Tissue-specific binding of nuclear factors to the 5′-flanking region of rat gene for calcium-binding protein regucalcin

The existence of nuclear factors which bind to the 5-flanking region of calcium-binding protein regucalcin gene in rats was investigated. We previously reported that rat regucalcin mRNA is expressed in a highly tissue-specific manner; the mRNA was mainly present in the liver but only slightly in the kidney. When the nuclear proteins extracted from the liver and kidney of rats were used in the gel mobility shift assays, a protein-DNA complex was uniquely formed with the DNA fragment containing the upstream region from the first exon of rat regucalcin gene. On the other hand, this complex was not found by using the nuclear extracts from rat brain, spleen, and heart. The nuclear proteins of these extracts, however, could specifically bind to the DNA fragment containing the first exon region of rat regucalcin gene, although Northern blot analysis did not show detectable amount of regucalcin mRNA levels in rat brain, spleen, and heart. The present study demonstrates that the existence of nuclear protein components which bind to the regucalcin gene. These identified components may be involved in the tissue-specific regulation of regucalcin gene expression.     

Nuclear factor A5 from the rat liver that requires a metal for specific interaction in vitro with distal element of the tyrosine aminotransferase gene promoter

To detect nuclear protein factors which might account for a tissue-specific and inducible expression of the rat tyrosine aminotransferase (TAT) gene promoter, extracts from rat liver and spleen nuclei have been fractionated by heparin-sepharose chromatography and the fractions assayed for sequence-specific binding to the distal TAT gene promoter element (sequence between -313 and -210). Gel retardation experiments carried out in the presence or absence of Mg2+, Ca2+, or Zn2+ ions showed that there are at least two nuclear factors (A3 and A4) binding to the distal promoter element only in the presence of the chelator (20 mM EDTA). Incubation of the protein fractions with Zn2+ or Ca2+ instead of commonly used Mg2+allowed: (i) to avoid 3 2P-DNA-probe degradation by "contaminating" endogenous nucleases; and (ii) to detect another sequence-specific nuclear factor, A5. No other specific binding activities were found in the rat-liver nuclear fractions tested under these conditions. As the metal ions became inaccessible to chelation in excess of EDTA and EGTA when protein factor A5 was complexed to DNA we assumed that factor A5 is metalloprotein which requires Zn or Ca to maintain a structure of its DNA-binding domain. To identify the polypeptide possessing this domain, a protein gel blotting procedure was employed. By incubating gel blots with the 3 2P-DNA-probe in the buffer containing Zn2+, specific binding to the only polypeptide with approximate Mr 30 kDa was clearly revealed. Both gel retardation and gel blotting assays consistently showed that nuclear factor A5 is present in the liver, but not in the spleen extracts.     

大鼠C3基因TATA区结合核蛋白与其转录活性关系的研究

大鼠Ｃ３基因的转录受雄激素调控且只在大鼠腹侧前列腺中表达。本文报道用凝胶电泳带漂移分析和离体ＤＮａｇｅＩ足迹法分析研究了大鼠Ｃ３基因ＴＡＴＡ区结合核蛋白与其组织专一的和雄激素调控的转录活性间的关系。结果表示：相同的（－５０到－１０）４ｂｐ的ＴＡＴＡ区ＤＮＡ片段在不同组织的细胞核中结合的核蛋白是不同的。通过与一个仅在ＴＡＴＡ区－２９．位核苷酸Ａ变为Ｇ的天然存在的不表达的Ｃ３（２）等位基因上此片段的核蛋白结合能力作比较后提示在Ｃ３基因唯一表达的组织—腹侧前列腺中，结合在此区的核蛋白是－２９位Ａ依赖的，而在所研究的不表达组织—肝和睾丸中则否。而且此类蛋白质还可能具有与雄激素调控此基因表达有关的其他反式作用因子相互作用的结构域．     

Noncovalent binding of poly(ADP-ribose) to nuclear matrix proteins: developmental changes and tissue specificity

Poly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functions via covalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis. The isolation and analysis of endogenous poly-(ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteins in vitro are also present under physiologic conditions in vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene

Summary A plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory element of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-1 is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-1 may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-1 binding.