PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex

PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.     

MLL targets SET domain methyltransferase activity to Hox gene promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.     

Haplotype analysis of the human beta-globin gene complex using multiple locus specific oligonucleotide probes

Three oligonucleotide probes complementary to specific DNA sequences of the six human globin genes (epsilon, G gamma, A gamma, psi beta, delta, beta) were synthesized. The oligonucleotides were used either singly or in combination as hybridization probes to determine the haplotype of the human beta-globin gene cluster employing the four conventionally used restriction endonucleases HincII, HindIII, AvaII, and BamHI, in addition to HpaI. Polymorphism in the epsilon- and psi beta-genes (HincII) can be simultaneously determined with a single probe mixture. One of the probes complementary to both the psi beta- and gamma-genes is useful for determining both HindIII and HincII polymorphisms. The advantages of these probes relative to conventional cDNA probes are discussed.     

Multi-protein complexes at the beta-globin locus.

Biochemical analysis of the beta-globin gene function has led to the identification of several multi-protein complexes at the locus control region (LCR), insulator and promoters. This review briefly summarizes these multi-protein complexes and discusses their contribution towards the regulation of the beta-globin gene expression.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Primary structure of the goat beta-globin locus control region

The goat beta-globin cluster is composed of a triplicated four-gene set. A locus control region (LCR) containing elements homologous to 5'DNase I hypersensitive sites (HS) 1, 2, and 3 of the human beta-globin LCR has been identified at the 5' end of this locus. We determined 10.2 kb of nucleotide sequence from the goat beta-globin locus control region. Self-comparison of this sequence by dot matrix analysis revealed the presence of six complete and three incomplete artiodactyl repeats. A novel repeated element, termed D repeat, was also identified. Southern blotting analysis demonstrated that these elements exist in the goat genome as a low to medium frequency interspersed repeat family. The absence of any other large region of self-homology (direct or inverted) in the goat LCR suggests that 5'HSs 1, 2, and 3 did not arise through duplication, but rather evolved independently. By comparing goat 5'HS 1 to those of human, rabbit, and mouse, we show a greater than 80% conservation in sequence between the four species. This level of evolutionary conservation suggests that 5'HS 1 plays an important role in the regulation of beta-globin loci.     

Substrate preferences of the EZH2 histone methyltransferase complex

Histone methylation plays an important role in chromatin dynamics and gene expression. Methylation of histone H3-lysine 27 by the EZH2 complex has been linked to the silencing of homeotic genes and the inactivation of the X chromosome. Here we report a characterization of the substrate preferences of the enzyme complex using a reconstituted chromatin and enzyme system. We found that the linker histone H1, when incorporated into nucleosomes, stimulates the enzymatic activity toward histone H3. This stimulatory activity may be explained by protein-protein interactions between H1 and components of the EZH2 complex. In addition, we found that the EZH2 complex exhibits a dramatic preference for dinucleosomes when compared with mononucleosomes and that the stimulation of H3 methylation by H1 requires dinucleosomes or oligonucleosome substrates. Furthermore, in contrast with a recent study suggesting that Embryonic Ectoderm Development EED isoforms may affect substrate specificity, we found that EZH2 complexes reconstituted with different EED isoforms exhibit similar substrate preference and specificity. Our work supports the hypothesis that linker histone H1 and chromatin structure are important factors in determining the substrate preference of the EZH2 histone methyltransferase complex.     

Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.     

H-33-A histocompatibility locus to the left of theH-2 complex

Another minor histocompatibility locus, calledH-33, was found on chromosome 17. This locus was revealed by skin graft experiments between BALB/c and a new congenic strain, BALB.TTF.     

Chromatin structure at the 3'-boundary of the human beta-globin locus control region hypersensitive site-2

Chromatin structure was examined at the 3′-boundary region of the human β-globin locus control region hypersensitive site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif—this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3′-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the hypersensitive site.