Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.

Satb1 is a first cell-type-specific transcription factor of a novel type that functions as a regulator of the transcription of large chromatin domains. We identified a close homologue of Satb1, Satb2, in a cDNA subtraction screening in a search for genes controlling neural differentiation. Satb2 showed 61% amino-acid homology to Satb1. Satb2 and Satb1 expression was detected in different cell subpopulations of developing mouse CNS in a mutually exclusive manner. In the electrophoretic mobility shift assay we demonstrate that nuclear extracts from the embryonic day 18.5 mouse developing neocortex, in contrast to basal ganglia, contain a protein complex interacting with matrix attachment region DNA elements (MARs) with high affinity. Endogenous Satb2 protein is a part of this complex. In the developing neocortex Satb2 was detected largely in the superficial layers. In the developing spinal cord Satb2 expression marks a subpopulation of Lbx1-positive neurons dorsally and a subgroup of Isl1-positive neurons ventrally. In the Lbx1 mutants Satb2 expression is greatly reduced. We suggest that Satb2 may regulate differentiation of subsets of neurons at the level of higher order chromatin structure via binding to MARs.

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We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

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Solitary long terminal repeats (LTRs) of the human endogenous retroviruses, scattered in several thousand copies throughout the human genome, are potentially capable of affecting the expression of closely located genes. To assess their regulatory potential, the LTR sequences of one of the most abundant HERV families (HERV-K) were screened for the presence of binding sites for the host cell nuclear factors using mobility shift and UV-crosslinking assays. It was shown that the LTR sequences of two subfamilies harbor a specific binding site for a complex consisting of at least three proteins, ERF1, ERF2 and ERF3 of 98, 91 and 88 kDa apparent molecular mass, respectively. This binding site is located in the 5P region of the LTR U3 element. The preservation of the specific protein binding site in different HERV-K LTR sequences suggests their possible role in regulation of nearby located genes.z 1998 Federation of European Biochemical Societies.

The spatial organization of an ∼170 kb region of human chromosome 19, including CD22 and GPR40–GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix.

We used the 4C-Seq technique to characterize the genome-wide patterns of spatial contacts of several CpG islands located on chromosome 14 in cultured chicken lymphoid and erythroid cells. We observed a clear tendency for the spatial clustering of CpG islands present on the same and different chromosomes, regardless of the presence or absence of promoters within these CpG islands. Accordingly, we observed preferential spatial contacts between Sp1 binding motifs and other GC-rich genomic elements, including the DNA sequence motifs capable of forming G-quadruplexes. However, an anchor placed in a gene/CpG island-poor area formed spatial contacts with other gene/CpG island-poor areas on chromosome 14 and other chromosomes. These results corroborate the two-compartment model of the spatial organization of interphase chromosomes and suggest that the clustering of CpG islands constitutes an important determinant of the 3D organization of the eukaryotic genome in the cell nucleus. Using the ChIP-Seq technique, we mapped the genome-wide CTCF deposition sites in the chicken lymphoid and erythroid cells that were used for the 4C analysis. We observed a good correlation between the density of CTCF deposition sites and the level of 4C signals for the anchors located in CpG islands but not for an anchor located in a gene desert. It is thus possible that CTCF contributes to the clustering of CpG islands observed in our experiments.