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.
Highlights
Chromatin is Nature’s elegant architectural solution to the challenge of packing approximately 3 billion base-pairs of human genomic DNA in an average nuclear volume of only about 500 cubic microns
Acetylation of lysine side-chains on H3 and H4 by the acetyltransferase p300, is necessary for efficient activator-driven transcription of both 11-nm chromatin[21] and 30-nm linker histone H1-containing heterochromatin, likely through mechanisms that include direct decompaction of chromatin upon H4K16 acetylation and 96 octamer eviction by the chromatin remodeler NAP1.22, 23. Due to their proposed opposing roles in gene transcription, we investigated the precise nature of biochemical crosstalk between histone sumoylation and histone acetylation by p300
We previously showed that H4K12su stimulates activity of 127 the H3K4me1/2 demethylase, LSD1, in the context of a LSD1-CoREST sub-complex.[17]
Summary
Chromatin is Nature’s elegant architectural solution to the challenge of packing approximately 3 billion base-pairs of human genomic DNA in an average nuclear volume of only about 500 cubic microns. Pioneering studies of the reconstitution of class II promoter-driven accurate eukaryotic transcription in both nuclear extracts and purified systems has led to insights into roles for 87 histone modifications in gene function.[18, 19] The ability to reconstitute chromatinized plasmid templates using chemically modified histones enables studies of the roles of specific histone modifications in transcription and investigations of their crosstalk with key enzymes associated 90 with transcription initiation and elongation.[20] Multiple proteins involved in gene transcription bind to and modify histone tails, which enables the remodeling of chromatin prior to and during transcription One such modification, acetylation of lysine side-chains on H3 and H4 by the acetyltransferase p300, is necessary for efficient activator-driven transcription of both 11-nm chromatin[21] and 30-nm linker histone H1-containing heterochromatin, likely through mechanisms that include direct decompaction of chromatin upon H4K16 acetylation and 96 octamer eviction by the chromatin remodeler NAP1.22, 23. Our observations provide the first unambiguous biochemical demonstration that sumoylated histone H4 directly inhibits RNAPII-mediated transcription from chromatin templates, and reveal its direct negative crosstalk with histone acetylation by p300 and methylation by Set1/COMPASS that are strongly associated with active gene transcription
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