Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

Abstract

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

Total chemical synthesis of sumoylated histone H4 reveals negative biochemical crosstalk with histone ubiquitylation

“…Because the specific enzymes for histone sumoylation in cells are presently unknown, we adopted the approach of genetically fusing SUMO-3 to the N-terminus of an H4 truncant to generate the protein construct HA-SUMO-3(ΔGG)-H4(Δ1–11), hereon abbreviated as SU-H4. 4 This genetic fusion places a non-hydrolyzable form of SUMO-3 close to the K12 side-chain in H4. Nuclear chromatin was isolated from HEK293T immortalized human embryonic kidney cells transfected with a plasmid encoding SU-H4, and mononucleosomes were generated by Micrococcal nuclease digestion (Fig.…”

“…2 Histone H4 sumoylation was first reported in human cell lines in 2003, 3 and site-specific sumoylation at Lys12 (H4K12su) was shown to directly inhibit gene transcription from chromatinized plasmid templates. 4 Despite the recent progress in understanding the roles of H4K12su, there is a need to understand its biochemical relationships, or crosstalk, with key histone PTMs associated with gene regulatory programs, such as methylation and ubiquitylation, that are misregulated in human diseases. 5 Our previously reported semisynthesis of H4K12su (Scheme 1A) led to the discovery of cross-talk between histone methylation 6 and acetylation.…”

“…5 Our previously reported semisynthesis of H4K12su (Scheme 1A) led to the discovery of cross-talk between histone methylation 6 and acetylation. 4 However, the efficiency of H4K12su semisynthesis remained limited by (1) the use of a ligation auxiliary that is thermodynamically reversible and precludes complete ligation (Scheme 1A), and (2) inefficient cleavage of an N-terminal Ni 2+ -affinity hexahistidine tag from a truncated form of histone H4 due to the poor solubility of protein in aqueous buffers, which necessitated multiple rounds of tag-cleavage and affinity purification (Scheme 1A). 4,7 Therefore, we sought to overcome these limitations by the total chemical synthesis of histone H4 and the use of a high-yielding Cys-mediated ligation to conjugate SUMO at H4K12 (Scheme 1B).…”

“…In biochemical assays with the purified COMPASS (complex of proteins associated with Set1) histone methyltransferase extended catalytic (eCM) module of 6 proteins from the Kluyveromyces lactis yeast, we discovered that H4K12su inhibits trimethylation at histone H3 (H3K4me3). 4 Our discovery was enabled by the fact that the COMPASS eCM is capable of nucleosome methylation in the absence of ubiquitylated H2B (H2BK120ub). 16 However, nucleosome methylation by the complete 8 protein COMPASS complex in cells is strictly dependent on the presence of H2BK120ub in chromatin.…”

“…Importantly, the negative crosstalk of H4K12su with H2BK120ub, which is associated with actively transcribed genes in cells, is consistent with the proposed role of H4 tail sumoylation in gene repression. 3,4…”