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Ubiquitination of Histone H2B Regulates Chromatin Dynamics by Enhancing Nucleosome Stability

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Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability Mahesh B. Chandrasekharan, Fu Huang, and Zu-Wen Sun1 Department of Biochemistry and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 Communicated by C. David Allis, The Rockefeller University, New York, NY, July 15, 2009 (received for review February 6, 2009) The mechanism by which ubiquitination of histone H2B (H2Bub1) (13, 15), but precisely how it causes this structural change has not regulates H3-K4 and -K79 methylation and the histone H2A-H2B yet been defined. In this study, the role of H2Bub1 in regulating chaperone Spt16-mediated nucleosome dynamics during transcrip- global chromatin structure was investigated. We found that tion is not fully understood. Upon investigating the effect of inducing the bulkier sumoylation at the H2B C terminus cannot H2Bub1 on chromatin structure, we find that contrary to the functionally replace ubiquitination to support H3 methylation. supposed role for H2Bub1 in opening up chromatin, it is important Thus, the SUMO or ubiquitin moiety, do not act as a mere for nucleosome stability. First, we show that H2Bub1 does not ‘‘wedge’’ to unfold chromatin. Using a repertoire of biochemical function as a ‘‘wedge’’ to non-specifically unfold chromatin, as and genetic analyses, we uncover an unexpected finding that replacement of ubiquitin with a bulkier SUMO molecule conju- H2Bub1 stabilizes nucleosomes. Collectively, our study provides gated to the C-terminal helix of H2B cannot functionally support a mechanism, wherein the transcriptional processes and histone H3-K4 and -K79 methylation. Second, using a series of biochemical chaperone-mediated chromatin dynamics are regulated by the analyses, we demonstrate that nucleosome stability is reduced or concerted action of H2Bub1 and its deubiquitination via the enhanced, when the levels of H2Bub1 are abolished or increased, stabilization and destabilization of the nucleosome, respectively. respectively. Besides transcription elongation, we show that H2Bub1 regulates initiation by stabilizing nucleosomes positioned Results over the promoters of repressed genes. Collectively, our study The Engineered H2B Sumoylation Mimics the Occurrence but Not the reveals an intrinsic difference in the property of chromatin assem- Function of H2Bub1 on Chromatin. If the bulky ubiquitin (7.5 kDa) bled in the presence or absence of H2Bub1 and implicates the moiety at the H2B C terminus acts as a ‘‘wedge’’ to non- regulation of nucleosome stability as the mechanism by which specifically unfold chromatin, we reasoned that attaching a H2Bub1 modulates nucleosome dynamics and histone methylation bulkier SUMO (12 kDa) might work similar to H2Bub1 in during transcription. mediating H3-K4 and -K79 methylation. To test this hypothesis, residues T122 and K123 in H2B were replaced by inserting two elongation ͉ methylation ͉ sumoylation consensus sumoylation sites (⌿KxE; ⌿, a hydrophobic residue; x, any amino acid) to obtain an engineered H2B [H2B(2SU); Fig. A onoubiquitination of histones H2A (H2Aub1) and H2B 1 ]. To determine sumoylation and/or ubiquitination of H2B(2SU) by Western blotting, whole cell extracts were pre- (H2Bub1) plays important roles in regulating gene expres- M pared from yeast strains containing Flag epitope-tagged H2B, sion (1). In yeast, Rad6 conjugates ubiquitin to lysine 123 (K123) H2B(2SU) or their mutant derivatives. In H2B(2SU) strain, two at the H2B C terminus (2), which in turn regulates the estab- ␣-Flag cross-reacting proteins migrating slower than H2Bub1 lishment of H3-K4 and -K79 methylation and gene silencing were seen in addition to the unmodified H2B(2SU) (Fig. 1B; (3–6). To explain this trans-histone cross-talk, it was postulated lanes 2 and 4). The faster migrating species is the ubiquitinated- that H2Bub1 might act as a ‘‘wedge’’ to non-specifically unfold H2B(2SU), as it is RAD6-dependent (Fig. 1B, lane 6). The slower chromatin for the methyltransferases (Set1 and Dot1) to gain migrating species might be sumoylated H2B(2SU), as it is access to their substrates or, H2Bub1 might function as a dependent on Siz1 (the SUMO E3 ligase) (Fig. 1C). Immuno- ‘‘bridge’’ to directly recruit them (6, 7). Two recent studies precipitation (IP) using ␣-Flag and Western blotting with implicate Swd2, a Set1-COMPASS complex subunit, as the link ␣-SUMO confirm that H2B(2SU) is indeed sumoylated (Fig. between H2Bub1 and H3-K4 methylation (8, 9). Swd2 seems to S1A). Sumoylation has been shown to occur at the N terminus regulate H3-K79 methylation by recruiting Dot1 (8). As viable of H2B (16), but it was not detected in H2B strain or its Swd2 mutants mainly affect H3-K4 trimethylation in vivo (9–10), derivatives after IP (Fig. S1A; lanes 2, 3, and 5), probably due to there might be other