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In some cases core histone genes in the mouse depend on intragenic sequence elements for high level expression [Gene 176 (1996) 1]. Here we report that the highly expressed gene for rat linker histone H1d also contains an intragenic activating region (IAR). Using transient transfection assays in mouse fibroblast NIH3T3 cells, we showed that rat H1d contains a downstream region (+21 to +116) that imparts a two- to threefold up-regulation of fused reporters. This region also activated expression when moved to the promoter region, though the effect was dependent on its distance from other promoter elements. The IAR contains sequence homologies to the core alpha and Omega elements identified as functional protein binding sites within the mouse H3.2 coding region activating sequence (CRAS). A pair of Omega elements (+32 and +66) accounts for the activating effect of the H1d intragenic region as shown by targeted mutations as well as stepwise deletions. The H1d and H3.2 Omega sequences bound similar and perhaps identical proteins by gel shift analysis. The H1d alpha-like sequence at +56 overlaps the translational start codon and was therefore not mutated. Like the mouse H3.2 alpha element, it bound transcription factor YY1 in gel shift assays. H1t, the gene for the testis-specific linker histone, did not demonstrate an IAR. While H1t has a similar alpha sequence and did bind YY1, it lacks the Omega homologies of H1d. Sequence comparison shows that the YY1/alpha site as well as the adjacent Omega site are likely present in genes for other standard H1 variants, but that the +32 Omega site in the 5' untranslated region (UTR) of H1d is unique. We conclude that the +32 and +66 Omega sequences of the rat H1d gene contribute significantly to its high-level expression.

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.

Transcription factor Sp1 is often described as an ubiquitous protein. However, a positive immunohistochemical signal for Sp1 in mouse testis was obtained only over the outer ring of cells in most seminiferous tubules, comprising Sertoli cells, spermatogonia, and early spermatocytes. Loss of Sp1 in maturing germ cells was confirmed by Western blots of purified spermatogonia, pachytene spermatocytes, and round spermatids. 5' RACE identified several alternative Sp1 Cap sites. One of these lies in intron 1-2, and identifies an alternative exon 1 (1P). PCR designed to amplify eDNA containing exon 1P generated primarily productss that contained intron 1P-2. RNase protection assays showed that, beginning with the appearance of pachytene cells in testis, mRNAs with the strandard exon 1 of Spl decrease sharply while those with exon 1P become prominent. Real-time PCR confirmed that the unspliced product from exon 1P is much more common in adult testis than the spliced version. The unspliced mRNA beginning at exon IP. contains potential initiation codons followed by stop codons in all reading frames, and is expected to be translationally non-functional. RT-PCR confirmed that testis contains mRNAs that contain exon IP joined to the full coding length of Spl. Accordingly, in pachytene a switch to a new promoter occurs that results in coding-length products that have a translationally defective sequence upstream of exon 2. Thus many measures of Sp1 mRNA in the testis will markedly over-estimate its functional level, and Sp1 protein is down-regulated in developing germ cells. [ABSTRACT FROM AUTHOR]