Molecular cloning and preliminary functional analysis of two novel human KRAB zinc finger proteins, HKr18 and HKr19.

cDNA clones encoding two novel human KRAB zinc finger proteins, HKr18 and HKr19, were isolated from a human testis cDNA library. Their corresponding genes were later identified in sequences originating from chromosomes 19 and 7, respectively. On the basis of the collected information from gene and cDNA sequences, Hkr18 was found to be a protein of 94 kDa with 20 zinc finger motifs in its C terminus. The HKr19 is a smaller protein, with a molecular weight of 56 kDa containing 11 zinc finger motifs. Both HKr18 and HKr19 contained a KRAB A as well as a KRAB B domain in their N termini. Northern blot analysis showed expression of HKr18 in all human tissues tested, indicating a ubiquitous expression pattern. In contrast, HKr19 showed a more restricted tissue distribution, with detectable expression primarily in testis and fetal tissues. The HKr19 protein is a member of the large ZNF91 subfamily of KRAB zinc finger genes. A PCR-based analysis of the expression of HKr19 and other closely related genes showed that lymphoid, myeloid, and nonhematopoietic cells expressed different sets of these genes. This latter finding indicates that some members of the ZNF91 family may be involved in regulating lineage commitment during hematopoietic development. Transfection of various parts of HKr19 into human embryonic kidney cells (HEK 293 cells) showed that the entire protein and its zinc finger region were toxic to these cells when expressed at high levels. In contrast, the KRAB domain and the linker region seemed to be well tolerated.

Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 beta.

The Krüppel-associated box (KRAB) domain, originally identified as a 75-aa sequence present in numerous Krüppel-type zinc-finger proteins, is a potent DNA-binding-dependent transcriptional repression domain that is believed to function through interaction with the transcriptional intermediary factor 1 (TIF1) beta. On the basis of sequence comparison and phylogenetic analysis, we have recently defined three distinct subfamilies of KRAB domains. In the present study, individual members of each subfamily were tested for transcriptional repression and interaction with TIF1 beta and two other closely related family members (TIF1 alpha and TIF1 gamma). All KRAB variants were shown, (i) to repress transcription when targeted to DNA through fusion to a heterologous DNA-binding domain in mammalian cells, and (ii) to interact specifically with TIF1 beta, but not with TIF1 alpha or TIF1 gamma. Taken together, these results implicate TIF1 beta as a common transcriptional corepressor for the three distinct subfamilies of KRAB zinc-finger proteins and suggest a high degree of conservation in the molecular mechanism underlying their transcriptional repression activity.

Expression of lactoferrin in the kidney: implications for innate immunity and iron metabolism.

BACKGROUND: Sequestering of free iron by lactoferrin (LF) is important in the defense against bacteria. In a screening for LF expression in various organs, high levels of LF mRNA were detected in human kidney. This indicated that LF is produced by the kidney and that it may participate in innate immunity of this organ. METHODS AND RESULTS: Antibody staining and in situ hybridization of paraffin-embedded kidney sections showed that LF is expressed in cells lining the distal collecting ducts of the medulla. High levels of both protein and mRNA were detected in these cells. However, a clear difference in the distribution of mRNA and protein within the tissue was observed. LF mRNA was detected along a relatively large portion of the tubuli, whereas LF antigen was found mainly in the very distal regions of the same tubuli. This indicates that LF is released by large regions of the tubuli and possibly reabsorbed in the most distal parts. Using enzyme-linked immunosorbent assay, only very low LF levels were detected in urine. CONCLUSION: The present study shows that LF is produced by the kidney and that both LF mRNA and protein are distributed in a highly ordered fashion. This latter finding, together with the very low levels of LF detected in urine, indicates that LF may contribute to the immune defense in the kidney by reduction of available free iron in the urine. Other possibilities are that LF may play a role in the iron metabolism by recovering free iron from urine and making it available for metabolic use, and that LF may participate in the antioxidant defense systems protecting the kidney against nonmicrobial oxidative injury, that is, ischemia, reperfusion and inflammation.

