ESPE Abstracts (2014) 82 HA1

Deciphering the Functional Mechanisms by which MKRN3 Regulates Puberty Initiation

Ana Paula Abreua,b, Victor Navarroa, Martha Boschc, Joy Lianga, Delanie Macedob, Serap Simavlia, Sekoni Noela, Iain Thompsona, Oline Ronnekleivc, Rona Carrolla, Ana Claudia Latronicob & Ursula Kaisera

aHarvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts, USA; bDisciplina de Endocrinologia e Metabologia, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, SP, Brazil; cOregon Reg Primate Rsrch Ctr, Portland, Oregon, USA

Background: We recently identified loss-of-function mutations in makorin ring finger 3 (MKRN3) as a cause of familial central precocious puberty (CPP). Analysis of Mkrn3 expression in the arcuate nucleus of mice showed high expression levels in juvenile mice, with a marked reduction prior to puberty onset, suggesting that MKRN3 inhibits puberty initiation. The function of MKRN3 is not known but based on its amino acid sequence, it is predicted to act as an ubiquitin ligase.

Objective and Hypotheses: To elucidate the mechanism by which MKRN3 regulates GnRH secretion.

Method: As a first step to analyze if MKRN3 acts as an ubiquitin ligase, we performed affinity purification of HEK293T cells co-transfected with MKRN3 and His-tagged ubiquitin (Ub-His). Secondly, we performed in situ hybridization (ISH) assays to analyze Mkrn3 expression and map the distribution of Mkrn3 in the hypothalamus of mice. Finally, to detect Mkrn3 expression in two neuronal populations critical for reproduction -GnRH and Kiss1 neurons- we performed single cell RT-PCR in cells derived from Kiss1-GFP and Gnrh-GFP female mice.

Results: MKRN3 was detected in immunoprecipitated Ub-His protein complexes suggesting that MKRN3 acts as a ubiquitin ligase. ISH detected high diffuse Mkrn3 expression in the hypothalamus at postnatal day (PND) 1, with more localized expression in the arcuate and ventromedial nuclei at PND10, which decreased to very low levels by PND15. Our single cell RT-PCR studies showed that Mkrn3 was expressed in ~35 and 20% of arcuate Kiss1 neurons at age PND13 and in adult mice, respectively; and in ~30% of GnRH neurons at PND13 but not in adults.

Conclusion: Our in vitro studies show that MKRN3 likely acts through ubiquitination. The temporal and spatial pattern of Mkrn3 expression and co-localization in Kiss1 and GnRH neurons suggests that MKRN3 acts as a ‘brake’ for GnRH secretion and corroborates our genetic findings in humans.