Danny Reinberg

Publications (122)1686.62 Total impact

• Article: Histone chaperone FACT action during transcription through chromatin by RNA polymerase II.

  Fu-Kai Hsieh, Olga I Kulaeva, Smita S Patel, Pamela N Dyer, Karolin Luger, Danny Reinberg, Vasily M Studitsky
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  ABSTRACT: FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)-nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT-H2A/H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.
  Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
• Article: SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors.

  Jin Zhang, Roberto Bonasio, Francesco Strino, Yuval Kluger, J Kim Holloway, Andrew J Modzelewski, Paula E Cohen, Danny Reinberg
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  ABSTRACT: SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1-LSD1-CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.
  Genes & development 04/2013; 27(7):749-766. · 12.08 Impact Factor
• Article: Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4.

  Shilpa S Dhar, Sung-Hun Lee, Pu-Yeh Kan, Philipp Voigt, Li Ma, Xiaobing Shi, Danny Reinberg, Min Gyu Lee
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  ABSTRACT: Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD(4-6)) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD(4-6) reduce PHD(4-6)'s binding ability and MLL4's catalytic activity. PHD(4-6)'s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD(4-6)'s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation.
  Genes & development 12/2012; 26(24):2749-62. · 12.08 Impact Factor
• Article: A chromatin link to caste identity in the carpenter ant Camponotus floridanus.

  Daniel F Simola, Chaoyang Ye, Navdeep S Mutti, Kelly Dolezal, Roberto Bonasio, Juergen Liebig, Danny Reinberg, Shelley L Berger
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  ABSTRACT: In many ant species, sibling larvae follow alternative ontogenetic trajectories that generate striking variation in morphology and behavior among adults. These organism-level outcomes are often determined by environmental rather than genetic factors. Therefore, epigenetic mechanisms may mediate the expression of adult polyphenisms. We produced the first genome-wide maps of chromatin structure in a eusocial insect and found that gene-proximal changes in histone modifications, notably H3K27 acetylation, discriminate two female worker and male castes in Camponotus floridanus ants and partially explain differential gene expression between castes. Genes showing coordinated changes in H3K27ac and RNA implicate muscle development, neuronal regulation, and sensory responses in modulating caste identity. Binding sites of the acetyltransferase CBP harbor the greatest caste variation in H3K27ac, are enriched with motifs for conserved transcription factors, and show evolutionary expansion near developmental and neuronal genes. These results suggest that environmental effects on caste identity may be mediated by differential recruitment of CBP to chromatin. We propose that epigenetic mechanisms that modify chromatin structure may help orchestrate the generation and maintenance of polyphenic caste morphology and social behavior in ants.
  Genome Research 12/2012; · 13.61 Impact Factor
• Article: The role of PR-Set7 in replication licensing depends on Suv4-20h.

  David B Beck, Adam Burton, Hisanobu Oda, Céline Ziegler-Birling, Maria-Elena Torres-Padilla, Danny Reinberg
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  ABSTRACT: PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation (H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2. PR-Set7 is required for proper cell cycle progression and is subject to degradation by the CRL4(Cdt2) ubiquitin ligase complex as a function of the cell cycle and DNA damage. This report demonstrates that PR-Set7 is an important downstream effector of CRL4(Cdt2) function during origin of DNA replication licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation (H4K20me3). Expression of a degradation-resistant PR-Set7 mutant in the mouse embryo that is normally devoid of Suv4-20 does not compromise development or cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an artificial locus results in recruitment of the origin recognition complex (ORC) in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20 methylation status plays a direct role in recruiting ORC through the binding properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes.
  Genes & development 11/2012; · 12.08 Impact Factor
• Article: SIRT3 Functions in the Nucleus in the Control of Stress-Related Gene Expression.

  Toshinori Iwahara, Roberto Bonasio, Varun Narendra, Danny Reinberg
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  ABSTRACT: SIRT3 is a member of the Sir2 family of NAD(+)-dependent protein deacetylases that promotes longevity in many organisms. The processed, short form of SIRT3 is a well-established mitochondrial protein whose deacetylase activity regulates various metabolic processes. However, the presence of full-length (FL) SIRT3 in the nucleus and its functional importance remains controversial. Our previous studies demonstrated that nuclear FL-SIRT3 functions as a histone deacetylase and is transcriptionally repressive when artificially recruited to a reporter gene. Here, we report that nuclear FL-SIRT3 is subjected to rapid degradation upon cellular stress, including oxidative stress and UV-irradiation, whereas the mitochondrial, processed form is unaffected. FL-SIRT3 degradation is mediated by the ubiquitin-proteasome pathway, at least partially through the E3 activity of SKP2. Finally, we show by chromatin immunoprecipitation that some target genes of nuclear SIRT3 are derepressed upon the degradation of SIRT3 caused by stress stimuli. Thus, SIRT3 exhibits a previously unappreciated role in the nucleus modulating the expression of some stress-related and nuclear-encoded mitochondrial genes.
  Molecular and cellular biology 10/2012; · 6.06 Impact Factor
• Article: Asymmetrically modified nucleosomes.