Comparative analysis of KRAB zinc finger proteins in rodents and man: evidence for several evolutionarily distinct subfamilies of KRAB zinc finger genes.

Although the KRAB zinc finger proteins probably constitute the single largest class of transcription factors within the human genome, almost nothing is known about their biological function. To increase our knowledge about this interesting and relatively unexplored family of potent transcriptional repressors, we here present the cloning, structural analysis, and expression study of three novel mouse KRAB zinc finger proteins. In addition, we present an extensive comparative analysis of various members of this gene family based on the structure of the common KRAB A motif. At least three larger subfamilies of KRAB zinc finger proteins are identified: one carrying the classical KRAB A motif only, another holding both a classical KRAB A and a classical KRAB B motif, and a third holding a classical KRAB A and a highly divergent KRAB B domain, named b. A large variation both in size and in primary amino acid sequence was observed in the linker region between the KRAB domain and the C-terminally located zinc finger repeats. This variability indicates that this region is of minor importance for the biological function of KRAB-containing zinc finger proteins. The fact that in many zinc finger genes, the entire or almost the entire linker region is composed of degenerate finger motifs substantiates this conclusion. The absence of identifiable KRAB A and B motifs in the genome of yeast, Saccharomyces cerevisiae, indicates a relatively late appearance of the KRAB domain in evolution and may suggest that the biological functions are restricted to multicellular organisms. In addition, we show that the expression of individual members of one subfamily of KRAB zinc finger genes is restricted to specific hematopoietic cell lineages. This finding suggests that KRAB zinc finger proteins may play a role in lineage commitment, possibly silencing leakage transcription from nonlineage-expressed genes.

Demethylation of ERV3, an endogenous retrovirus regulating the Krüppel-related zinc finger gene H-plk, in several human cell lines arrested during early monocyte development.

The activation of H-plk (Human-proviral linked Krüppel), a human Krüppel-related zinc finger gene in organs such as placenta, adrenal cortex, and testis, is probably due to insertion of an endogenous retrovirus, ERV3, upstream of the gene. Several differently spliced transcripts originate from this locus, e.g., a transcript encoding the retroviral envelope protein and a few differentially spliced transcripts encoding both the env and the zinc finger protein. During a screening for zinc finger proteins expressed during monocyte differentiation, two H-plk encoding cDNA clones were isolated from the human monoblast cell line U-937. Northern blot analysis of a panel of human hematopoietic cell lines showed high levels of constitutive expression of this zinc finger transcript in two monocytic cell lines (U-937 and THP-1) but not in any of the other cell lines or tissues tested. In addition, the H-plk transcript was upregulated by the phorbolester PMA in U-937 and in an additional monocytic cell line, MonoMac 6. Genomic Southern blot analysis of a panel of hematopoietic cell lines, after cleavage with the methylation sensitive enzyme Xho I, led to the detection of tissue specific demethylation in all three monocytic cell lines. The Xho I site was mapped to a position just downstream of the regulatory region of the endogenous retrovirus. By analysis of the U-937 cell line with two additional restriction enzymes, Nar I and Sma I, the demethylation was shown to affect at least three independent CpG dinucleotides in this region of the gene. In summary, the present data provide evidence for specific demethylation of this genomic region, in cells of monocytic origin, resulting in enhanced transcription of the genetic regions derived from both the env region of the retrovirus and the Krüppel-related zinc finger gene.

Isolation of cDNA clones for 42 different Krüppel-related zinc finger proteins expressed in the human monoblast cell line U-937.

