Chip-chip from HeLa S3 expressing epitope-tagged H2Abbd, macroH2A and H2A histones: H2Abbd versus H2A, and macroH2A versus H2A, distributions on 13 regions of the HG18 assembly
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE28041 | GEO | 2011/03/19
SECONDARY ACCESSION(S): PRJNA137953
REPOSITORIES: GEO
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Project description:The distribution of histone variants H2Abbd and macroH2A in 13 regions of the HG18 assembly have been studied using a variant of the ChIP-on-Chip technique. HeLa S3 cell lines expressing tagged histones H2A, H2Abbd or macroHA were obtained using retroviral transfer. DNA fractions associated with tagged histones were isolated using a two-step purification procedure that involved affinity chromatography on a column with anti-FLAG antibodies, followed by affinity chromatography on a Ni-agarose column. The obtained genomic DNA samples were analyzed by hybridization with custom NimbleGene genomic microarrays. Two samples. Test sample 1 is HeLa S3 cells expressing epitope-tagged histone H2Abbd and test sample 2 is HeLa S3 cells expressing epitope-tagged histone macroH2A . The control for both test sample 1 and test sample 2 is HeLa S3 expressing epitope-tagged histone H2A. Two copies of each probe per array were made.
2011-03-19 | E-GEOD-28041 | biostudies-arrayexpress
Project description:Fusarium equiseti HG18 Transcriptome
| PRJNA1076825 | ENA
Project description:Fusarium equiseti strain:HG18 Genome sequencing
| PRJNA1076715 | ENA
Project description:MacroH2As core histone variants have a unique structure that includes C-terminal nonhistone domain. MacroH2As are highly conserved in vertebrates, and are thought to regulate gene expression. However the nature of genes regulated by macroH2As and the biological significance of macroH2As for the organism remain unclear. Our gene expression studies indicate that macroH2A.1 and macroH2A.2 work together to regulate specific genes. In these studies we examine the distributions of macroH2A.1 and macroH2A.2 nucleosomes to determine if they are localized to the genes that show altered expression in macroH2A knockout mouse liver. MacroH2A.1 and macroH2A.2 nucleosomes prepared from ~ 50 fetal mouse livers were purified by thio-affinity chromatography. Five samples were sequenced: Thiopropyl Sepharose, Normal Liver - contains mononucleosomal DNA from macroH2A.1-containing nucleosomes; Activate Thiol Sepharose, Normal Liver - contains mononucleosomal DNA primarily from macroH2A.2-containing nucleosomes. Starting Material, Normal Liver - this is a reference samplefor the first two samples. It contains mononucleosomal DNA from bulk fetal liver chromatin. Activated Thiol Sepharose, Knockout Livers - this is a control sample that contains mononucleosomal DNA from non-macroH2A nucleosomes that contaminate the macroH2A.2 nucleosomes. This fraction was prepared from macroH2A1/2 double knockout fetal livers; Starting Material, Knockout Liver - this is a reference sample for the fourth sample. It contains mononucleosomal DNA from bulk chromatin prepared from macroH2A1/2 double knockout fetal livers.
2014-10-15 | E-GEOD-57796 | biostudies-arrayexpress
Project description:The fundamental unit of eukaryotic chromatin is the nucleosome core particle, a protein/DNA complex that binds ∼147 base pairs of DNA to a histone octamer. These histones-H3, H4, H2A, H2B-form the nucleosome core through a stacked interaction in which two H2A-H2B dimers flank the (H3-H4)2 tetramer. In vivo, genetic accessibility can be modulated by the substitution of canonical histones with variant proteins that contain the same structural motif but a different amino acid sequence, such as the transcriptional repression-associated macroH2A variant. Previously, Chakravarthy and Luger published a crystal study that showed that H2A substitution is not necessarily required of both H2A moieties, but that in vitro recombination of nucleosomes in the presence of both macroH2A and H2A histone folds results in a hybrid macroH2A-H2A nucleosome with one dimer of each type. Here, we present molecular dynamics simulations of this hybrid construct and compare the results to our previous study on homogeneous H2A- and macroH2A-containing nucleosomes. We find that the hybrid contains a unique set of dynamics that stabilize the interactions between protein constituents and create an altogether more stable nucleosome, both in terms of protein-DNA and protein-protein binding. While dimer-tetramer interactions are asymmetric, as the difference in moieties would suggest, we observe that it is the canonical dimer that is pulled further into the nucleosome core, resulting in more secure dimer-tetramer bonds and a more stable histone core, and we also find significantly more interaction between the dimer subunits. Together, these models provide evidence for hybrid H2A-macroH2A nucleosome formation being not only possible but actually energetically more favorable than a homogeneous construct, with dynamics that are unique from their homogeneous H2A or macroH2A nucleosome counterparts. These effects of hybrid substitution likely propagate into higher-order chromatin structures to hinder transcriptional activity.
| S-EPMC6584036 | biostudies-literature
Project description:Bapx1 was endogenously tagged with S-Peptide and dissected vertebral columns from these tagged embryos at E12.5 were subjected to immunoprecipitation by S-peptide antibody. Simultaneously V5 tagged Bapx1 was also over expressed in NIH_3T3 cells and immunoprecipitated with V5 antibody. Concurrently vertebral column fro E12.5 mouse embryos were subjected to immunoprecipitation with Sox9 antobody to compare binding sites of Sox9 and BApx1 in the vertebral column of developing mouse embryo
2015-03-19 | GSE36672 | GEO
Project description:Interaction proteomics in cell extracts on tagged macroH2A, either expressed in cells or using recombinant dimers with H2B.
