Comment[ArrayExpressAccession] E-GEOD-51737 MAGE-TAB Version 1.1 Public Release Date 2013-10-26 Investigation Title Kinetoplastid-specific histone variant functions are conserved in Leishmania major Comment[Submitted Name] Kinetoplastid-specific histone variant functions are conserved in Leishmania major Experiment Description Protein-coding genes in kinetoplastid protists are transcribed from polycistronic arrays, yielding RNA precursors which are processed to form mature transcripts bearing a 5M-bM-^@M-^Y spliced leader (SL) and 3M-bM-^@M-^Y poly(A) tract. Regions of transcription initiation and termination lack known eukaryotic promoter and terminator elements, and current data suggest that transcription is instead regulated predominantly through epigenetic mechanisms. Several epigenetic marks, including histone modifications, histone variants, and an atypical DNA modification known as base J have been localized to regions of transcription initiation or termination in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Despite this conservation, the phenotypes of base J mutants vary significantly across trypanosomatids, suggesting that the specific epigenetic networks governing transcription initiation and termination have diverged significantly during evolution. In this light, we sought to characterize and compare the roles of the histone variants H2A.Z, H2B.V, and H3.V in L. major. As in T. brucei, the histone variants H2A.Z and H2B.V were shown to be essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast and again similar to T. brucei, H3.V is not essential in Leishmania as H3.V-null lines grew normally, resembled WT, and remained infectious. Using SL-primed RNA-seq, we found that H3.V-null parasites have steady-state transcript levels comparable to WT parasites and display no defects in the efficiency of transcription termination at convergent strand switch regions (SSRs). Our results show a conservation of histone variant phenotypes between L. major and T. brucei, in contrast to the phenotypes associated with the epigenetic DNA base J modification. Total RNA from Four LmjF samples were analyzed using RNA-Seq. One of them is wildtype parasites, one is single knockout for H3V gene and two independent double knockouts for H3V gene. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Ramasamy Anderson Wong Baugh Ramasamy Myler Beverley Person First Name Gowthaman Britta Iris Loren Gowthaman Peter Stephen Person Mid Initials A L J M Person Email Gowthaman.Ramasamy@seattlebiomed.org Person Affiliation SEATTLE BIOMEDICAL RESEARCH INSTITUTE Person Phone 206-256-7188 Person Address GHBC, SEATTLE BIOMEDICAL RESEARCH INSTITUTE, 307 WESTLAKE AVE N, SEATTLE, WASHINGTON, USA Person Roles submitter Protocol Name P-GSE51737-3 P-GSE51737-2 P-GSE51737-1 Protocol Description Basecalls performed using CASAVA version 1.3 Quality of the reads in fastq files were determined using fastqc and internal tools SL RNA-Seq reads were aligned against Leishmania major Friedlin genome Version 4.1 downloaded from www.TriTrypDB.org Samtools v0.1.19 was used to convert alignment files from one format to another Internal tools were used to estimate splice leader counts against each gene Genome_build: LmjF V4.1 Supplementary_files_format_and_content: Gene level splice leader read count data generated using custom scripts. The read counts are median normalzied within each library Logarithmically-growing promastigotes from WT L. major FV1, one H3V/HYG transfectant, and two M-NM-^Th3v transfectants were collected and resuspended at a concentration of 5x108 cells/mL in TriZOL (Invitrogen). The aqueous phase was isolated by adding 0.2 mL chloroform (Fisher Scientific) and centrifugation at 12,000 x g for 15 minutes at 4M-0C. The aqueous phase was isolated and RNA was precipitated by adding 1 volume of 100% isopropanol (Fisher Scientific) and centrifugation at 12,000 x g for 10 minutes at 4M-0C. The RNA pellet was washed with 75% ethanol (Pharmco) and was resuspended in nuclease-free water (Ambion). Purified RNA was treated with 20 units of DNAse I (Ambion) and was precipitated using 1/10 volumes 3M sodium acetate (Sigma) and 3 volumes 100% ethanol. RNA was pelleted by centrifugation at 15,000 g-1 and the resulting pellet was washed with cold 75% ethanol and resuspended in RNAse-free water (Ambion). Next Generation Sequencing (NGS) libraries were constructed by priming ~1 M-5g of total RNA from either the purine-starved or purine-replete sample with a random hexamer primer (TCCGATCTCTNNNNNNN) for first strand cDNA synthesis and an oligonucleotide primer (TCAGTTTCTGTA) derived from the Leishmania splice leader (SL) sequence for second-strand cDNA synthesis. The resultant cDNA was amplified by PCR for 12-15 cycles using the oligonucleotides that overlapped with the initial primers and containing adapters for subsequent cluster generation and sequencing using Solexa sequencing by synthesis (SBS) technology. All studies used derivatives of Leishmania major Friedlin V1 (MHOM/JL/81/Friedlin), grown at 26M-0C in M199 medium (US Biologicals) supplemented with 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesuphonic acid (HEPES) pH 7.4 (Fisher Scientific), 100 uM adenine (Sigma), 1 M-5g mL-1 biotin (Sigma), 10 M-5g mL-1 hemin (Sigma), 2 M-5g mL-1 biopterin (Schircks Laboratories), 50 units/mL penicillin (Gibco), 50 M-5g/mL streptomycin (Gibco), and 10% (v/v) heat inactivated fetal calf serum (HyClone). Cell density was determined by using a model Z1 Coulter counter (logarithmic phase) or hemocytometer (stationary phase). Metacyclic promastigotes were purified from stationary phase day 4 cultures using a negative peanut agglutination assay [22]. Semisolid M199 medium was prepared using supplemented M199 medium with 1% (w/v) Difco noble agar (BD Diagnostic Systems) Protocol Type normalization data transformation protocol nucleic acid library construction protocol growth protocol Experimental Factor Name GENOTYPE Experimental Factor Type genotype Comment[SecondaryAccession] GSE51737 Comment[GEOReleaseDate] 2013-10-26 Comment[ArrayExpressSubmissionDate] 2013-10-25 Comment[GEOLastUpdateDate] 2013-10-28 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE51737_LmjF_HistoneVariant_analysis_MedianNormalizedReadCounts.csv Comment[SecondaryAccession] SRP032094 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1016916-SRR1016919 SDRF File E-GEOD-51737.sdrf.txt