Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.

Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities.     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.     

Affinity purification and characterization of CIF, an insect immunoresponsive factor with NF-kappa B-like properties.

1. A rapid DNA affinity purification procedure was worked out for the purification of the Cecropia Immunoresponsive Factor (CIF) from the pupae of Hyalophora cecropia. 2. CIF consists of a single polypeptide chain of 65 kDa and is present as a homodimer under native conditions. 3. CIF binds to the kappa B-like sequences upstream of the H. cecropia immune genes with the following order of affinity: attacin kappa B greater than lysozyme kappa B greater than cecropin A kappa B greater than cecropin B kappa B. 4. The purified CIF also strongly binds to the kappa B sequences from both the immunoglobulin kappa light chain gene and the MHC class I gene. 5. The DNA binding of CIF can be inhibited by antisera directed against NF-kappa B-related proteins. 6. The cytoplasmic factor Cl, co-purified from the affinity column, contains two polypeptide chains, one of which has the same molecular weight as CIF.     

EVX2, a human homeobox gene homologous to the even-skipped segmentation gene, is localized at the 5' end of HOX4 locus on chromosome 2.

We isolated and mapped three new human homeoboxes located on chromosome 2 upstream from the reported seven HOX4 homeobox sequences. Two of them, HOX41 and HOX4H, clearly belong to the HOX gene family, in particular to homology groups 1 and 2, and possibly represent the most 5' HOX4 homeoboxes. A third homeobox 13 kb upstream from HOX41 was identified. Sequencing data show that this is the human homolog of the murine Evx-2 homeobox. Both homeoboxes are closely related to the murine Evx-1 and to the frog Xhox-3 homeoboxes. The four genes represent vertebrate homologs of Drosophila even-skipped (eve), a segmentation gene of the pair-rule class. Human EVX2 sequences belong to an active gene because they are transcribed and properly processed in cells and tissues. We have identified for the first time a homeogene of a different class at a HOX locus. These findings are relevant to the understanding of the evolution of HOX gene clusters and their regulation.     

A key role for EZH2 in epigenetic silencing of HOX genes in mantle cell lymphoma

The chromatin modifier EZH2 is overexpressed and associated with inferior outcome in mantle cell lymphoma (MCL). Recently, we demonstrated preferential DNA methylation of HOX genes in MCL compared with chronic lymphocytic leukemia (CLL), despite these genes not being expressed in either entity. Since EZH2 has been shown to regulate HOX gene expression, to gain further insight into its possible role in differential silencing of HOX genes in MCL vs. CLL, we performed detailed epigenetic characterization using representative cell lines and primary samples. We observed significant overexpression of EZH2 in MCL vs. CLL. Chromatin immune precipitation (ChIP) assays revealed that EZH2 catalyzed repressive H3 lysine 27 trimethylation (H3K27me3), which was sufficient to silence HOX genes in CLL, whereas in MCL H3K27me3 is accompanied by DNA methylation for a more stable repression. More importantly, hypermethylation of the HOX genes in MCL resulted from EZH2 overexpression and subsequent recruitment of the DNA methylation machinery onto HOX gene promoters. The importance of EZH2 upregulation in this process was further underscored by siRNA transfection and EZH2 inhibitor experiments. Altogether, these observations implicate EZH2 in the long-term silencing of HOX genes in MCL, and allude to its potential as a therapeutic target with clinical impact.     

Differential binding of quadruplex structures of muscle-specific genes regulatory sequences by MyoD, MRF4 and myogenin

Four myogenic regulatory factors (MRFs); MyoD, Myf-5, MRF4 and Myogenin direct muscle tissue differentiation. Heterodimers of MRFs with E-proteins activate muscle-specific gene expression by binding to E-box motifs d(CANNTG) in their promoters or enhancers. We showed previously that in contrast to the favored binding of E-box by MyoD-E47 heterodimers, homodimeric MyoD associated preferentially with quadruplex structures of regulatory sequences of muscle-specific genes. To inquire whether other MRFs shared the DNA binding preferences of MyoD, the DNA affinities of hetero- and homo-dimeric MyoD, MRF4 and Myogenin were compared. Similarly to MyoD, heterodimers with E47 of MRF4 or Myogenin bound E-box more tightly than quadruplex DNA. However, unlike homodimeric MyoD or MRF4, Myogenin homodimers associated weakly and nonpreferentially with quadruplex DNA. By reciprocally switching basic regions between MyoD and Myogenin we demonstrated dominance of MyoD in determining the quadruplex DNA-binding affinity. Thus, Myogenin with an implanted MyoD basic region bound quadruplex DNA nearly as tightly as MyoD. However, a grafted Myogenin basic region did not diminish the high affinity of homodimeric MyoD for quadruplex DNA. We speculate that the dissimilar interaction of MyoD and Myogenin with tetrahelical domains in muscle gene promoters may differently regulate their myogenic activities.