Aggregation of the Aβ1-40 peptide is linked to the development of extracellular plaques characteristic of Alzheimer’s disease. While previous studies commonly show the Aβ1-40 is largely unstructured in solution, we show that Aβ1-40 can adopt a compact, partially folded structure. In this structure, the central hydrophobic region of the peptide forms a 310 helix from H13 to D23 and the N- and C-termini collapse against the helix due to the clustering of hydrophobic residues. Helical intermediates have been predicted to be crucial on-pathway intermediates in amyloid fibrillogenesis, and the structure presented here presents a new target for investigation of early events in Aβ1-40 fibrillogenesis.
Histone modification patterns and their combinatorial readout have emerged as a fundamental mechanism for epigenetic regulation. Here we characterized Spindlin1 as a histone effector that senses a cis-tail histone H3 methylation pattern involving trimethyllysine 4 (H3K4me3) and asymmetric dimethylarginine 8 (H3R8me2a) marks. Spindlin1 consists of triple tudor-like Spin/Ssty repeats. Cocrystal structure determination established concurrent recognition of H3K4me3 and H3R8me2a by Spin/Ssty repeats 2 and 1, respectively. Both H3K4me3 and H3R8me2a are recognized using an ''insertion cavity'' recognition mode, contributing to a methylation statespecific layer of regulation. In vivo functional studies suggest that Spindlin1 activates Wnt/b-catenin signaling downstream from protein arginine methyltransferase 2 (PRMT2) and the MLL complex, which together are capable of generating a specific H3 ''K4me3-R8me2a'' pattern. Mutagenesis of Spindlin1 reader pockets impairs activation of Wnt target genes. Taken together, our work connects a histone ''lysine-arginine'' methylation pattern readout by Spindlin1-to-Wnt signaling at the transcriptional level.
SUMMARY Crosstalk between H2B ubiquitylation (H2Bub) and H3 K4 methylation plays important roles in coordinating functions of diverse cofactors during transcription activation. The underlying mechanism for this trans-tail signaling pathway is poorly defined in higher eukaryotes. Here, we show that 1) ASH2L in the MLL complex is essential for H2Bub-dependent H3 K4 methylation. Deleting or mutating K99 of the N-terminal winged helix (WH) motif in ASH2L abrogates H2Bub-dependent regulation; 2) crosstalk can occur in trans and does not require ubiquitin to be on nucleosomes or histones to exert regulatory effects; 3) Trans-regulation by ubiquitin promotes MLL activity for all three methylation states; and 4) MLL3, an MLL homolog, does not respond to H2Bub, highlighting regulatory specificity for MLL family histone methyltransferases. Altogether, our results potentially expand the classic histone crosstalk to nonhistone proteins, which broadens the scope of chromatin regulation by ubiquitylation signaling.
SUMMARY PRDM16 is a transcription cofactor that plays critical roles in development of brown adipose tissue (BAT) as well as maintenance of adult hematopoietic and neural stem cells. Here we report that PRDM16 is a histone H3 K4 methyltransferase on chromatin. Mutation in the N-terminal PR-domain of PRDM16 completely abolishes the intrinsic enzymatic activity of PRDM16. We show that the methyltransferase activity of PRDM16 is required for specific suppression of MLL leukemogenesis both in vitro and in vivo. Mechanistic studies show that PRDM16 directly activates the SNAG family transcription factor GFI1b, which in turn down regulates the HOXA gene cluster. Knockdown GFI1b represses PRDM16-mediated tumor suppression while GFI1b overexpression mimics PRDM16 overexpression. In further support of the tumor suppressor function of PRDM16, silencing PRDM16 by DNA methylation is concomitant with MLL-AF9 induced leukemic transformation. Taken together, our study reveals a previously uncharacterized function of PRDM16 that depends on its PR-domain activity.
We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8 M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2 M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted from the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirm that reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro.
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