Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb.

SUMMARY MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density, from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveal that homologous recombination and nonhomologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology; either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving 10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.

By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, Ϸ240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of Ϸ50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.Transcription initiation at a eukaryotic protein-encoding gene involves assembly on promoter DNA of a complex consisting of RNA polymerase II (RNAPII) and six general transcription factors: IIA, IIB, IID (or TATA-element binding protein, TBP), IIE, IIF, and IIH (1-3). A subcomplex containing TBP, IIB, IIF, RNAPII, and promoter DNA is stable (4, 5), and, under certain conditions (e.g., conditions that promote DNA melting), is fully competent for transcription initiation (6-11). Results of DNA footprinting experiments indicate that the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C involves interactions both upstream and downstream of the transcription start (positions Ϫ42 to ϩ17; H. Lu and D.R., unpublished data) and involves unmelted DNA (11). Thus, the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C appears to correspond to the RNA polymerase-promoter ''intermediate complex'' characterized in studies of Escherichia coli RNA polymerase (RP i or RP c2 ; refs. 12-15).The TBP-IIB-IIF-RNAPII-promoter complex contains at least 14 distinct polypeptides (one in TBP, one in IIB, two in IIF, and at least 10 in RNAPII) and has a molecular mass in excess of 700 kDa (1-3). High-resolution structures have been determined for the TBP-DNA and TBP-IIB-DNA complexes (16-18). However, the TBP-IIB-IIF-RNAPII-promoter complex is too large for high-resolution structure determination by current methods. Therefore, information about the structure of the TBP-IIB-IIF-RNAPII-promoter complex must rely on low-resolution structure determination (19,20) supplemented by biochemical and imaging data.In the work in this report, we have used site-specific protein-DNA photocrosslinking and electron microscopy to define protein-DNA interactions within the human TBP-IIB-IIF-RNAPIIpromoter complex. MATERIALS AND METHODSDerivatized Promoter DNA Fragments. Derivatized promoter DNA fragments were prepared essentially as in ref. 21. Oligodeoxyribonucleotides containing phosphorothioate 5Ј to the third nucleotide were synthesized by using solid-phase ␤-cyanoethylphosphoramidite chemistry and tetraethylthiuram disulfide (Applied Biosystems), purified on OPC (Applied Biosystems), and derivatized with azidophenacyl bromide (Sigma; ref. 22). Derivatized oligodeo...