A simple and versatile system for the ATP-dependent assembly of chromatin.

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins.

The prenucleosome, a stable conformational isomer of the nucleosome.

Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼ 80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone-DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone-DNA species in the cell.

Prenucleosomes and Active Chromatin.

Chromatin consists of nucleosomes as well as nonnucleosomal histone-containing particles. Here we describe the prenucleosome, which is a stable conformational isomer of the nucleosome that associates with ∼80 bp DNA. Prenucleosomes are formed rapidly upon the deposition of histones onto DNA and can be converted into canonical nucleosomes by an ATP-driven chromatin assembly factor such as ACF. Different lines of evidence reveal that there are prenucleosome-sized DNA-containing particles with histones in the upstream region of active promoters. Moreover, p300 acetylates histone H3K56 in prenucleosomes but not in nucleosomes, and H3K56 acetylation is found at active promoters and enhancers. These findings therefore suggest that there may be prenucleosomes or prenucleosome-like particles in the upstream region of active promoters. More generally, we postulate that prenucleosomes or prenucleosome-like particles are present at dynamic chromatin, whereas canonical nucleosomes are at static chromatin.

ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling.

Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly. DOI:http://dx.doi.org/10.7554/eLife.00863.001.

Biochemical analysis of histone deacetylase-independent transcriptional repression by MeCP2.

MeCP2 is an abundant methyl-cytosine-guanine (CG)-binding protein and transcriptional repressor. We developed a biochemical system that exhibits CG methylation-specific transcriptional repression by purified human MeCP2. MeCP2 represses transcription by histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Our system appears to recreate the HDAC-independent component of MeCP2-mediated repression and occurs via inhibition of the assembly of transcription preinitiation complexes. At a ratio of approximately one molecule of MeCP2 per two methyl-CG dinucleotides, as found in mammalian neurons, the magnitude of methylation-specific repression was greater than 10-fold. Notably, the HDAC inhibitor trichostatin A had no effect on MeCP2-mediated repression with either naked DNA or chromatin templates. We designed a CG-deficient core promoter that is resistant to MeCP2-mediated repression when placed in a plasmid lacking CG dinucleotides. By using this CG-deficient reporter as a reference, we found that eight CG dinucleotides in the core promoter region are sufficient for strong methylation-specific repression by MeCP2. In contrast, MeCP2 does not repress a construct with 13 CG dinucleotides located ∼1.7 kbp upstream of the promoter. Furthermore, by analysis of C-terminally truncated MeCP2 proteins, we found that binding of MeCP2 to methyl-CG dinucleotides is not sufficient for transcriptional repression. Hence, MeCP2-mediated repression is not due to the simple steric blockage of the transcriptional machinery. These experiments suggest that MeCP2 can function as a global methyl-CG-specific, HDAC-independent repressor. This HDAC-independent mechanism of MeCP2-mediated repression may be important in cells, such as mammalian neurons, that have high levels of CG methylation and MeCP2.