Isolation of a cDNA clone encoding the RNase-superfamily-related gene highly expressed in chicken bone marrow cells.

The RNase gene superfamily combines functionally divergent proteins which share statistically significant sequence similarity. Known members assigned to this family include secretory and nonsecretory RNases; angiogenin; eosinophil cationic protein; eosinophil-derived neurotoxin; sialic-acid binding lectin and anti-tumor protein P-30. We report the cDNA cloning of the chicken RNase Super Family Related (RSFR) gene that is specifically overexpressed in normal bone marrow cells and bone marrow-derived AMV transformed monoblasts. It codes for a 139 amino acid protein with a putative signal peptide and remarkable conservation of active-site residues, other residues known to be important for substrate binding and catalytic activity and half-cystine residues common for all RNase family members. Phylogenetic tree analysis shows that RSFR defines a new group of genes within the family. We also conclude that an amino acid sequence block CKXXNTF(X) 11C is a "shortest RNase superfamily signature" which is both necessary and sufficient to identify all previously recognized family members as well as chicken RSFR.     

A differentially methylated imprinting control region within the Kcnq1 locus harbors a methylation-sensitive chromatin insulator

The mechanisms underlying the phenomenon of genomic imprinting remain poorly understood. In one instance, a differentially methylated imprinting control region (ICR) at the H19 locus has been shown to involve a methylation-sensitive chromatin insulator function that apparently partitions the neighboring Igf2 and H19 genes in different expression domains in a parent of origin-dependent manner. It is not known, however, if this mechanism is unique to the Igf2/H19 locus or if insulator function is a common feature in the regulation of imprinted genes. To address this question, we have studied an ICR in the Kcnq1 locus that regulates long range repression on the paternally derived p57Kip2 and Kcnq1 alleles in an imprinting domain that includes Igf2 and H19. We show that this ICR appears to possess a unidirectional chromatin insulator function in somatic cells of both mesodermal and endodermal origins. Moreover, we document that CpG methylation regulates this insulator function suggesting that a methylation-sensitive chromatin insulator is a common theme in the phenomenon of genomic imprinting.     

Multiple nucleosome positioning sites regulate the CTCF-mediated insulator function of the H19 imprinting control region

The 5' region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.     

Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF-binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c-myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF-binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF-binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.