Abstract
The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.
Highlights
The human genes coding for growth hormone and placental lactogen are clustered on chromosome 17 in the following order 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'
As a model system for analyzing the regulation of gene transcription, our laboratory is studying the family of genes coding for prolactin, growth hormone (GH), and placental lactogen
All constructs were made in pBLNHCAT2, a vector derived from pBLCAT2 [35] and comprising the herpesvirus thymidine kinase (TK) promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene
Summary
The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. The five genes are clustered over a 66-kb segment on human chromosome 17, in the order 5' human GH (hGH)-N hCS-L hCS-A hGH-V hCS-B 3' Despite their homology, these genes are expressed in different cell types, i.e., the somatotrophs of the anterior pituitary for the hGH-N gene and the placental syncytiotrophoblast for the other four [3, 8, 39]. The placental genes are expressed at very different levels, in the order hGH-V < hCS-L < hCS-B
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