cDNA clones encoding leucine-zipper proteins which interact with G-CSF gene promoter element 1-binding protein.

The gene expression of granulocyte colony-stimulating factor (G-CSF) is induced by lipopolysaccharide (LPS). GPE1, a cis-controlling element of the G-CSF gene, functions as an LPS-responsive element. GPE1-binding protein (GPE1-BP), a leucine-zipper protein, did not independently activate G-CSF gene expression. Protein blot analysis with biotinylated GPE1-BP revealed that there were nuclear proteins that interact specifically with GPE1-BP. Three leucine-zipper proteins were isolated from mouse cDNA expression libraries by this method: NF-IL6, ATF4, and a novel ATF4-related ATFx. The interactions of these proteins with GPE1-BP may play key roles in G-CSF gene expression.     

Transformation of a novel direct-repeat repressor element into a promoter and enhancer by multimerisation.

Studies on the regulation of interferon (IFN) responsive genes have mainly been centred on the highly conserved IFN stimulated responsive elements (ISREs) which can mediate type I and II IFN inducibility. To date little is known about other functional cis-acting regulatory motifs in IFN responsive genes. We report here on the identification of a repressor element in the human MxA gene defined to a 19 base pair (bp) region which houses a 9 bp direct repeat. DNA-specific protein binding on this element is not affected by IFN treatment and is distinct from ISRE binding proteins. Remarkably, contrary to expectations, when the repressor element is multimerised and spliced, in either orientation, to a reporter gene it behaves like a functional, constitutive promoter. Positioning the multimerised element in front of the SV40 enhancerless promoter also led to enhanced expression. The same protein(s) seem to bind to both the single repressor element and its multimerised form. This discovery of phenotypic reversal on a repressor element via multimerisation may have important implications in vivo.     

Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins

In previous investigations, cell fusion was found to silence either the endogenous rat growth hormone (GH) gene or a transfected rat GH gene promoter, implying that repression plays a role in regulation of this gene. To search the rat GH gene promoter for repressor sequences, a series of 5'-deleted GH-CAT constructs was analyzed by transient expression in GH3 rat pituitary cells. Deletion of either a distal region between positions -307/-244 or a proximal sequence between -169/-152 increased CAT enzymatic activity by 3-4-fold. Since the action of the proximal repressor element (PRE) at -169/-152 was serum-independent, and the element is located between two strong positive elements, the PRE and its cognate binding proteins were further analyzed. A 5-base pair sequence centered at -163 is critical for PRE repressor activity, since mutation of this sequence in GH-CAT constructs yielded 6-11-fold increases in expression in GH3 cells. Although the PRE is adjacent to the GH thyroid hormone (T3) response region, they are distinct elements, since the PRE mutation has little effect on the T3 response of GH-CAT constructs. Nuclear extracts of 10 cell lines were searched by DNA mobility shift for protein(s) binding specifically to a double-stranded PRE probe. No such protein was detected in any of four rodent pituitary cell lines or three human cell lines. However, three different rodent non-pituitary cell lines yielded a common shifted band, corresponding to a DNA sequence-specific PRE-binding protein (PREB). Similar analysis with the coding strand of the PRE detected no shifted band in any of these cell lines. However, the PRE noncoding strand yielded a common shifted band in all of the cell lines, corresponding to a ubiquitous, strand-specific, single-stranded PRE-binding protein (ssPREB). Mutation of the PRE permitted ssPREB binding to the coding strand, implying that the wild-type coding strand somehow excludes ssPREB binding. That PREB and ssPREB are distinct proteins was confirmed by the inability of their DNA binding sites to cross-compete binding of the proteins.     

Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway

The efficient assembly of newly replicated and repaired DNA into chromatin is essential for proper genome function. Based on genetic studies in Saccharomyces cerevisiae, the histone chaperone anti-silencing function 1 (Asf1) has been implicated in the DNA repair response. Here, the human homologs are shown to function synergistically with human CAF-1 to assemble nucleosomes during nucleotide excision repair in vitro. Furthermore, we demonstrate that hAsf1 proteins can interact directly with the p60 subunit of hCAF-1. In contrast to hCAF-1 p60, the nuclear hAsf1 proteins are not significantly associated with chromatin in cells before or after the induction of DNA damage, nor specifically recruited to damaged DNA during repair in a bead-linked DNA assay. A model is proposed in which the synergism between hAsf1 and CAF-1 for nucleosome formation during DNA repair is achieved through a transient physical interaction allowing histone delivery from Asf1 to CAF-1.