H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing

Abstract

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non‐coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2‐dependent H3K27me3 and SETD8‐dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3‐specific intracellular antibody or H3K27me3‐mintbody. By combining live‐cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP‐seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.

Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

“…RNA was extracted from the hybridoma cells using TRIzol (Thermo Fisher Scientific) and subjected to RNA-seq to determine the DNA sequence encoding the IgG heavy and light chains, as described previously (Kuniyoshi et al, 2016). The variable regions of heavy (VH) and light (VL) chains of 42B3 were amplified and connected, as described previously (Sato et al, 2018;Tjalsma et al, 2021), using sets of primers specific for VH (VH_s, VH_as), VL (VL_s, VL_as), and the linker (LINK primer1 and LINK primer2), which are listed in Table S1. The resulting scFv fragments were further amplified using the 5' and 3' primers (scFv primer_s and scFv primer_as) and cloned into the psfGFP-N1 vector (Addgene #54737, deposited by Michael Davidson and Geoffrey Waldo; Pédelacq et al, 2006) to generate a 42B3-sfGFP expression vector.…”

“…In this study, we developed a genetically encoded probe for visualizing Ser2-phosphorylated RNAP2 in living cells based on the specific antibody. The expression of functional scFv depends on the folding and stability of the framework regions (Cattaneo and Biocca, 1999;Ewert et al, 2004;Sato et al, 2016;Tjalsma et al, 2021). The original 42B3 clone showed a moderate level of nuclear enrichment, an indication of the functionality of scFv that targets a nuclear protein or its modification.…”

“…Introducing several amino acid substitutions improved the nuclear enrichment. Although no universal framework is currently available (Kabayama et al, 2020), some framework sequences are more stable than others and a substitution of the Met residue in a hydrophobic core with a more hydrophobic amino acid, such as Leu and Ile, often increases scFv stability (Tjalsma et al, 2021). In 42B3, Met95 substitution to Val or Ile also improved the nuclear accumulation, implying that such substitutions are a good option to improve the folding and stability of scFv in general.…”

“…To visualize and track RNAP2 Ser2ph conveniently without protein loading, we developed a genetically encoded live-cell probe, which is a modification-specific intracellular antibody, or mintbody, that consists of the single chain variable fragment (scFv) of the specific antibody and superfolder GFP (sfGFP) (Sato et al, 2013;Sato et al, 2016;Tjalsma et al, 2021). Using high-resolution microscopy, we analyzed the relative localization of RNAP2 Ser2ph-mintbody with proteins involved in RNAP2 phosphorylation, elongation, and transcription activation, such as CDK9, CDK12, a Paf1 complex component Leo1, splicing appeared to reach a plateau at ~4,000-5,000, which is close to the number of factories in HeLa cells (Jackson et al, 1998;Pombo et al, 1999).…”

“…Structural modeling showed that the R78K/A80T and M95I mutations likely contribute to intramolecular ionic and hydrophobic interactions, respectively. As the hydrophobic cores of scFv are critical for folding and/or stability Tjalsma et al, 2021), we examined another M95 mutant with Val substitution and this 42B3(R78K/A80T/M95V)-mCherry exhibited the highest N/C ratio (Fig. 1, B and C).…”