regulator(s) mediating H3-K4 mono- and its low abundance as compared to H2B(2SU)su. dimethylation. Nevertheless, the question as to whether H2Bub1 Each lysine in the inserted sumoylation sites was replaced with plays a structural (wedge) or signaling (bridge) role in modu- a leucine to test whether sumoylation occurs at one or both sites lating Swd2 or any other regulator(s) remains unanswered. (K1L and K2L; Fig. 1A). The K1L mutation prevented ubiq- Recent studies revealed that H2Bub1 regulates transcription uitination, but not sumoylation; and only ubiquitination was elongation independent of H3 methylation (11–13). In vitro detected in the H2B(2SU)-K2L strain (Fig. 1B). Thus, ubiquiti- transcription elongation experiments have shown that H2Bub1 nation and sumoylation mainly occur on K1 and K2 in promotes the function of human H2A-H2B chaperone, FACT H2B(2SU), respectively. Next, we tested whether H2B(2SU)su is (hFACT), in stimulating Pol II passage through a nucleosomal associated with chromatin. First, fractionation of yeast sphero- template by displacing an H2A-H2B dimer (12, 14). Addition- plasts to separate proteins localized to cytoplasm or nucleus ally, H2Bub1 and Spt16 (a subunit of the yeast FACT) function revealed that H2B(2SU)su partitioned with the chromatin- cooperatively in nucleosome reassembly in the wake of elongat- ing Pol II (15). Collectively, these findings imply an important role for H2Bub1 in modulating nucleosome dynamics during Author contributions: M.B.C. and Z.-W.S. designed research; M.B.C. and F.H. performed transcription. research; M.B.C., F.H., and Z.-W.S. analyzed data; and M.B.C. and Z.-W.S. wrote the paper. Since ubiquitination leads to a bulky moiety addition onto The authors declare no conflict of interest. histones, it is expected to exert a dramatic effect on chromatin 1To whom correspondence should be addressed. E-mail: [email protected]. structure and transcription. Indeed, H2Bub1 has been proposed This article contains supporting information online at www.pnas.org/cgi/content/full/ to exert its regulatory functions by affecting chromatin structure 0907862106/DCSupplemental. 16686–16691 ͉ PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907862106 Downloaded by guest on October 3, 2021 H2B …VSEGTRAVTKYSSSTQA rad6 K1L K2L A 123 A H2B(2SU) H2B(2SU) H2B(2SU) H2B(2SU) …VSEGTRAV G YSSSTQA H2B(2SU) IKQE IKQE Marker MNase K1 K2 H2B(2SU)-K1L …VSEGTRAVILQEGIKQEYSSSTQA H2B(2SU)-K2L …VSEGTRAVIKQEGILQEYSSSTQA B 6 rad 2B(2SU)/ H2B no H2Btag H2B-K123RH2B(2SU)H2B/rad6H H2B(2SU)-K1LH2B(2SU)-K2L * H2B(2SU)su B H2B-K123R ubp8 H2B(2SU)ub1 H2B H2Bub1 -Flag Marker 0 1 2.5 5 10 0 1 2.5 5 10 0 1 2.5 5 10 Time H2B(2SU) (min) H2B 15678243 C 2B(2SU)-K1L H2B no H2Btag H2B(2SU)H2B(2SU)-K1LH2B H2B(2SU)H * H2B(2SU)su Fig. 2. H2Bub1 levels can differentially affect the sensitivity of chromatin to H2B(2SU)ub1 micrococcal nuclease digestion. (A) Nuclei isolated from the indicated strains -Flag H2Bub1 were treated with increasing MNase concentrations. Undigested or partially H2B(2SU) H2B digested DNA was resolved in a 1.8% agarose gel. (B) Nuclei isolated from the 1456723 indicated strains were treated with MNase (5 U/mL) for increasing incubation SIZ1 siz1 time. Arrowheads indicate the nucleosomal ladder. H2B(2SU)-K2L D 6 H2B(2SU) H2Bub1 (17), H2B(2SU)su is present in the 5Ј ORF of consti- 4 tutively expressed genes (PMA1 and DMA2) and in an ORF-free 2 intergenic region on chromosome V, but is absent from the H2B(2SU)su Fold change in right-end telomeric region of chromosome VI (Fig. 1D). 0 PMA1 DMA2 INT-V TEL-VI-R As only sumoylation was detected in H2B(2SU)-K1L, this mutant was used to determine whether sumoylation is sufficient E to mediate H3-K4 and -K79 methylation similar to H2Bub1. In ad6 r contrast to H2B(2SU) and H2B(2SU)-K2L, H3-K4 di- and 2SU)-K2L trimethylation were totally abolished in the H2B(2SU)-K1L 2B(2SU)-K1L H2B noH2B tag H2B-K123RH2B(2SU)H2B/rad6H2B(2SU)/H H2B( mutant (Fig. 1E). Likewise, H3-K79 methylation levels in -H3K4me1 H2B(2SU)-K1L are similar to those in H2B-K123R and rad6⌬ -H3K4me2 strains (Fig. 1E). Therefore, although H2B(2SU)su is associated -H3K4me3 with transcriptionally active euchromatin and excluded from -H3 heterochromatin-like regions (Fig. 1D), it cannot support H3-K4 -H3K79me1 and -K79 methylation. -H3K79me2 -H3K79me3 H2Bub1 Regulates Global Chromatin Structure. We wondered -H3 whether the bulky SUMO at the H2B(2SU) C terminus exerts 123 45678 any effect on bulk chromatin structure. To this end, we used Fig. 1. Sumoylation induced at the H2B C terminus cannot functionally micrococcal nuclease (MNase), an enzyme that preferentially replace H2Bub1 in mediating H3-K4 and -K79 Methylation. (A) Amino acid cleaves the linker region between the nucleosomes, but can also sequence of the C-terminal region of yeast histone H2B from valine (114) to nick the nucleosomal DNA (18).
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  • DNA Condensation and Packaging

