Unique Trophoblast Chromatin Environment Mediated by the PcG Protein SFMBT2 [ChIP-seq]

Sfmbt2 is a paternally imprinted gene critical for the establishment and maintenance of trophoblast stem cells in mice (Miri K et al., 2013). Inheritance of a gene trap null allele (Sfmbt2gt/gt) results in embryonic lethality by e12.5 due to reduced numbers in all trophoblast cell types. SFMBT2 is a mammalian Polycomb group protein (PcG) and is assumed to act as a master transcriptional regulator. ChIP-seq was performed against endogenous and FLAG-SFMBT2 in trophoblast stem cells (TSCs) to complement the RNA-seq dataset acquired from extraembryonic tissues of wildtype and Sfmbt2gt/gt mice. We aim to determine the binding profile of SFMBT2 in TSCs and how it contributes to the observed transcriptomic profile. Trophoblast stem cells derived from C57Bl6/Castaneus mice were cultured as described (Tanaka S et al., 1998). A stable FLAG-SFMBT2 TSC line was generated through lentiviral transfection using Polybrene transfection reagent. Endogenous SFMBT2 and FLAG-SFMBT2 ChIP-seq samples were isolated from wildtype and a stably transfected FLAG-SFMBT2 TSC cell line respectively. To reduce cellular differentiation of TSCs resulting from dissociation of colonies, the weight of 10^7 TSCs was calculated and all subsequent TSCs to be used for ChIP-seq were aliquoted into tubes of 0.23 g. All TSCs were crosslinked and quenched prior before they were aliquoted. ChIP-seq fragmentation was achieved through either sonication (Raga-Iglesias et al., 2011) or micrococcal nuclease digestion (Tsankov AM et al., 2015). To reduce technical variability between samples, all subsequent steps post-immunoprecipitation was performed as published by Raga-Iglesias et al (2011). Of note, FLAG-SFMBT2 crosslinked complexes were immunoprecipitated using FLAG-conjugated magnetic beads (Sigma Aldrich #M8823), while endogenous complexes are immunoprecipitated using protein G Dynabeads (ThermoFisher Scientific #100009D) coated with anti-SFMBT2 generated inhouse (Miri K et al., 2013). To reduce phenol contamination, aqueous fractions were heated at 55oC for 5 mins then cooled to room temperature for 2 mins prior to the ethanol-based DNA precipitation. DNA quantification was performed using PicoGreen and 10 ng of input and ChIP DNA was submitted to The Centre for Applied Genomics (TCAG) for library preparation and next-generation sequencing. All samples were prepared in duplicates except for input DNA. Of note, the sonicated endogenous SFMBT2 ChIP and input DNA library was prepared using the Illumina TruSeq protocol and subject to paired-end sequencing (2 x 100 bp) at an earlier date on the Illumina HiSeq 2500 platform. All other DNA libraries were prepared using the NEB Ultra DNA protocol to account for the low DNA concentration in micrococcal nuclease-digested samples, and subject to paired-end sequencing (2 x 126 bp) on the Illumina HiSeq 2500 platform.