  Philipp Voigt, Gary Leroy, William J Drury, Barry M Zee, Jinsook Son, David B Beck, Nicolas L Young, Benjamin A Garcia, Danny Reinberg
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  ABSTRACT: Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.
  Cell 09/2012; 151(1):181-93. · 32.40 Impact Factor
• Article: Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity.

  Inês Pinheiro, Raphaël Margueron, Nicholas Shukeir, Michael Eisold, Christoph Fritzsch, Florian M Richter, Gerhard Mittler, Christel Genoud, Susumu Goyama, Mineo Kurokawa, Jinsook Son, Danny Reinberg, Monika Lachner, Thomas Jenuwein
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  ABSTRACT: Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.
  Cell 08/2012; 150(5):948-60. · 32.40 Impact Factor
• Article: Genome-wide and Caste-Specific DNA Methylomes of the Ants Camponotus floridanus and Harpegnathos saltator.

  Roberto Bonasio, Qiye Li, Jinmin Lian, Navdeep S Mutti, Lijun Jin, Hongmei Zhao, Pei Zhang, Ping Wen, Hui Xiang, Yun Ding, Zonghui Jin, Steven S Shen, Zongji Wang, Wen Wang, Jun Wang, Shelley L Berger, Jürgen Liebig, Guojie Zhang, Danny Reinberg
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  ABSTRACT: Ant societies comprise individuals belonging to different castes characterized by specialized morphologies and behaviors. Because ant embryos can follow different developmental trajectories, epigenetic mechanisms must play a role in caste determination. Ants have a full set of DNA methyltransferases and their genomes contain methylcytosine. To determine the relationship between DNA methylation and phenotypic plasticity in ants, we obtained and compared the genome-wide methylomes of different castes and developmental stages of Camponotus floridanus and Harpegnathos saltator. In the ant genomes, methylcytosines are found both in symmetric CG dinucleotides (CpG) and non-CpG contexts and are strongly enriched at exons of active genes. Changes in exonic DNA methylation correlate with alternative splicing events such as exon skipping and alternative splice site selection. Several genes exhibit caste-specific and developmental changes in DNA methylation that are conserved between the two species, including genes involved in reproduction, telomere maintenance, and noncoding RNA metabolism. Several loci are methylated and expressed monoallelically, and in some cases, the choice of methylated allele depends on the caste. These first ant methylomes and their intra- and interspecies comparison reveal an exonic methylation pattern that points to a connection between DNA methylation and splicing. The presence of monoallelic DNA methylation and the methylation of non-CpG sites in all samples suggest roles in genome regulation in these social insects, including the intriguing possibility of parental or caste-specific genomic imprinting.
  Current biology: CB 08/2012; 22(19):1755-64. · 10.99 Impact Factor
• Article: Phylogenetic and transcriptomic analysis of chemosensory receptors in a pair of divergent ant species reveals sex-specific signatures of odor coding.

  Xiaofan Zhou, Jesse D Slone, Antonis Rokas, Shelley L Berger, Jürgen Liebig, Anandasankar Ray, Danny Reinberg, Laurence J Zwiebel
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  ABSTRACT: Ants are a highly successful family of insects that thrive in a variety of habitats across the world. Perhaps their best-known features are complex social organization and strict division of labor, separating reproduction from the day-to-day maintenance and care of the colony, as well as strict discrimination against foreign individuals. Since these social characteristics in ants are thought to be mediated by semiochemicals, a thorough analysis of these signals, and the receptors that detect them, is critical in revealing mechanisms that lead to stereotypic behaviors. To address these questions, we have defined and characterized the major chemoreceptor families in a pair of behaviorally and evolutionarily distinct ant species, Camponotus floridanus and Harpegnathos saltator. Through comprehensive re-annotation, we show that these ant species harbor some of the largest yet known repertoires of odorant receptors (Ors) among insects, as well as a more modest number of gustatory receptors (Grs) and variant ionotropic glutamate receptors (Irs). Our phylogenetic analyses further demonstrate remarkably rapid gains and losses of ant Ors, while Grs and Irs have also experienced birth-and-death evolution to different degrees. In addition, comparisons of antennal transcriptomes between sexes identify many chemoreceptors that are differentially expressed between males and females and between species. We have also revealed an agonist for a worker-enriched OR from C. floridanus, representing the first case of a heterologously characterized ant tuning Or. Collectively, our analysis reveals a large number of ant chemoreceptors exhibiting patterns of differential expression and evolution consistent with sex/species-specific functions. These differentially expressed genes are likely associated with sex-based differences, as well as the radically different social lifestyles observed between C. floridanus and H. saltator, and thus are targets for further functional characterization. Our findings represent an important advance toward understanding the molecular basis of social interactions and the differential chemical ecologies among ant species.
  PLoS Genetics 08/2012; 8(8):e1002930. · 8.69 Impact Factor
• Article: Fcp1 dephosphorylation of the RNA polymerase II C-terminal domain is required for efficient transcription of heat shock genes.

