Psnapf

Full-length cDNA of mouse macroH2A1.1, macroH2A1.2 and macroH2A2 were cloned from WT DFs. MacroH2A isoforms were subcloned into pEGFP-N1 to add a C-terminal GFP tag. Tagged macroH2A isoforms were subcloned into lentiviral vectors pLVX (Clontech) for dox-inducible expression or pHAGE2 for constitutive expression under human EF-1α promoter. GFP and GFP-H2A (human) were subcloned into the same lentiviral vectors. Mouse H3.1 and macroH2A isoforms were subcloned into pSNAPf (NEB) to generate N-terminally SNAP-tagged H3.1 and C-terminally SNAP-tagged macroH2A isoforms. SNAP-tagged histones were subcloned into pHAGE2 for constitutive expression. Full-length and truncated macroH2A2s (histone fold region and non-histone region with NLS) were N-terminally GFP-tagged and subcloned into lentiviral vectors for constitutive expression. sgRNAs for Tks4 TSS deletion were designed using CRISPR Design Tool (http://crispr.mit.edu) and cloned into pLKO.1-sgRNA-mCherry. A scrambled sgRNA was used as control. sgRNA targeting Hoxc13 promoter was designed with Cas9 Activator Tool (http://sam.genome-engineering.org/database) and cloned into lenti-sgRNA(MS2)-Zeo. pLKO.1 based shRNA constructs (TRC lentiviral shRNA, Open Biosystems) were used for SPT16 knockdown. A scrambled shRNA (shScr) was used as control. Targeting sequences of all sgRNAs and shRNAs are listed in Supplementary Table 3.

The pHaloTag–GR has been previously described [11 (link)]. Briefly, the construct expresses the rat GR with HaloTag protein (Promega, Madison, WI, USA) fused in the C-terminal domain under the CMVd1 promoter. The SNAP-tag–GR expresses the rat GR with SNAP-tag protein fused in the C-terminal domain under the CMV promoter. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pSNAPf (N9183S, New England Biolabs, Ipswich, MA, USA) backbone with NheI and AgeI sites. The CLIP-tag–GR expresses the rat GR with CLIP-tag fused in the C-terminal domain under the CMV promoter. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [48 (link)], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab. Finally, pEGFP-NF1[49 (link)] was used as an homogenous nuclear marker in Figure 3.