To study the complexity and structural characteristics of zinc finger proteins expressed during human hematopoiesis and to isolate novel regulators of blood cell development, a degenerate oligonucleotide probe specific for a consensus zinc finger peptide domain was used to isolate 63 cDNA clones for Krüppel-related zinc finger genes from the human monoblast cell line U-937. By extensive nucleotide sequence and Northern blot analysis, these cDNA clones were found to originate from approximately 42 different genes (HZF 1-42) of which only 8 have previously been described. Northern blot analysis showed that a majority of these genes were expressed at comparable levels in U-937 and HeLa cells. The large number of individual genes represented among the 63 clones and their apparent non-cell-type-specific expression suggest that the majority of the Krüppel-related zinc finger genes are likely to be expressed in most human tissues. In contrast, some of the genes displayed a restricted expression pattern, indicating that they represent potential regulators of monocyte differentiation or proliferation. Detailed structural analysis of the first 12 cDNAs (HZF 1-10) and a partial characterization of HZF 11-42 revealed that a common feature of human Krüppel-related zinc finger proteins is the presence of tandem arrays of zinc fingers ranging in number from 3 to over 20 that are preferentially located in the carboxy-terminal regions of the proteins. In addition, several novel KRAB-containing zinc finger genes and a novel conserved sequence element were identified.

Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage.

Three human monocytic cell lines, U-937, THP-1 and Mono Mac 6 have, because of their morphology and staining properties, been classed as cell lines frozen in a window of the monocyte differentiation lineage corresponding to monoblasts and/or immature monocytes. These cell lines were analyzed for expression of a panel of hematopoietic differentiation markers by Northern blot analysis. They were all found to express one or several biochemical markers characteristic of immature cells in monocytic development, including myeloperoxidase, N-elastase, cathepsin G, myeloblastin, and azurocidin. Normal peripheral blood monocytes did not express these markers. Moreover, several markers expressed at high levels in mature monocytes, such as lysozyme, CD14, MHC class II and alpha-1 antitrypsin were either not expressed or were expressed only at low levels in the three cell lines analyzed. These results show that arrested differentiation at a relatively early stage of monoblast development is a common denominator for these human monocytic cell lines. Thus, transforming mutations acting at such an immature differentiation stage may frequently lead to neoplastic transformation, whereas similar mutations occurring at a more mature differentiation stage never give rise to any leukemias due to the loss of proliferative potential in committed cells.

Expression of a mast cell tryptase in the human monocytic cell lines U-937 and Mono Mac 6.

Expression of a mast cell tryptase mRNA was detected in two human monocytic cell lines, the U-937 and the Mono Mac 6, and in normal human peripheral blood (PB) monocytes. In the U-937 cell line but not in normal PB monocytes, the tryptase expression was upregulated 3-50 fold following phorbol ester (PMA)-induced differentiation, but no such induction was seen after retinoic acid, interferon-gamma or vitamin D3 exposure. The tryptases expressed in PMA-induced and non-induced U-937 and in Mono Mac 6 were characterized by PCR amplification and nucleotide sequence analysis. The U-937 cell line was found to express a tryptase identical to one of the previously cloned mast-cell beta tryptases (Tryptase I), and the tryptase expressed in Mono Mac 6 was found to be nearly identical to the previously cloned alpha tryptase. By northern blot analysis with oligonucleotide probes specific for the alpha and beta tryptases both cell lines were found to express only one type of tryptase. Densitometric quantifications of tryptase mRNA levels, in the two cell lines, showed approximately 80 times higher mRNA levels in Mono Mac 6 compared to non-induced U-937. Immunohistochemical staining for tryptase showed a marked heterogeneity in the Mono Mac 6 cell line. Only one out of 10 cells were positive for the protein but the levels in these cells were very high, equivalent, or even higher than the levels seen in the human mast cell line HMC-1. This shows that the expression of a single tryptase, in this case the alpha tryptase, is sufficient for the production of a stable protein and probably also a stable proteolytically active tetramer. The family of human mast-cell tryptases has been considered to represent a class of proteases specifically expressed in mast cells and basophilic leucocytes. The expression of tryptases in two monocytic cell lines and in normal PB monocytes indicate that in humans, the lineage specificity of these serine proteases is less restricted than earlier expected. The cloning of a full length cDNA for the murine counterpart to the human mast cell tryptases, the MMCP-6, is presented. No expression of the MMCP-6 was detected in a panel of mouse monocyte or macrophage cell lines indicating a species difference in the lineage specificity of the 'mast cell tryptases'.