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Regulation of gene expression is a complicated process, subject to different mechanisms operating at different levels. At the genomewide level, work in this thesis describes the use of chromatin immunoprecipitation and next-generation sequencing to interrogate the coincident and allele-specific binding of the proteins CTCF, cohesin, ATRX and MeCP2. Identifying regions co-binding these proteins helps generate a model to understand how these proteins influence gene expression, particularly at imprinted loci. Using these tools we are able to understand more about the proteins that participate in the genomic landscape around imprinted loci and drive this unusual mode of gene regulation. These loci act as models for studying DNA binding proteins and their roles in transcription. At the level of the individual locus, mechanisms of gene regulation were investigated using imprinted retrogenes as a model. Retrogenes are transcriptionally active intronless genes located within an intron of a ‘host’ gene. The role of epigenetic factors influencing gene transcription were investigated at the H13/Mcts2 locus. Expression of an intronic retrogene can cause premature termination of a ‘host’ transcript. Our hypothesis is that this premature termination can be caused by transcription of the retrogene interfering with host gene transcription. We have designed a construct based on this locus, which allows us to regulate the expression through the retrogene to study this in more detail. These studies provide a mechanistic component to our whole genome analyses.

Lens epithelium-derived growth factor (LEDGF) is a critical co-factor of lentiviral integration that accounts for the characteristic bias of the retroviral genus towards integration within active transcription units of the host genome. It’s well-characterised role in viral replication notwithstanding, cellular functions of LEDGF are poorly understood. In this study gene expression profiling and proteomic analysis have been used to elucidate cellular functions of LEDGF. Gene expression profiling of mouse embryonic fibroblasts (MEFs) derived from Psip1-/- (LEDGF-null) embryos revealed that less than 200 genes were significantly up- or down-regulated compared to wild type (WT) MEFs. The changes in gene expression of a subset of affected genes were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. However, these changes could not be reversed by re-introduction of LEDGF into knockout MEFs. Furthermore, in vitro knockout of Psip1 did not reproduce the effects of in utero knockout, suggesting that LEDGF ablation per se does not account for the observed perturbations in gene expression. Collectively, these data strongly indicate that in spite of its apparent ubiquitous association with active transcription units, LEDGF does not play an essential nor direct role in the regulation of gene expression. Tandem affinity purification (TAP) of a cTAP-tagged truncation mutant of LEDGF (residues 326-530) identified the heterodimeric S-phase kinase, termed cell division cycle 7 (Cdc7):activator of S-phase kinase (ASK), as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with LEDGF from human cells, and endogenous Cdc7 and LEDGF proteins displayed nuclear co-localisation in G1 and S phases of the cell cycle. Moreover, Cdc7:ASK was enriched on chromatin when ectopically co-expressed with LEDGF in human cells, suggesting that LEDGF may affect chromatin binding of the kinase. Truncation analyses identified the integrase-binding domain (IBD) of LEDGF as essential and minimally sufficient for the interaction with Cdc7:ASK. Reciprocally, the interaction required auto-phosphorylation of the kinase and the presence of fifty C-terminal residues of ASK. LEDGF potently stimulated the kinase activity of Cdc7:ASK, increasing phosphorylation of one of its substrates, mini-chromosome maintenance 2 (MCM2), in vitro by more than 10-fold. The effect strictly depended on the interaction, as LEDGF mutants unable to bind Cdc7:ASK also failed to promote its kinase activity. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyper-active kinase. These results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7:ASK kinase activity, imposed by the C-terminus of ASK. While more work is required to fully elucidate the natural cellular functions of LEDGF, this study has provided some insight into its role in cell biology. In particular, understanding the role of LEDGF in Cdc7:ASK kinase regulation and the implications of this interaction during S phase of the cell cycle are promising avenues of investigation highlighted in this study.

This portfolio thesis comprises of four parts: a systematic literature review paper, an empirical paper, a reflective statement and appendices.Part one is a systematic literature review which examines whether personal therapy is an effective method of professional development for therapists. Quantitative and qualitative literature is critically reviewed. A model of the reported benefits of personal therapy for therapists is proposed. Implications for clinical practice are discussed.Part two is an empirical paper examining the relationship between stage of development and behaviour in clinical supervision for trainee clinical psychologists. Forty trainee clinical psychologists, from three years of a training course, completed a questionnaire (the SLQ-R[A]) measuring their stage of development as supervisees. A subsample submitted DVD-recordings of their supervision sessions which were coded using the Teacher's PET to analyse the supervision behaviours. Comparisons were made between the supervision behaviour of first (n = 8) and third (n = 3) year trainee clinical psychologists and their supervisors. Correlations between questionnaire responses and supervision behaviours were examined. Results are discussed in the context of the Integrated Developmental Model of Supervision. Implications for clinical practice are highlighted.Part three is a reflective statement which considers the process of conducting the research and developing this portfolio thesis.Part four is the appendices.