BRFU, a TFIIB-like factor, is directly recruited to the TATA-box of polymerase III small nuclear RNA gene promoters through its interaction with TATA-binding protein.

The human snRNA genes transcribed by RNA polymerase II (pol II) and III (pol III) have different core promoter elements. Both gene types contain similar proximal sequence elements (PSEs) but differ in the absence (pol II) or presence (pol III) of a TATA-box, which, together with the PSE, determines the assembly of a pol III-specific pre-initiation complex. BRFU is a factor exclusively required for transcription of the pol III-type snRNA genes. We report that recruitment of BRFU to the TATA-box of these promoters is TATA-binding protein (TBP)-dependent. BRFU in turn stabilizes TBP on TATA-containing template and extends the TBP footprint both upstream and downstream of the TATA element. The core domain of TBP is sufficient for BRFU.TBP.DNA complex formation and for interaction with BRFU off the template. We have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. BRFU has no specificity for sequences flanking the TATA-box and also forms a stable complex on the TATA-box of the pol II-specific adenovirus major late promoter (AdMLP). Furthermore, pol III-type transcription can initiate from an snRNA gene promoter containing an AdMLP TATA-box and flanking sequences. Therefore, the polymerase recruitment is not simply determined by the sequence of the TATA-box and immediate flanking sequences.

Early gene expression of both RNA polymerase I transcription factors UBF1 and UBF2 precedes ribosomal RNA synthesis during lymphocyte mitogenic stimulation.

Phytohaemagglutinin stimulates lymphoid cells to initiate active cell division which is tightly coordinated with transcription of ribosomal RNA genes. Nuclear Run-On assays demonstrated that treatment of peripheral blood lymphocytes with PHA (10 microg/ml) resulted in maximal rRNA synthesis after 64 hrs. In contrast, mRNA levels for upstream binding factor (UBF)1 and UBF2, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, increased relatively rapidly within 3 to 6 hrs. and remained elevated for at least the next 60 hrs. We further showed that exponentially growing cells of promyelocytic leukemia line HL-60 contained the same amounts of UBF1 and UBF2 mRNAs as phytohaemagglutinin (PHA)-stimulated lymphocytes for 6 hrs. Growth arrest of HL-60 cells, caused by 10 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic/macrophage-like differentiation for 72 hrs., has been accompanied by a 50% decrease in UBF1/2 mRNAs expression. The lowest concentrations of UBF1/2 mRNAs were revealed in non-dividing terminally differentiated granulocytes. Regardless the activity of RNA polymerase I transcription and cell division rate, UBF1 mRNA levels prevailed over UBF2 mRNA levels in all human blood cell populations tested. Our results suggest that UBF gene expression is an important regulatory mechanism involved in the acceleration and possibly deceleration of rDNA transcription observed during mitogenic stimulation and inhibition of blood cells.

Granulocytic protein p25 is a DNA-binding subunit of protein M(r) = 50,000: subcellular localization, cell and species specificity.

We have previously reported the presence and isolation of the novel protein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) subunit association, (c) partial protein sequencing, (d) subcellular localization, (e) cellular and species specificity and (f) stability in the presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hematopoietic cells, SDS-PAGE/Western blotting and immunohistochemical staining were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using Southwestern blotting. Selective inhibition of granulocytic proteinases was performed. Granulocytic protein p25 was found to be a product of oxidative cleavage of disulfide bridges in the p50 dimer. It was shown that neither protein p50 nor the p25 subunit is a degradation product of a protein of higher molecular weight. The N-terminal amino acid sequence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DNA without significant sequence specificity was revealed and nuclear localization of some fraction of p50 dimer was established. The data concerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p25 originates from splitting of the p50 dimer. This subunit shows no identity with proteins already sequenced. DNA-binding of p25 is not sequence specific. It is concluded that the protein p50 is localized in the nuclei and cytoplasmic granules of mature human polymorphonuclear leukocytes or granulocytes of species high on the evolutionary tree. The functions of this protein remain to be determined.

Differential expression of nuclear HMG1, HMG2 proteins and H1(zero) histone in various blood cells.

Changes in the levels of chromosomal high-mobility group proteins HMG1, HMG2 and histone H1 zero were investigated in blood cells of various types, proliferation activity and stage of differentiation. The relative amounts of proteins HMG1, HMG2 and histone H1 zero were evaluated densitometrically by SDS-PAGE of 5 per cent w/v perchloric acid extracts of blood cells. Concerning the HMG1 and HMG2, the main conclusions were: the expression of these HMG proteins was higher in malignant cells, namely leukemia cell lines, then in lymphocytes or granulocytes and the distribution of HMG1 and HMG2 was highly cell-specific. In comparison with lymphoid cells, the levels of HMG1/2 were higher in myeloid cells. The results revealed that in myeloid cells HMG2 prevails over HMG1. There was no direct correlation between HMG1/2 expression and proliferation activity. The levels of HMG1/2 did not depend on the transcription of chromatin either. However, there was some connection between irreversibly differentiated nonproliferating cells and a loss of HMG1/2 proteins. Reversibly differentiated leukemic cells retain their HMG1/2 levels. Similarly to HMG1/2,H1 zero showed a strong cell specificity. The level of H1 zero was different in the various blood cell types. As compared with lymphoid cells, the level of H1 zero was several-fold higher in myeloid cells, regardless of whether they were normal or malignant. Moreover, there was an accumulation of H1 zero in differentiating HL-60 cells accompanied by only a slight decline in cell proliferation; this agrees with the idea that H1 zero expression is not directly associated with the inhibition of cell growth. Rather higher expression of H1 zero is related to changes during cell differentiation.