone octamer. A desthiobiotin affinity tag was introduced into the nucleosomal DNA by 5′-primer modification used in PCR amplification of nucleosomal DNA. Nucleosomes were subsequently assembled by mixing of core histone octamers and desthiobiotin-labeled DNA with a nucleosome positioning sequence. Chromatosomes were obtained by addition of site-specifically modified H1.2 variants. ( B ) SDS-PAGE gel of assembled core histone octamer shows the presence of all core histones in equal parts. ( C ) Agarose gel of PCR reaction after amplification of nucleosomal DNA shows specific production of the desired product (207 bp) in high quantities. ( D ) Native PAGE gel of assembled nucleosomes shows quantitative formation of nucleosomes upon addition of core histone octamer to nucleosomal DNA. ( E ) Native PAGE gel of intact chromatosome variants demonstrates quantitative conversion of nucleosome into chromatosome H1.2 variants.

Article Snippet: Oligonucleotides for the PCR of nucleosomal DNA were purchased from biomers.net.

Techniques: Modification, Amplification, Labeling, Sequencing, SDS Page, Agarose Gel Electrophoresis, Clear Native PAGE

Journal: Nucleic Acids Research

Article Title: Interactome of intact chromatosome variants with site-specifically ubiquitylated and acetylated linker histone H1.2

doi: 10.1093/nar/gkad1113

Figure Lengend Snippet: Interactome of H1-variant specific chromatosomes. ( A ) Overview of AE-MS workflow for H1.2 variant-specific chromatosome interactomes. Chromatosomes containing unmodified H1.2 or H1.2 variants are immobilized on streptavidin beads via a desthiobiotin tag on the nucleosomal DNA. Interacting proteins are enriched from HEK 293T cell lysates followed by washing steps to remove unspecific binders. After gentle elution of intact chromatosomes and enriched interactors with biotin, proteins are subjected to a tryptic digest followed by identification by LC-MS/MS and label-free quantification using DIA. Data analysis and statistical validation reveals PTM-specific interactions. ( B ) Heatmap of significantly enriched interactors for nucleosomes and chromatosome H1-variants. Shown are the mean z-score normalized values for each sample (ANOVA S 0 = 0.1, FDR = 0.05; t -test S 0 = 0.1 FDR = 0.05). ( C ) Classification of enriched proteins by GO-terms using PANTHER for the nucleosome (green) and chromatosome (blue) cluster.

Article Snippet: Oligonucleotides for the PCR of nucleosomal DNA were purchased from biomers.net.

Techniques: Variant Assay, Gentle, Liquid Chromatography with Mass Spectroscopy