Epigenetics
SmcHD1 is a novel epigenetic modifier critically involved in X inactivation
ME Blewitt, DJ Hilton in collaboration with E Whitelaw, G Kay (Queensland Institute of Medical Research), SL Dunwoodie, DB Sparrow (Victor Chang Cardiac Research Institute, Sydney), JM Craig (Murdoch Children’s Research Institute), A-V Gendrel, N Brockdorff (Medical Research Council Clinical Sciences Centre, London UK) Pub ref: 13
X chromosome inactivation is the dosage compensation mechanism by which the dose of X-linked genes is made equal between XY males and XX females. This inactivation is achieved by the epigenetic silencing of one of the two X chromosomes in females. We previously performed an ENU mutagenesis screen for modifiers of epigenetic reprogramming, utilising a variegating GFP transgene. In this screen we identified MommeD1 (Modifier of Murine Metastable Epialleles) as a semi-dominant suppressor of variegation. Interestingly, MommeD1 displays female specific mid-gestation lethality and hypomethylation of an X-linked gene, suggestive of a defect in X inactivation. We have now identified the causative point mutation in the MommeD1 mice in an uncharacterised gene called Structural Maintenance of Chromosomes Hinge Domain containing 1 (SmcHD1), and confirmed this using a gene-trap allele of SmcHD1. Hinge domains are normally found in the SMC proteins, which are components of the cohesin and condensin complexes.
We have studied the X inactivation defect in the SmcHD1MommeD1/MommeD1 females, and found SmcHD1 is not required for the correct initiation of X inactivation. Rather, SmcHD1 localises to the inactive X and is required for the maintenance of X inactivation and the hypermethylation of CpG islands associated with the inactive X. This finding links a group of proteins normally associated with structural aspects of chromosome biology with epigenetic gene silencing.
MommeD1 homozygous females display activation of an X-linked GFP transgene from E7.5 onwards. We tested the transcriptional activity of the X chromosomes in MommeD1 homozygous females using an X-linked GFP transgene. To simplify analysis, we used a paternally inherited X-GFP and maternally inherited Xist knockout allele, so that normal embryos should not display any GFP fluorescence, since the X chromosome with the GFP transgene is the obligate inactive X. We find that E7.5 (A) and E10.5 (B) SmcHD1MommeD1/MommeD1 female embryos display GFP fluorescence in the embryonic and extra-embryonic tissues indicating that they have 2 active X chromosomes. Similar results were found by measuring the expression of endogenous X-linked genes.
Mof acetylates histone 4 lysine 16 and is required for the maintenance of uncondensed chromatin
T Thomas, MP Dixon, AJ Kueh, AK Voss. Pub ref: 140
Acetylation of histone tails is a hallmark of transcriptionally active chromatin. Mof (Myst1, Kat8), a MYST histone acetyltransferase, was originally discovered as an essential component of the X-chromosome dosage compensation system in Drosophila. We examined the role of Mof in mammals in vivo. In contrast to flies, male and female mouse embryos require Mof to develop beyond the expanded blastocyst stage and to acetylate H4K16. Mof–/– cell nuclei exhibit abnormal chromatin aggregation, while still negative for indicators of apoptosis. Our results show that, while the molecular function is conserved, Mof has a different physiological role in mammals as compared to flies.
Investigating tumour cell death using RNA interference
TA Willson, RA Dickins in collaboration with A Strasser, DCS Huang, S Cory, J Adams (Molecular Genetics of Cancer Division) and WS Alexander (Cancer and Haematology Division)
The advent of RNA interference (RNAi) heralds a new era of experimental genetics, where specific gene products can be reversibly inhibited in a high throughput manner. We have recently developed technology allowing inducible and reversible gene knockdown in cultured cells and transgenic animal models. Using novel reagents that effectively knock down genes encoding various known or putative drug targets, we aim to mimic targeted therapeutics in several primary tumour cell lines and tumour-prone mouse models. In vitro and in vivo RNAi screening will also be used to identify mediators of therapy-induced tumour cell death in various genetic contexts.
Analysis of Phf6, mutated in Börjeson-Forssman-Lehmann Syndrome
AK Voss, R Gamble, C Collin, T Thomas in collaboration with C Shoubridge, M Corbett, J Gécz (Women’s and Children’s Hospital, SA) Pub ref: 145
The plant homeodomain finger gene 6 (PHF6) is the gene mutated Börjeson-Forssman-Lehmann Syndrome (BFLS), an X-linked mental disability disorder. The PHF6 protein contains two imperfect plant homeodomain (PHD) fingers similar to the third PHD finger in members of the trithorax family of transcriptional regulators. We determined strongest Phf6 gene expression and nuclear localisation of Phf6 protein in the developing central nervous system, the anterior pituitary gland, primordia of facial structures and limb buds. In the adult brain moderate Phf6 expression is maintained in cerebrocortical and cerebellar projection neurons. Phf6 gene expression and nuclear localisation of Phf6 protein correlate with clinical symptoms in BFLS patients.