In a screen for ATP-dependent chromatin remodeling proteins in Tetrahymena thermophila, we identified and partially characterized two conserved Chromodomain Helicase DNA-binding (Chd) proteins of the SNF2 superfamily, Chd3 and Chd7. Tetrahymena Chd proteins have essential and diverse functions in growth and development. These proteins contain chromo-, bromo-, and PHD- domains and classically bind DNA. Chromodomain proteins and other ATP-dependent chromatin remodelers can also bind mRNAs, as well as ncRNAs during transcriptional silencing. These nucleic acid binding complexes are called DNA- and RNA-binding Proteins (DRBPs). DRBPs likely play a role in promoting recognition and removal of Internal Eliminated Sequences (IES) and irreversible genome silencing. A bioinformatic analysis of developmental-specific Tetrahymena proteins with DNA and RNA binding domains identified Chd3 and Chd7 among candidate DRBPs. To study three-way DNA:RNA:protein interaction, a three-way immunoprecipitation protocol was used. Protein Immunoprecipitation (PIP), Chromatin Immunoprecipitation (ChIP), and RNA Immunoprecipitation (RIP) were used to identify DRBP interactions in an experimental setup called IP3.
FZZ-tagged constructs of Chd3 and Chd7 were transformed into Tetrahymena for expression analysis, localization, and purification. Indirect immunofluoresence in vegetative growth localized these proteins to the macronucleus (MAC). During development, Chd3 and Chd7 expression was lost in the old MAC, and localized exclusively in the developing MAC from 6-8 hours of conjugation. There was no localization to micronuclei (MIC) at any time. This is indicative of zygotic expression in anlagen, consistent with microarray and RNA-seq expression data. Affinity purification/mass spectrometry (APMS) of Chd3 and Chd7 in vegetative and conjugating cells indicate interaction with core histone proteins, heat shock protein 70, and an RNA-recognition motif protein. We have focused on and will discuss the interaction of a previously uncharacterized MIZ Ring zinc finger (Znf) protein with Chd3. RNA immunoprecipitation and Urea-PAGE show Chd3 and Chd7 bind diverse RNAs, including multiple long RNAs, a 100nt ssRNA and an 80nt ssRNA species. Experiments in progress to be presented include RNAseq to identify RNA species immunoprecipitated, ChIP analysis to identify loci the DNA-binding chromatin remodelers are recruited to, and protein microarray analysis to identify histone modifications bound by Chd proteins.