Abstract
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.
Highlights
Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena
We show that the Drosophila nucleoplasmin-like protein (dNLP)-imitation switch (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 minimal chromatin assembly process is mediated by a combination of an ATPutilizing motor protein, such as ACF, Chd1, or RSF, and a core histone chaperone, such as NAP1 or dNLP
Summary
We sought to establish and characterize a simple, reliable, and versatile system for the ATP-dependent assembly of periodic nucleosome arrays. S1 and S2, we observed the most distinct pattern of MNase digestion products (“MNase ladder”) with the complete reaction containing dNLP, ISWI, core histones, and ATP These experiments revealed that purified bacterially synthesized dNLP, ISWI, and core histones can be assembled efficiently into periodic nucleosome arrays. We tested whether the dNLP-ISWI system could be used to assemble histone H1– containing chromatin To this end, we purified native histone H1 from Drosophila embryos as well as recombinant human histone H1 variant H1.0 (Fig. 4A). We further analyzed the histone H1– containing chromatin by extensive MNase digestion into mononucleosomes followed by native gel electrophoresis (Fig. 4D) In these experiments, we found that the H1-containing chromatin yields chromatosomes (mononucleosomes ϩ H1), as seen previously with chromatin isolated from cells or assembled in vitro with ACF and dNAP1 [6, 26, 27].
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