  Nicholas J Fuda, Martin S Buckley, Wenxiang Wei, Leighton J Core, Colin T Waters, Danny Reinberg, John T Lis
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  ABSTRACT: Fcp1 dephosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II) to recycle it into a form that can initiate a new round of transcription. Previously, we identified Drosophila Fcp1 as an important factor in optimal Hsp70 mRNA accumulation after heat shock. Here, we examine the role of Fcp1 in transcription of heat shock genes in vivo. We demonstrate that Fcp1 localizes to active sites of transcription including the induced Hsp70 gene. The reduced Hsp70 mRNA accumulation seen by RNA interference (RNAi) depletion of Fcp1 in S2 cells is a result of a loss of Pol II in the coding region of highly transcribed heat shock-induced genes: Hsp70, Hsp26, and Hsp83. Moreover, Fcp1 depletion dramatically increases phosphorylation of the non-chromatin-bound Pol II. Reexpression of either wild-type or catalytically dead versions of Fcp1 demonstrates that both the reduced Pol II levels on heat shock genes and the increased levels of phosphorylated free Pol II are dependent on the catalytic activity of Fcp1. Our results indicate that Fcp1 is required to maintain the pool of initiation-competent unphosphorylated Pol II, and this function is particularly important for the highly transcribed heat shock genes.
  Molecular and cellular biology 06/2012; 32(17):3428-37. · 6.06 Impact Factor
• Article: PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription.

  David B Beck, Hisanobu Oda, Steven S Shen, Danny Reinberg
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  ABSTRACT: Histone post-translational modifications impact many aspects of chromatin and nuclear function. Histone H4 Lys 20 methylation (H4K20me) has been implicated in regulating diverse processes ranging from the DNA damage response, mitotic condensation, and DNA replication to gene regulation. PR-Set7/Set8/KMT5a is the sole enzyme that catalyzes monomethylation of H4K20 (H4K20me1). It is required for maintenance of all levels of H4K20me, and, importantly, loss of PR-Set7 is catastrophic for the earliest stages of mouse embryonic development. These findings have placed PR-Set7, H4K20me, and proteins that recognize this modification as central nodes of many important pathways. In this review, we discuss the mechanisms required for regulation of PR-Set7 and H4K20me1 levels and attempt to unravel the many functions attributed to these proteins.
  Genes & development 02/2012; 26(4):325-37. · 12.08 Impact Factor
• Article: PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.

  Zhonghua Gao, Jin Zhang, Roberto Bonasio, Francesco Strino, Ayana Sawai, Fabio Parisi, Yuval Kluger, Danny Reinberg
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  ABSTRACT: The heterogeneous nature of mammalian PRC1 complexes has hindered our understanding of their biological functions. Here, we present a comprehensive proteomic and genomic analysis that uncovered six major groups of PRC1 complexes, each containing a distinct PCGF subunit, a RING1A/B ubiquitin ligase, and a unique set of associated polypeptides. These PRC1 complexes differ in their genomic localization, and only a small subset colocalize with H3K27me3. Further biochemical dissection revealed that the six PCGF-RING1A/B combinations form multiple complexes through association with RYBP or its homolog YAF2, which prevents the incorporation of other canonical PRC1 subunits, such as CBX, PHC, and SCM. Although both RYBP/YAF2- and CBX/PHC/SCM-containing complexes compact chromatin, only RYBP stimulates the activity of RING1B toward H2AK119ub1, suggesting a central role in PRC1 function. Knockdown of RYBP in embryonic stem cells compromised their ability to form embryoid bodies, likely because of defects in cell proliferation and maintenance of H2AK119ub1 levels.
  Molecular cell 02/2012; 45(3):344-56. · 14.61 Impact Factor
• Article: BRD4 jump-starts transcription after mitotic silencing.

