ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of > or =29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter.

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin in vivo, yet the structural consequences of acetylation leading to increased gene activity are only poorly defined. We employed a new approach to characterize the effects of histone acetylation: A Drosophila embryo-derived cell-free system for chromatin reconstitution under physiological conditions was programmed with exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unmodified chromatin at similar nucleosome density with respect to its sensitivity toward microccal nuclease, its nucleosomal repeat length, and the incorporation of the linker histone H1. In contrast, DNA in acetylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational f lexibility upon temperature shift pointing to profound alterations of DNA͞histone interactions. This successful reconstitution of accessible and f lexible chromatin outside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.

The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trx Z11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.The SET domain is an ϳ130-amino-acid (aa) sequence which was initially identified in the protein products of three regulatory genes in Drosophila, whose names account for the name SET, i.e, SU(VAR)3-9, enhancer of Zeste [E(Z)], and trithorax (Trx). SET domains are usually located at the carboxyl termini of proteins, although in some proteins, such as ASH1 (another regulator belonging to the TrxG family), the motif is located in the middle of the protein (Fig. 1A), and in the proteins RIZ and BLIMP this motif maps to the N termini. In addition to the highly conserved SET motif, less conserved ϳ50-to 80-aa pre-SET and post-SET regions may be present at the amino and carboxyl boundaries, respectively, of the SET domain (Fig. 1A) (reviewed in reference 12). Genes encoding SET domains are widely represented in eukaryotic genomes (Ͼ300 database entries). Based on the homology of their SET motifs, the most characterized human proteins were classified into four major families, SET1, SET2, SUV39, and RIZ (15). A recently discovered function of the SET domain is the methylation of lysine residues in nucleosomal histones. SET domains of different proteins target specific lysines, and these modifications have different consequences on gene expression.For example, the methylation of Lys 4 of histone H3 is usually associated with activation, while the m...