H5 gene specific trans-activation by nuclear extracts from avian erythroid cells.

Abstract

Nuclear extracts from chicken erythroid cells selectively stimulate transcription of the chicken histone H5 gene (and not of other chicken histone genes) after coinjection into frog oocytes. This effect is shown to involve an enhancerlike activity, and a region of the H5 gene sufficient to mediate trans-activation is defined.H5 is a linker histone variant (1, 13) which, in the chicken, is found only in erythroid cells (19). Levels of H5 protein increase during the differentiation and maturation of these cells (11,17,21), and this increase is correlated with chromatin condensation and repression of replication and transcription (17). We have previously isolated and characterized the single-copy chicken H5 gene (9) and demonstrated that transcription of this gene is accurately initiated in Xenopus oocytes (22). Our current interest lies in the transcriptional regulation of this gene.We report here our use of the frog oocyte to identify factors in erythroid-cell extracts that are involved in transcription of the H5 gene. Coinjection of DNA and cell extracts into frog oocytes has previously been used in the identification of regulatory factors for sea urchin histone genes (5,12,20) and in transcriptional studies with the adenovirus ElA protein (7,15).Effect of erythroid-cell extracts on the level of H5 transcripts in coinjected oocytes. Chromatin salt-wash fractions (CSWFs) were isolated from a chicken erythroid cell line by the methods of Stunnenberg and Birnstiel (20). The cells used (ts34 AEV LSCC HD3 [2]) (AEV cells) are transformed by a temperature-sensitive avian erythroblastosis virus, and they express H5 mRNA and protein (19).To test the fractions for possible effects on H5 gene expression, CSWF samples were coinjected, with the H5 gene and control genes, into batches of frog oocytes. The injected DNA was a mixture of pH.5/2.6 DNA, containing the H5 gene from -1,200 to +1,360 in pBR322 (Fig. la), and pH1/H23 DNA, containing two chicken histone genes, Hi and H2B (from pCH7.OE [3]), in pAT153. As shown in Fig. 2a, the CSWFs were injected into the oocyte cytoplasm, followed by nuclear injection of the DNA mixture. Oocyte manipulations were carried out according to Gurdon (6). DNA (5 to 10 ng) and CWSF (protein) (25 to 100 ng) were injected into each oocyte, and after incubation, total RNA was isolated from pooled batches of 25 oocytes (14). Figure 2b shows the result of quantitative primer extension analysis (10) of the oocyte RNA with synthetic 26-base primers. Extension on all three histone transcripts was carried out in the same reaction; each transcript generated two or more major extension products (due to cap site heterogeneity). Track 1 shows the result obtained when buffer alone was coinjected with the DNA, and track 2 shows coinjection of an AEV cell CSWF with the DNA.By comparing the two tracks, it can be seen that injection of the CSWF did not affect the level of Hi and H2B * Corresponding author.transcripts produced. In contrast, injection of the CSWF resulted in a dramatic increase (of at least 10...

Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

“…Except for in vitro binding activity, little is known on the trans-active elements controlling these cis-acting signals. In the present study, we addressed this question by transactivation experiments in the Xenopus oocyte system which has been adapted for functional tests of cell type-specific trans-active proteins (Mous et al, 1985;Wigley and Wells, 1987;Wu et al, 1987;Rungger et al, 1990). The transcription machinery of the oocyte may be complemented with the gene and the putative transcription factors (TFs) to be tested.…”

Basal expression of the histone H5 gene is controlled by positive and negativecis-acting sequences

“…While this work was in progress, two surveys of the regulatory regions of the H5 gene have appeared. Using microinjection in Xenopus oocytes, Wigley and Wells (8) have suggested that the region -85 to +313 (according to the +1 cap site determined by these authors) contains an enhancer-like element responsive to erythroid trans-acting factors. On the other hand, Engel and co-workers (9) have proposed the existence of a downstream enhancer element, the activity of which appears to be tissue-and stage-specific and independent of the promoter proximal DNA sequences.…”

Transcription of the chicken histone H5 gene is mediated by distinct tissue-specific elements within the promoter and the 3' enhancer.

“…This activity was detected in a microinjection assay in Xenopus laevis oocytes when plasmids containing the histone H5 gene (and other histone genes) were coinjected with various nuclear extract protein fractions prepared from avian erythroblastosis virustransformed erythroid progenitor cells. The enhancerlike activity could reside within the 45-bp segment of the H5 gene, which differs between the test genes used in the assays reported here and the previously published assays (33), or the discrepancy may be the result of the differences between the two assay systems used. We are unable to explain these differences but do not feel that the low-level expression of the H5 promoter deletion mutations could be due entirely to lack of an enhancer element, since the enhancers (both H5E and RSVE) artificially restructured into the reporter gene plasmids tested here should complement the removal of any other (putative 5') H5E element.…”

“…An enhancerlike activity was reported to exist in the H5 gene between positions -90 and +313 (33). This activity was detected in a microinjection assay in Xenopus laevis oocytes when plasmids containing the histone H5 gene (and other histone genes) were coinjected with various nuclear extract protein fractions prepared from avian erythroblastosis virustransformed erythroid progenitor cells.…”