  Philipp Voigt, Danny Reinberg
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  ABSTRACT: The chromatin adapter BRD4 may be crucial for transmitting epigenetic information by acting as a histone acetylation-dependent gene bookmark and accelerating post-mitotic transcriptional reactivation.
  Genome biology 11/2011; 12(11):133. · 6.63 Impact Factor
• Article: L3MBTL2 protein acts in concert with PcG protein-mediated monoubiquitination of H2A to establish a repressive chromatin structure.

  Patrick Trojer, Alina R Cao, Zhonghua Gao, Yan Li, Jin Zhang, Xiaoqin Xu, Guohong Li, Regine Losson, Hediye Erdjument-Bromage, Paul Tempst, Peggy J Farnham, Danny Reinberg
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  ABSTRACT: We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb repressive complex 1 (PRC1)-like 4 (PRC1L4), given the copresence of PcG proteins RING1, RING2, and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/HP1γ, known to function in transcriptional repression. PRC1L4-mediated repression necessitated L3MBTL2 that compacted chromatin in a histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT domain-containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genes that exhibited a significant reduction in H2A lysine 119 monoubiquitination. Our findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks.
  Molecular cell 05/2011; 42(4):438-50. · 14.61 Impact Factor
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  Available from: Luis Alejandro Rojas

  Article: The C-terminal domain of RNA polymerase II is modified by site-specific methylation.

  Robert J Sims, Luis Alejandro Rojas, David Beck, Roberto Bonasio, Roland Schüller, William J Drury, Dirk Eick, Danny Reinberg
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  ABSTRACT: The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1(-/-) mouse embryo fibroblasts. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types.
  Science 04/2011; 332(6025):99-103. · 31.20 Impact Factor
• Article: Epigenetic inheritance: uncontested?

  Bing Zhu, Danny Reinberg
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  ABSTRACT: "Epigenetics" is currently defined as "the inheritance of variation (-genetics) above and beyond (epi-) changes in the DNA sequence". Despite the fact that histones are believed to carry important epigenetic information, little is known about the molecular mechanisms of the inheritance of histone-based epigenetic information, including histone modifications and histone variants. Here we review recent progress and discuss potential models for the mitotic inheritance of histone modifications-based epigenetic information.
  Cell Research 02/2011; 21(3):435-41. · 8.19 Impact Factor
• Article: Chromatin higher-order structures and gene regulation.

  Guohong Li, Danny Reinberg
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  ABSTRACT: Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin to fit inside the nucleus. The dynamics of higher-order chromatin compaction play a crucial role in transcription and other biological processes inherent to DNA. Many factors, including histone variants, histone modifications, DNA methylation, and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. In this review, we focus on the recent progress in elucidating the structure of the 30-nm chromatin fiber. We also discuss the structural plasticity/dynamics and epigenetic inheritance of higher-order chromatin and the roles of chromatin higher-order organization in eukaryotic gene regulation.
  Current opinion in genetics & development 02/2011; 21(2):175-86. · 8.99 Impact Factor
• Article: Histone tails: ideal motifs for probing epigenetics through chemical biology approaches.

  Philipp Voigt, Danny Reinberg
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  ABSTRACT: Post-translational modifications (PTMs) on histone proteins have emerged as a central theme in the regulation of gene expression and other chromatin-associated processes. The discovery that certain protein domains can recognize acetylated and methylated lysine residues of histones has spurred efforts to uncover and characterize histone PTM-binding proteins. In this task, chromatin biology has strongly benefited from synthetic approaches stemming from chemical biology. Peptide-based techniques have been instrumental in identifying histone mark-binding proteins and analyzing their binding specificities. To explore how histone PTMs carry out their function in the context of chromatin, reconstituted systems based on recombinant histones carrying defined modifications are increasingly being used. They constitute promising tools to analyze mechanistic aspects of histone PTMs, including their role in transcription and their transmission in replication. In this review, we present strategies that have been used successfully to investigate the role of histone modifications, concepts that have emerged from their application, and their potential to contribute to current developments in the field.
  ChemBioChem 01/2011; 12(2):236-52. · 3.94 Impact Factor
• Article: A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo.

  Eerappa Rajakumara, Julie A Law, Dhirendra K Simanshu, Philipp Voigt, Lianna M Johnson, Danny Reinberg, Dinshaw J Patel, Steven E Jacobsen
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  ABSTRACT: Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci.
  Genes & development 01/2011; 25(2):137-52. · 12.08 Impact Factor