Molecular basis for chromatin assembly and modification by multiprotein complexes

Abstract: Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post‐translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.     

Spatiotemporal coordination of transcription preinitiation complex assembly in live cells

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A nonhistone protein-protein interaction required for assembly of the SIR complex and silent chromatin

Budding yeast silent chromatin, or heterochromatin, is composed of histones and the Sir2, Sir3, and Sir4 proteins. Their assembly into silent chromatin is believed to require the deacetylation of histones by the NAD-dependent deacetylase Sir2 and the subsequent interaction of Sir3 and Sir4 with these hypoacetylated regions of chromatin. Here we explore the role of interactions among the Sir proteins in the assembly of the SIR complex and the formation of silent chromatin. We show that significant fractions of Sir2, Sir3, and Sir4 are associated together in a stable complex. When the assembly of Sir3 into this complex is disrupted by a specific mutation on the surface of the C-terminal coiled-coil domain of Sir4, Sir3 is no longer recruited to chromatin and silencing is disrupted. Because in sir4 mutant cells the association of Sir3 with chromatin is greatly reduced despite the partial Sir2-dependent deacetylation of histones near silencers, we conclude that histone deacetylation is not sufficient for the full recruitment of silencing proteins to chromatin and that Sir-Sir interactions are essential for the assembly of heterochromatin.     

The complexities of eukaryotic transcription initiation: regulation of preinitiation complex assembly.

Individual steps in the assembly of RNA polymerase II and general initiation factors into a preinitiation complex serve as points of control for activators, whose functions require additional cofactors. The full range of induction by activators appears to involve both the reversal of negative constraints and net positive effects on promoter interactions with general factors.     

Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation

The metazoan nucleus is disassembled and re-built at every mitotic cell division. The nuclear envelope, including nuclear pore complexes, breaks down at the beginning of mitosis to accommodate the capture of massively condensed chromosomes by the spindle apparatus. At the end of mitosis, a nuclear envelope is newly formed around each set of segregating and de-condensing chromatin. We review the current understanding of the membrane restructuring events involved in the formation of the nuclear membrane sheets of the envelope, the mechanisms governing nuclear pore complex assembly and integration in the nascent nuclear membranes, and the regulated coordination of these events with chromatin de-condensation.     

The essential ATP-binding cassette protein RLI1 functions in translation by promoting preinitiation complex assembly

RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors. RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes. Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size. There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal. A mutation in the second ATP-binding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts. These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation. RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells. Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis.     

Methods for chromatin assembly and remodeling

Packaging of the DNA in nucleosomes restricts its accessibility to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a short linear DNA fragment allows gel fractionation of nucleosomes according to their translational positions, whose locations are dependent on the underlying DNA sequence. Nucleosome mobilization by chromatin remodeling factors is easily detected by observing band disappearance in gel, which in turn provides evidence for histone octamer displacement. Here, we provide methods for chromatin assembly that we have been using in our analysis for nucleosome mobilization by chromatin remodeling factors. These methods are straightforward and easy to follow. Thus, they may provide a good starting assay system for analysis of nucleosome movements by other chromatin remodeling machines.