Positive and negative regulatory elements in the late lactation protein-A gene promoter from the tammar wallaby ( Macropus eugenii)

Abstract

Little is known about the regulation of the marsupial-specific late lactation protein-A (LLP-A) gene, first expressed at mid-lactation in the mammary gland of the tammar wallaby. A genomic clone of LLP-A was sequenced and shown to include seven exons. The LLP-A promoter region of 1969 bp ligated to a secreted alkaline phosphatase (SEAP) gene reporter was co-transfected into CHO-K1 cells with prolactin (PRL) receptor cDNA. Transfected cells cultured with insulin, cortisol and PRL did not secrete SEAP into media. Similarly, this construct was not expressed in the mammary gland of eight lines of transgenic mice. In contrast, when the LLP-A promoter region was reduced to 850 bp, the expression of the SEAP reporter in CHO-K1 cells was constitutive and PRL-independent, despite the presence of two low affinity Stat5 binding sites. The 1969 bp promoter was analyzed using nine serial deletions ligated to the SEAP gene. The expression of these constructs was PRL-independent. Five putative inhibitory elements were identified between −1969 and −1796, −1404 and −1184, −1184 and −992, −992 and −757, and −591 and −425, and a putative enhancer or core transcription element between −425 and−239. These studies indicate that the complex temporal regulation of the LLP-A gene involves elements in its 5′-regulatory region.