Deadline for full application: December 15th, 2013
Interviews: March, 2014
Start of the Ph.D.: October 1st, 2014
Department: Developmental and Stem Cell Biology
Title of the PhD project: Development of novel tools to understand mammalian sex determination.
Name of the lab: Human Developmental Genetics
Head of the lab: Ken McElreavey
PhD advisor: Anu Bashamboo
Email address: email@example.com
Doctoral school affiliation and University:
Presentation of the laboratory and its research topics:
The objective of the unit is to understand reproductive processes by analysing the genetic and epigenetic mechanisms associated with the development of both somatic and germ cell lineages of the mammalian gonad. This is achieved by complimentary approaches. We have a large collection of biological material from patients with urogenital anomalies (n>2,000) or infertility (n>3,700), through collaborations with clinical centres worldwide.
Much of our research activities on determination of sex (i.e. the somatic cells of the gonad) centre on the use of this unique biological resource. Analysis of patients with DSD (Disorder of Sex Development) phenotypes using comparative genomic hybridization (CGH) and the functional characterisation of pathogenic mutations revealed by Sanger and Next Generation sequencing approaches is aimed at both identifying new genetic variants associated with urogenital anomalies and to use this information to understand the mechanism of gonad formation and the choice of somatic sex. Since the mechanism of sex determination is not conserved through the evolution, there is a need to develop an appropriate model system to understand the in-vivo consequences of mutations associated with DSD. We are modifying protocols to direct pluripotent cells towards the sterodiogenic lineages of the testis to understand patient-specific progression of the pathology.
The specification and differentiation of primordial germs cells (PGCs) into male germ cells is a dynamic process involving a series of synchronised genetic and epigenetic changes that are poorly defined. To understand germ cell development we conduct detailed genetic/epigenetic analyses on infertile men. Our data show that 20% of infertile men have imprinting anomalies in their sperm. Using genome-wide methylation profiling and RNAseq we are determining if these epigenetic modifications are associated with specific genetic changes. To further understand the molecular mechanism of PGC specification and differentiation we are using an ex-vivo murine ES cell model system.
Description of the project:
(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))
The initial events of mammalian sex determination are genetically determined. SRY is the master regulator switch that triggers the formation of the testes. Errors in this process are common and can result in a range of phenotypes from complete sex-reversal to minor genital anomalies. Although many of the early cellular and morphological events that occur downstream of SRY action have been characterized, the mechanisms involved in mammalian sex determination remain poorly understood. The research focus of the unit is to understand reproductive processes by analysing the genetic and epigenetic mechanisms associated with the development of both somatic and germ cell lineages of the mammalian gonad. This is being achieved by the analysis of patients with DSD (Disorder of Sex Development) phenotypes. The strategy involves the functional characterisation of pathogenic mutations revealed by exome sequencing approaches using both sporadic and familial cases of DSD. These mutations can reveal novel insights into the mechanism of gonad formation and how choice of somatic sex is established and maintained. The project will involve the bioinformatic analyses of data generated by exome sequencing using publicly available software and ad-hoc scripts to identify novel genetic mutations associated with anomalies of human sex determination. Potentially pathogenic mutations in genes that will be identified by these procedures and that have not previously associated with DSD phenotypes will be analysed for their expression profiles using in silico and animal models. The putative candidate mutations will be further studied for their effect(s) on the biological activity of the protein using appropriate assays, which may include the analysis of -
(a) DNA-binding activity of mutated proteins.
(b) Transcriptional modulation of the mutant proteins on reporter constructs and profiling the effects of the mutant proteins on endogenous targets.
(c) Protein-protein interactions using the pull-down assays and Far-Western approaches.
(d) The effects of mutations on the post-translational modifications of the protein
A major focus of the project is to understand the mechanisms involved in human sex-determination. This will be achieved by using patient derived somatic cells. The project involves establishing iPS lines using the lymphocytes/fibroblasts isolated from the patients that carry mutations as well as unaffected family members. iPS cells will then be directed to differentiate into steroidogenic lineages of the gonad using protocols and tools that are being established in the laboratory. In parallel, direct transdifferentiation of somatic cells, obtained from patients, using a combination of male- specific transcription factors will be done. This will provide insights into the fundamental causes and cellular-molecular mechanisms involved in sex-determination and provide an unprecedented tool for screening therapeutic agents influencing the development and progression of the pathology in the individual.
Expected profile of the candidate (optional):