2023-10-24 | PXD045696 | Pride
Project description:To investigate the relationship between histones, chaperone function, and cataracts, we performed RNA-seq, isothermal titration calorimetry (ITC), size-exclusion chromatography, and gel electrophoresis of histones. The RNA-seq of postnatal lenses from 2-day-old cryaa -R49C mice revealed increased histone gene expression, suggesting that a α-crystallin mutation regulates histones via a transcriptional mechanism .
2017-08-20 | GSE98027 | GEO
Project description:This track depicts high throughput sequencing of long RNAs (>200 nt) from RNA samples from tissues or subcellular compartments from ENCODE cell lines. The overall goal of the ENCODE project is to identify and characterize all functional elements in the sequence of the human genome. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Sample preparation and sequencing: K562 and GM12878 total cell, total RNA: Standard Illumina Pair-end kit with the sole exception that a "tagged" random hexamer was used to prime the 1st strand synthesis: 5'-ACTGTAGGN6-3'. The addition of this tag is what permits us to make strand assignments for the reads. The sequence of the tag is reported in the 5' end of the read. Asymmetric PCR can place the tag on either the 1st or 2nd read depending on which strand it used as a template. Strand assignments are made by looking for the tag at the 5' end of either read 1 or read 2. Read 1 is physically linked to read 2. Therefore, if a tag is present on one end strand assignments are made for both ends. We noted during analysis that the tags are generally 5' truncated. We only "strand" reads that contain ACTGTAGG, CTGTAGG, TGTAGG, GTAGG. Between 63-68% of reads could be stranded in these libraries. It is possible to cull additional stranded reads that contain non-templated TAGG, AGG, GG, or G sequences at their 5' end. The peak in insert size distribution is between 200-250 nucleotides. K562 cytosol, polyA+ RNA: Oligo-dT selected poly-A+ RNA was RiboMinus-treated according to the manufacturer's protocol (Invitrogen). The RNA was treated with tobacco alkaline pyrophosphatase to eliminate any 5' cap structures and hydrolyzed to ~200 bases via alkaline hydrolysis. The 3' end was repaired using calf intestinal alkaline phosphatase, and poly-A polymerase was used to catalyze the addition of Cs to the 3' end. The 5' end was phosphorylated using T4 PNK, and an RNA linker was ligated onto the 5' end. Reverse transcription was carried out using a poly-G oligo with a defined 5' extension. The inserts were then amplified using oligos targeting the 5' linker and poly-G extension. This cloning protocol generated stranded reads that were read from the 5' ends of the inserts. The library was sequenced on a Solexa platform for a total of 36 cycles; however, the reads underwent post-processing, resulting in trimming of their 3' ends. Consequently, the mapped read lengths are variable. Analysis: K562 and GM12878 total cell, total RNA: Tags were removed from the 5' ends of the reads in accordance to their lengths and strand assignments made. Subsequently, the reads were trimmed from their 3' ends to a final length of 50 nucleotides and were mapped using NexAlign, a program developed by Timo Lassman, RIKEN. We allowed up to 2 mismatches across the entire length and only report reads that mapped to a single/unique locus in the assembled hg18 genome. K562 cytosol, polyA+ RNA: Reads were mapped to the human (hg18, March 2006) assembly using Nexalign, with only uniquely mapping (one loci), exactly matching (no mis-matches) aligned reads reported in the processed files, as follows: 1) Collect the read sequences from Illumina non-filtered output files. 2) Filter out all reads that contain undefined nucleotides ('N'). 3) Perform iterative alignment/C-tail chopping algorithm (below). On each alignment step, the reads are aligned to the genome with 100% identity. All reads that align to a single locus are withdrawn from the alignment pool and only the reads that could not be aligned continue to the next step. a) Align to the hg18 genome using Nexalign 1.3.3 (© Timo Lassmann) without chopping off any nucleotides. b) Chop off any C-blocks (until the first non-C) at the ends of the reads. c) Align to the genome -> remove and save those that align. d) Chop off any non-Cs until the next C. e) Chop off C-block until the next non-C. f) Align to the genome -> remove and save those that align. g) Repeat steps d, e, and f until the reads align to the genome, or chopping results in the reduction of the reads' lengths to below 16 (default), or there are no non-Cs left.
2011-10-12 | E-GEOD-32931 | biostudies-arrayexpress
Project description:Eukaryotic chromatin structure is highly conserved, with the canonical histone proteins revealing only small sequence changes across species. Yet, all vertebrates exhibit three much larger histone H2A variants, macroH2A. A distinctive feature of these atypical histones is the globular macrodomain module, which can bind metabolites and is connected to the histone fold through a flexible linker. MacroH2A variants impact heterochromatin organization, transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how these large H2A variants are incorporated into chromatin and the identity of any chaperones required for histone deposition have remained elusive. Here, we developed a split-GFP-based cellular readout for histone incorporation and conducted a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We identified and validated the histone chaperone ANP32B as a regulator of macroH2A chromatin deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetraso me. In cells, depletion of ANP32B in cells strongly affects global macroH2A deposition, revealing ANP32B as a macroH2A chaperone. Our study highlights the power of haploid cell functional genomics coupled with cellular imaging to identify factors that are required for chromatin plasticity and diversity.
2023-11-07 | GSE241387 | GEO