Structural and Biochemical Studies on the Chromo-barrel Domain of Male Specific Lethal 3 (MSL3) Reveal a Binding Preference for Mono- or Dimethyllysine 20 on Histone H4

Abstract

We have determined the human male specific lethal 3 (hMSL3) chromo-barrel domain structure by x-ray crystallography to a resolution of 2.5 Å (r ‫؍‬ 0.226, R free ‫؍‬ 0.270). hMSL3 contains a canonical methyllysine binding pocket made up of residues Tyr-31, Phe-56, Trp-59, and Trp-63. A six-residue insertion between strands ␤ 1 and ␤ 2 of the hMSL3 chromobarrel domain directs the side chain of Glu-21 into the methyllysine binding pocket where it hydrogen bonds to the NH group of a bound cyclohexylamino ethanesulfonate buffer molecule, likely mimicking interactions with a histone tail dimethyllysine residue. In vitro binding studies revealed that both the human and Drosophila MSL3 chromo-barrel domains bind preferentially to peptides representing the mono or dimethyl isoform of lysine 20 on the histone H4 N-terminal tail (H4K20Me 1 or H4K20Me 2 ). Mutation of Tyr-31 to Ala in the hMSL3 methyllysine-binding cage resulted in weaker in vitro binding to H4K20Me 1 . The same mutation in the msl3 gene compromised male survival in Drosophila. Combined mutation of Glu-21 and Pro-22 to Ala in hMSL3 resulted in slightly weaker in vitro binding to H4K20Me 1 , but the corresponding msl3 mutation had no effect on male survival in Drosophila. We propose MSL3 plays an important role in targeting the male specific lethal complex to chromatin in both humans and flies by binding to H4K20Me 1 . Binding studies on the related dMRG15 chromo-barrel domain revealed that MRG15 prefers binding to H4K20Me 3 . Nuclear histone acetyltransferase (HAT)3 enzymes are found in multiprotein complexes that acetylate specific lysine residues on the N-terminal tails of histone proteins, thereby regulating nucleosome structure, chromatin packaging, and gene expression (1-15). MOF, a conserved member of the MYST (Moz, Ysb2, Sas2, Tip60) family of HAT enzymes, functions as the catalytic subunit in a number of distinct HAT complexes that target gene promoters (8), large contiguous domains of chromatin (3, 4, 14, 15), or non-histone proteins such as p53 (5-7). The precise targeting and substrate specificity of MOF relies on the presence of components distinct from the catalytic subunit (3,4,7,8). Specifically, in the MOFcontaining Drosophila male specific lethal (MSL) complex, the MSL3 protein is required for chromatin targeting, nucleosome binding, histone tail substrate recognition, and maximal MOF HAT activity (16 -20).The most well studied MOF-containing complex is the Drosophila melanogaster male specific lethal or MSL complex that binds selectively to large regions of the X-chromosome in male flies (14,15,(21)(22)(23)(24)(25)(26) where it is enriched at the 3Ј ends of actively transcribed genes (27-30) and acetylates lysine 16 on histone H4 (H4K16Ac) (22, 31), thereby balancing male Xchromosomal gene expression. The Drosophila MSL complex contains the dMSL1, dMSL2, and dMSL3 proteins, the RNA/ DNA helicase MLE, the HAT enzyme MOF, and one of two apparently functionally redundant non-coding RNAs (roX1 and roX2) (reviewed in Refs. 14, 15, and 21). The...

A Dual Role for the Histone Methyltransferase PR-SET7/SETD8 and Histone H4 Lysine 20 Monomethylation in the Local Regulation of RNA Polymerase II Pausing

“…In comparison, very little is known about the recruitment mechanism of the human MSL complex, which mediates autosomal gene up-regulation. Recently, the isolated chromodomains of Drosophila and human recombinant MSL3 proteins have been shown to preferentially bind H4K20me1 and H4K20me2 peptides in vitro (40,41). This study provides biological evidence that PR-SET7-mediated H4K20me1, but not H4K20me2, is specifically required for the targeting of the MSL complex in human cells.…”

MRG15 is required for pre-mRNA splicing and spermatogenesis

“…Some other proteins or modifications might participate in the regulation of splicing events of Tpm1, Prm1, and Prm2 mRNAs. Because MRG15 can interact with HAT, HDAC complexes, and other histone modifications, such as methylated H4K20, localization of MRG15 on the genome may be regulated by not only H3K36me3 but also, other histone modifications and/or protein complexes (13)(14)(15)(16)(17)(18)(19)(20)(21)26). PTBPs are known to antagonize exon definition; therefore, those retained sequences could be recognized as an exon on the absence of MRG15 (44).…”

“…H3K36me3 and H4K20me3, both of which are recognized by MRG15, were not changed by lack of MRG15, indicating that MRG15 is not essential for meiotic homologous recombination (SI Appendix, Fig. S3) (25,26).…”

SUV420H2-Mediated H4K20 Trimethylation Enforces RNA Polymerase II Promoter-Proximal Pausing by Blocking hMOF-Dependent H4K16 Acetylation

“…Indeed, recent biochemical and structural data suggest that the chromoshadow domain of the MSL3 component of the MSL complex can bind H4K20me1/2 but cannot bind H4K20me3 (30,40). Thus, the local conversion to H4K20me3 by SUV420H2 could potentially block MSL complex recruitment by precluding MSL3 binding.…”