The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6
S. Branden Van OssMargaret K. ShirraAlain R. BatailleAdam D. WierKuangyu YenVinesh VinayachandranIn‐Ja L. ByeonChristine E. CucinottaA. HérouxJongcheol JeonJaehoon KimAndrew P. VanDemarkB. Franklin PughKaren M. Arndt
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Histone H2B
Transcription
Histone code
Eukaryotic transcription
Post-translational modifications (e.g., ubiquitylation) of histones play important roles in dynamic regulation of chromatin. Histone ubiquitylation has been speculated to directly influence the structure and dynamics of nucleosomes. However, structural information for ubiquitylated nucleosomes is still lacking. Here we report an alternative strategy for total chemical synthesis of homogenous histone H2B-K34-ubiquitylation (H2B-K34Ub) by using acid-cleavable auxiliary-mediated ligation of peptide hydrazides for site-specific ubiquitylation. Synthetic H2B-K34Ub was efficiently incorporated into nucleosomes and further used for single-particle cryo-electron microscopy (cryo-EM) imaging. The cryo-EM structure of the nucleosome containing H2B-K34Ub suggests that two flexible ubiquitin domains protrude between the DNA chains of the nucleosomes. The DNA chains around the H2B-K34 sites shift and provide more space for ubiquitin to protrude. These analyses indicated local and slight structural influences on the nucleosome with ubiquitylation at the H2B-K34 site.
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Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.
Histone H2B
Histone code
Histone Methylation
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DNA in nucleosomes has restricted nucleosome dynamics and is refractory to DNA-templated processes. Histone post-translational modifications play important roles in regulating DNA accessibility in nucleosomes. Whereas most histone modifications function either by mitigating the electrostatic shielding of histone tails or by recruiting 'reader' proteins, we show that ubiquitylation of H2B K34, which is located in a tight space protected by two coils of DNA superhelix, is able to directly influence the canonical nucleosome conformation via steric hindrances by ubiquitin groups. H2B K34 ubiquitylation significantly enhances nucleosome dynamics and promotes generation of hexasomes both with symmetrically or asymmetrically modified nucleosomes. Our results indicate a direct mechanism by which a histone modification regulates the chromatin structural states.
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Chromatosome
Histone Methylation
Linker DNA
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Histone Methylation
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Nucleosome variability is essential for their functions in compacting the chromatin structure and regulation of transcription, replication and cell reprogramming. The DNA molecule in nucleosomes is wrapped around an octamer composed of four types of core histones (H3, H4, H2A, H2B). Nucleosomes represent dynamic entities and may change their conformation, stability and binding properties by employing different sets of histone variants or by becoming post-translationally modified. There are many variants of histones H2A and H2B. Specific H2A and H2B variants may preferentially associate with each other resulting in different combinations of variants and leading to the increased combinatorial complexity of nucleosomes. In addition, the H2A-H2B dimer can be recognized and substituted by chaperones/remodelers as a distinct unit, can assemble independently and is stable during nucleosome unwinding. In this review we discuss how sequence and structural variations in H2A-H2B dimers may provide necessary complexity and confer the nucleosome functional variability.
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Contributions of DNA, histone chaperones and chromatin remodeling enzymes to nucleosome positioning.
Author(s): Partensky, Peretz | Advisor(s): Narlikar, Geeta | Abstract: The eukaryotic genome is packaged by wrapping ~147 bp units of DNA around histone octamers to form chains of nucleosomes. The packaging of the DNA within a nucleosome reduces access of the DNA to most transcription factors and polymerases. In between nucleosomes, there are regions of more accessible DNA, called linker regions that vary from a few base pairs to several hundred base pairs. Thus within any given cell type, the precise partitioning of the genome into nucleosome-bound and nucleosome-free DNA regions can have large consequences on gene regulation and help define a particular cellular state. Recent studies suggest that the genome plays a large role in encoding its own packaging through differences in affinity of the underlying sequence for the histone octamer. Nucleosome locations are also regulated by several different ATP-dependent chromatin remodeling enzymes, which enable rapid rearrangements in chromatin structure in response to developmental cues. Thirdly, the in vivo nucleosome assembly process involves proteins called histone chaperones. No individual factor is capable of playing a dominant role in generating the immense specificity required to regulate transcription in eukaryotes. This gives rise to the question of what are the relative contributions to nucleosome positions due to each of these factors. This question has been investigated with biochemical reconstitutions and activity assays, which tracked nucleosome position distributions and kinetics in the presence of various factors. Our data support a model in which remodeling enzymes move nucleosomes to new locations by a general sequence-independent mechanism. However, consequent to the rate-limiting remodeling step, the local DNA sequence promotes a collapse of remodeling intermediates into highly resolved positions that are dictated by thermodynamic differences between adjacent positions. Analogously, histone chaperones have been found to reduce thermodynamic equilibration among all available nucleosome positions, but leads to local equilibration after a rapid histone deposition step. Future understanding of how these factors coordinate their activities in vivo and in the presence of transcription factors, will hopefully lead to better predictive models of gene regulation.
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Summary Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning and transcription. Our results indicate that precise positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding, and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous pioneer transcription factors. We find no evidence for recruitment of RSC by pioneer factors, but show instead that the strength and directionality of RSC action on nucleosomes depends upon the arrangement of two specific DNA motifs that promote its binding and nucleosome displacement activity at promoters. Thus, despite their widespread co-localization, RSC and pioneer factors predominantly act independently to generate accessible chromatin. Our results provide insight into how promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.
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Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo . We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo .
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Deubiquitinating enzyme
Ubiquitin-conjugating enzyme
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