    DNA Condensation and Packaging

    DNA condensation and packaging October 13, 2009 Professor Wilma K. Olson Viral DNA - chain molecules in confined spaces Viruses come in all shapes and sizes Clockwise: Human immuno deficiency virus (HIV); Aeromonas virus 31, Influenza virus, Orf virus, Herpes simplex virus (HSV), Small pox virus Image from U Wisconsin Microbial World website: http://bioinfo.bact.wisc.edu DNA packaging pathway of T3 and T7 bacteriophages • In vivo pathway - solid arrows Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.” J. Mol. Biol. 384, 1384-1399 Cryo EM images of T3 capsids with 10.6 kbp packaged DNA • Labels mark particles representative of different types of capsids • Arrows point to tails on capsids Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.”” J. Mol. Biol. 384, 1384-1399 Cryo EM images of representative particles • (b) 10.6 kbp DNA • (c) 22 kbp DNA • (d) bacteriophage T3 Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.” J. Mol. Biol. 384, 1384-1399 3D icosohedral reconstructions of cryo-EM-imaged particles Threefold surface views and central cross sections • (b) 10.6 kbp DNA • (c) 22 kbp DNA • (d) bacteriophage T3 Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.” J. Mol. Biol. 384, 1384-1399 Top-down views of λ phage DNA toroids captured in cryo-EM micrographs Note the circumferential winding of DNA found in collapsed toroidal particles produced in the presence of multi-valent cations. Hud & Vilfan (2005) “Toroidal DNA condensates: unraveling the fine structure and the role of nucleation in determining size.” Ann.
  • Staining, and in Situ Digestion with Restriction Endonucleases

    Staining, and in Situ Digestion with Restriction Endonucleases

    Heredity66 (1991) 403—409 Received 23 August 1990 Genetical Society of Great Britain An analysis of coho salmon chromatin by means of C-banding, AG- and fluorochrome staining, and in situ digestion with restriction endonucleases R. LOZANO, C. RUIZ REJON* & M. RUIZ REJON* Departamento de Biologia Animal, Ecologia y Genética. E. /ngenierIa T. AgrIcola, Campus Universitario de Almeria, 04120 AlmerIa and *Facu/tad de Ciencias, 18071 Granada, Universidad de Granada, Spain Thechromosome complement of the coho salmon (Oncorhynchus kisutch) has been analysed by means of C-banding, silver and fluorochrome staining, and in situ digestion with restriction endo- nucleases. C-banding shows heterochromatic regions in the centromeres of most chromosomes but not in the telomeric areas. The fifteenth metacentric chromosome pair contains a large block of constitutive heterochromatin, which occupies almost all of one chromosome arm. This region is also the site where the ribosomal cistrons are located and it reacts positively to CMA3/DA fluorochrome staining. The NORs are subject to chromosome polymorphism, which might be explicable in terms of an amplification of ribosomal cistrons. The digestion banding patterns produced by four types of restriction endonucleases on the euchromatic and heterochromatic regions are described. Two kinds of highly repetitive DNAs can be distinguished and the role of restriction endonucleases as a valuable tool in chromosome characterization studies, as well as in the analysis of the structure and organization of fish chromatin, are also discussed. Keywords:C-banding,coho salmon, fluorochrome staining, restriction endonuclease banding. (Oncorhynchus kisutch), as well as applying conven- Introduction tional banding techniques, we have analysed the Theuse of restriction endonucleases (REs) is becom- mitotic chromosomes using DNA base-pair-specific ing common not only in molecular biology but also as fluorochromes and in situ digestion with restriction an important tool in molecular cytogenetics.