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Melatonin Mediated Transcriptional Mechanisms in the Ovine Pars Tuberalis

West, Alexander

[Thesis]. Manchester, UK: The University of Manchester; 2013.

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Abstract

In seasonal mammals the duration of nocturnal melatonin secretion accurately reflects the environmental photoperiod. The endocrine rhythm is decoded by a specialised portion of the pituitary gland (the pars tuberalis, PT) which then relays this information to the pars distalis and hypothalamus, precipitating huge annual changes in physiology and behaviour. However how the PT decodes the melatonin signal is currently unknown. Melatonin influences gene transcription in the ovine PT at its onset and offset, and the phase relationship of these two groups is believed to form the underlying mechanism by which the PT integrates seasonal time. The transcripts induced at melatonin offset are understood to be under the control of a seasonally gated cAMP mechanism. Conversely processes involved in melatonin-mediated gene induction are currently not understood.The work in this thesis ultimately aims to reveal how the seasonal melatonin signal is decoded by the PT. To this end melatonin-mediated gene induction has been characterised through RNAseq, the highly displaced cohorts submitted to bioinformatic promoter analysis and the paradigm tested though in vitro modelling techniques.In this study a 1.5 h infusion with melatonin acutely regulated 219 transcripts in the ovine PT (115 induced, 104 repressed, >1.5 fold change), confirming previous association of several genes (including Cry1, MT1, Gadd45g, Nampt and Npas4) to rapid melatonin control. Gross promoter analysis of these groups indicated that the induced gene cohort was significantly enriched for GC content and CpG islands suggesting the involvement of epigenetic mechanisms of transcriptional control. Further bioinformatic analysis specifically implicated the importance of transcription factors ZFP161 and PAX5 in melatonin-mediated gene induction in the PT. Several immortalised cell lines were screened for the presence of a functional melatonin receptor. Two strains (MCF7 oMT1 and NES2Y) exhibited significant attenuation of forskolin-mediated cAMP accumulation when co-treated with melatonin, a hallmark of melatonin Gαi-coupled protein receptor signalling. These lines were subsequently evaluated as models of melatonin-mediated gene induction of the sheep PT through ovine promoter reporter assays of Cry1, Nampt, NeuroD1 and Npas4. However, treatment with melatonin failed to evoke a reporter response suggesting that the cell line models were inadequately equipped to reflect PT biology. Subsequently a protocol was established to culture ovine PT explants culture which faithfully recapitulated melatonin mediated transcriptional dynamics in vitro, providing a possible tool for the future investigation of the PT. Lastly, previous work has shown the transcriptional profile of Npas4 to peak highly and transiently, pre-empting the expression of other melatonin-induced genes. Using a COS7 cell line model, heterologously-expressed NPAS4 was shown to form functional heterodimeric partnerships with ARNT and ARNTL and transactivate both Cry1 and Nampt promoter reporters through novel binding sites. Collectively these data indicated NPAS4 to act as an immediate activator of melatonin regulated circuits

Layman's Abstract

None

Bibliographic metadata

Type of resource:
Content type:
Administered thesis
Form of thesis:
Traditional
Type of submission:
Doctoral level ETD - final
Thesis title:
Melatonin Mediated Transcriptional Mechanisms in the Ovine Pars Tuberalis
Degree programme:
PhD Medicine (Human Development) 4yr
Publication date:
2013-03-26T18:02:38
Institution:
The University of Manchester
Location:
Manchester, UK
Total pages:
189
Abstract:
In seasonal mammals the duration of nocturnal melatonin secretion accurately reflects the environmental photoperiod. The endocrine rhythm is decoded by a specialised portion of the pituitary gland (the pars tuberalis, PT) which then relays this information to the pars distalis and hypothalamus, precipitating huge annual changes in physiology and behaviour. However how the PT decodes the melatonin signal is currently unknown. Melatonin influences gene transcription in the ovine PT at its onset and offset, and the phase relationship of these two groups is believed to form the underlying mechanism by which the PT integrates seasonal time. The transcripts induced at melatonin offset are understood to be under the control of a seasonally gated cAMP mechanism. Conversely processes involved in melatonin-mediated gene induction are currently not understood.The work in this thesis ultimately aims to reveal how the seasonal melatonin signal is decoded by the PT. To this end melatonin-mediated gene induction has been characterised through RNAseq, the highly displaced cohorts submitted to bioinformatic promoter analysis and the paradigm tested though in vitro modelling techniques.In this study a 1.5 h infusion with melatonin acutely regulated 219 transcripts in the ovine PT (115 induced, 104 repressed, >1.5 fold change), confirming previous association of several genes (including Cry1, MT1, Gadd45g, Nampt and Npas4) to rapid melatonin control. Gross promoter analysis of these groups indicated that the induced gene cohort was significantly enriched for GC content and CpG islands suggesting the involvement of epigenetic mechanisms of transcriptional control. Further bioinformatic analysis specifically implicated the importance of transcription factors ZFP161 and PAX5 in melatonin-mediated gene induction in the PT. Several immortalised cell lines were screened for the presence of a functional melatonin receptor. Two strains (MCF7 oMT1 and NES2Y) exhibited significant attenuation of forskolin-mediated cAMP accumulation when co-treated with melatonin, a hallmark of melatonin Gαi-coupled protein receptor signalling. These lines were subsequently evaluated as models of melatonin-mediated gene induction of the sheep PT through ovine promoter reporter assays of Cry1, Nampt, NeuroD1 and Npas4. However, treatment with melatonin failed to evoke a reporter response suggesting that the cell line models were inadequately equipped to reflect PT biology. Subsequently a protocol was established to culture ovine PT explants culture which faithfully recapitulated melatonin mediated transcriptional dynamics in vitro, providing a possible tool for the future investigation of the PT. Lastly, previous work has shown the transcriptional profile of Npas4 to peak highly and transiently, pre-empting the expression of other melatonin-induced genes. Using a COS7 cell line model, heterologously-expressed NPAS4 was shown to form functional heterodimeric partnerships with ARNT and ARNTL and transactivate both Cry1 and Nampt promoter reporters through novel binding sites. Collectively these data indicated NPAS4 to act as an immediate activator of melatonin regulated circuits
Layman's abstract:
None
Keyword(s):
Thesis main supervisor(s):
Thesis co-supervisor(s):
Thesis advisor(s):
Funder(s):
Degree grantor:
Language:
en

Institutional metadata

University researcher(s):
Academic department(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:190400
Created by:
West, Alexander
Created:
26th March, 2013, 18:02:40
Last modified by:
West, Alexander
Last modified:
10th April, 2013, 11:07:02

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