Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial
Introduction
The tammar wallaby (Macropus eugenii) has a short pregnancy of ∼26.5 days and gives birth to an altricial young. A long and complex ∼300-day lactation period follows during which the composition of milk proteins, carbohydrates and lipids change dynamically so as to provide appropriate nutrition for the growth and development of the pouch young (Green, 1984, Nicholas et al., 1997, Sharp et al., 2009). In contrast, apart from the secretion of colostrum for the first 24–36 h postpartum (pp), the changes in mature eutherian milk are generally less dramatic (Jenness, 1974, McSweeney and Fox, 2013).
In most mammals, the major casein and whey protein genes including: α- and β-casein (CSN1, CSN2) and α-lactalbumin and β-lactoglobulin (LALBA and LGB) respectively, are induced at parturition and expressed throughout lactation (McSweeney and Fox, 2013, Nicholas et al., 1997). Unlike in eutherians, several key milk protein genes are temporally-expressed in marsupials. For the tammar, these include: early lactation protein (ELP), whey acidic protein (WAP) and late lactation proteins A and B (LLPA and LLPB). Early lactation protein is expressed at low levels from day 10 of pregnancy, upregulated at parturition and expressed during the first third of lactation until ∼125 days postpartum (pp) (Phase 2A, early lactation) (Nicholas et al., 1997, Simpson et al., 1998). The cessation of ELP expression and secretion coincides with the maturation of the acquired immune system and the gut-associated lymphoid tissue of the young (Joss et al., 2009). The presence of a single bovine pancreatic trypsin inhibitor (BPTI)/Kunitz domain suggests that ELP may prevent the degradation of immunoglobulins that are transferred from mother to young during this period (Piotte and Grigor, 1996, Simpson et al., 1998). Unlike ELP, WAP is expressed during Phase 2B (mid-lactation) (Simpson et al., 2000) and LLPA and LLPB are expressed during late Phase 2B-Phase 3 (late lactation) and Phase 3, respectively (Nicholas et al., 1997, Trott, 1999). The stage-specific expression of these genes suggests that they are controlled by sophisticated regulatory mechanisms.
The endocrine regulation of tammar ELP has been investigated using a mammary gland explant culture system (Pharo, 2014, Simpson, 1998). ELP is maximally responsive to the lactogenic hormones insulin (I), hydrocortisone (HC) and prolactin (PRL) in vitro (Pharo, 2014, Simpson, 1998). This is consistent with the in vivo induction of ELP at parturition when hormones such as prolactin are elevated (Hinds, 1988; reviewed in Shaw and Renfree, 2006). Likewise, the major tammar casein and whey protein genes, i.e. CSN1, CSN2, LALBA and LGB are up-regulated at parturition (Nicholas et al., 1997, Sharp et al., 2009). Notably, ELP can only be induced in explants in vitro if the gene is already expressed in vivo, i.e. in tissues from Phase 1 (pregnancy) and Phase 2A (early lactation) (Pharo, 2014, Simpson, 1998). However, ELP is unresponsive to lactogenic hormones in mid-and late lactation explants (Pharo, 2014). Interestingly, ELP expression can be maintained in Phase 1 explants treated with either insulin or insulin and hydrocortisone (Pharo, 2014), unlike many milk protein genes which require lactogenic hormones, or prolactin at least, to initiate gene transcription (Brisken and Ataca, 2015, Qian and Zhao, 2014, Rosen et al., 1999).
There are many factors that influence the expression of milk protein genes. These include: cis- and trans-acting transcription factors, distal and/or intragenic enhancers, non-coding RNAs (ncRNA), post-transcriptional regulation, extracellular matrix (ECM) factors, cell-cell interactions and hormones (e.g. insulin, glucocorticoids and prolactin) [see reviews (Qian and Zhao, 2014, Rijnkels et al., 2010, Rosen et al., 1999)]. Global and/or local chromatin organisation also plays an important role in the stage and tissue-specific expression of mammary gland genes (Rijnkels et al., 2010, Rijnkels et al., 2013).
Although the mechanisms that regulate many milk protein genes in eutherians have been investigated (Qian and Zhao, 2014, Rijnkels et al., 2010, Rosen et al., 1999), those that control the temporal, mammary gland-specific expression of ELP are unknown. We used primer extension analysis, promoter reporter assays and comparative genomics analyses of ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene (Pharo et al., 2012) to identify conserved transcription factor binding sites that may regulate the temporal expression of tammar ELP.
Section snippets
Animals
Tammar wallabies (Macropus eugenii) were kept in grass paddocks with ad libitum access to food, water and shelter in accordance with the National Health and Medical Research Council guidelines (NHMRC, 2013). The collection of tammar mammary gland tissues was approved by The University of Melbourne Animal Experimentation Ethics Committee.
Primer extension analysis of tammar ELP
The transcription start site (TSS) of the tammar ELP gene was identified using the Primer Extension System AMV Reverse Transcriptase kit (Promega Corporation)
Tammar ELP has two transcription start sites
The tammar ELP promoter was identified by primer extension analysis of mammary gland total RNA (Fig. 1). Two different ELP transcription start sites, 35 bp apart were identified in both the Phase 1 and Phase 2A mammary gland (Fig. 1A and B; Supplementary file 5). In contrast, ELP was not detected in the Phase 3 RNA or minus RNA samples (negative controls). Transcription of ‘long’ ELP (transcript 1) commenced at guanine 96 (numbering based upon the start site of the ELP reverse primer; Fig. 1B)
Discussion
Tammar ELP gene expression is specific to the mammary gland and early lactation and may function to protect immunoglobulins transferred via milk (Simpson et al., 1998). However, the mechanisms controlling its temporal, tissue-specific expression have been elusive. We identified the tammar ELP promoter, created promoter reporter constructs and investigated their activity in 2D cell culture models. The inability to reproduce the lactogenic hormone-responsiveness of ELP in explants in 2D cell
Conclusions
This study has provided new insights into the regulation of the tammar ELP gene. ELP transcription is tightly controlled and involves both hormone-dependent and hormone-independent factors. During pregnancy, NF-κB may suppress ELP transcription. At parturition, the spike of prolactin, the influence of glucocorticoids and insulin, the open chromatin conformation of the ELP gene and the binding of MAF/ETS, AP2γ and C/EBP to the proximal promoter provide a plausible model for tammar ELP gene
Acknowledgements
This research was funded by the Cooperative Research Centre for Innovative Dairy Products (CRC-IDP) and the Department of Zoology, The University of Melbourne, Australia. EAP was the recipient of an Australian Postgraduate Award (APA) and KNC, a Dairy Australia Postgraduate Student Award and both EAP and KNC received a scholarship top-up from the CRC-IDP. We thank Professor Kevin Nicholas for his helpful advice, Dr. Hong-Jian Zhu for the use of his GloMax-96 Microplate Luminometer, Josie Iaria
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