Generation and analysis of a conditional mutation of the HirA gene
Lead Research Organisation:
University College London
Department Name: Institute of Child Health
Abstract
Lay Summary The chromosomes within our cells contain the genetic instructions for life on the strands of DNA. In order to form the chromosomes the DNA is wrapped very tightly around proteins known as histones. The DNA and histone together is known as chromatin. However, the chromatin is not merely an ingenious way of getting a long length of DNA into a small space. Chromatin is dynamic, i.e. it is constantly changing, and alters its structure during important events such as fertilization of the egg by sperm, division of the cells, aging of cells and the switching on/off the genes themselves. HirA is a key protein for certain aspects of making and changing chromatin. We already know it is vital for life, without HirA DNA from the sperm cannot correctly join up with the egg at fertilization (at least in flies), and mouse embryos without HirA die in the middle of the pregnancy. However, since embryos lacking HirA die so early making it is very difficult to do certain experiments to work out how HirA is involved in development and regulating genes and cells. This project will allow us to make the mutation of HirA occur at different times and in different cells at will, providing an exquisite probe of HirA function. We hope to shed light on developmental pathways and cellular functions of HirA and will collaborate actively with other groups interested in these phenomena.
Technical Summary
HirA is a WD40 domain protein we first identified while searching for the gene haploinsufficient in DiGeorge syndrome. While not a major player in that condition we have gone on to report HirA protein interactions with bicoid homeodomain transcription factors and histone H3.3, clone the Fugu and Drosophila homologues, and created a targeted mutation of HirA in mouse. With Tom Misteli we recently showed altered chromatin dynamics and differentiation of murine embryonic stem cells. Other groups have shown, in a variety of organisms, that Hira is involved in cellular senescence, chromatin remodelling immediately post fertilization, and epigenetic control of gene expression. HirA is required for embryonic viability with embryos dying shortly after gastrulation. This makes it exceeding difficult to define the role of Hira is specific developmental processes, senescence at the level of the whole organism, and even examine the cell cycle due to inability to culture mouse embryonic fibroblasts successfully. We request support to create a conditional mutation of the HirA gene in order to identify the timing requirements for HirA during development using a ubiquitously expressed, tamoxifen inducible, Cre. The targeting strategy will borrow heavily from our successful creation of a constitutive null allele and single loxP knock in (for chromosome engineering). We will create MEFs and analyse the cell cycle and cellular senescence in the absence of HirA. Following on from our observations concerning Hira expression and the phenotype of the constitutive null, we will examine the requirements for Hira specifically in the developing heart and neural crest. Given the recent interest in Hira from various quarters, we will immediately make mice available to a number of groups who have expertise in specific areas and where it would be unrealistic for us to propose equivalent experiments in a three year time frame.
People |
ORCID iD |
Peter Scambler (Principal Investigator) |
Publications
Goldberg AD
(2010)
Distinct factors control histone variant H3.3 localization at specific genomic regions.
in Cell
Dilg D
(2016)
HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3.
in PloS one
Saleh RNM
(2018)
HIRA directly targets the enhancers of selected cardiac transcription factors during in vitro differentiation of mouse embryonic stem cells.
in Molecular biology reports
Description | The chromosomes within our cells contain the genetic instructions for life on the strands of DNA. In order to form the chromosomes the DNA is wrapped very tightly around proteins known as histones. The DNA and histone together is known as chromatin. However, the chromatin is not merely an ingenious way of getting a long length of DNA into a small space. Chromatin is dynamic, i.e. it is constantly changing, and alters its structure during important events such as fertilization of the egg by sperm, division of the cells, aging of cells and the switching on/off the genes themselves. HirA is a key protein for certain aspects of making and changing chromatin. We already know it is vital for life, without HirA DNA from the sperm cannot correctly join up with the egg at fertilization (at least in flies), and mouse embryos without HirA die in the middle of the pregnancy. However, since embryos lacking HirA die so early making it is very difficult to do certain experiments to work out how HirA is involved in development and regulating genes and cells. We have now developed a system whereby mutation of HirA can be regulated at different times and in different cells at will, providing an exquisite probe of HirA function. We have identified that HirA is required at the very earliest stages of the process during which a cell that has the potential to make any specialized cell type (a stem cell) decides to become one of those specialized types (e.g. heart, brain, liver) . This has long term implications for developing stem cells for use in regenerative therapies. SCIENTIFIC PROGRESS REPORT While the primary aim of creating the conditional mutant line has been achieved, we had insufficient time to accomplish all the in vivo work we wished. On the other hand, the mutant ES lines have proven extremely useful as a tool for dissecting Hira function, and will certainly direct the in vivo work we are currently initiating. Hira constitutive mutants have defects immediately following gastrulation. We mimicked the early steps of gastrulation by differentiating embryonic stem cells towards embryoid bodies using the hand-drop classic differentiation protocol (removal of LIF). We analysed undifferentiated ES cells by gene array, cell cycle profile and proliferation assays and found no phenotype, an important result given the subsequent differentiation steps. We were able to recapitulate some of the phenotypes that we observed in the mice, such as an abnormal higher expression of Brachyury (T) in the Hira null cells compared respective WT ES cells during differentiation. Indeed, in the mouse embryo at E8.5, the Hira null embryos show an abnormal extension of the allantois, and a defect of placentation. The abnormal big allantois is actually so big that it bulges out of the yolk sac, and then is not directed as it should be to the chorion to give the chorio-allantoic fusion, a prelude to placentation. We then showed in the Hira null embryos that this extra-growth of the allantois is associated with abnormal and extended expression of the Brachyury gene compared to their WT littermates. At this stage Brachyury is induced directly by Wnt3A pathway in the embryo. On stimulation of differentiated ES cells with commercial available Wnt3A, and we observed a similar phenotype, namely a higher expression of Brachyury in the Hira null cells than in the respective WT ES cells. We dissected this by looking at this Wnt activation pathway more in detail. Hira is also known to be associated with the specific deposition of the variant histone H3.3. Accordingly, we developed a collaboration with a lab. specialising in chromatin biology. Dr. Chapgier obtained an EMBO short term fellowship (coupled with a no cost extension of the BBSRC grant) to work towards this collaboration. With them we described the differences of the deposition of H3.3 in the Hira null versus WT undifferentiated ES cells (published in Cell). Current work is directed at examining the dynamics of global expression changes during ES differentiation in the presence and absence of Hira. Array analysis has been conducted at 12hrs, 1, 2, 3 and 4 days with interesting variation of the transcriptome. The major changes appear early, interesting in light of the negligible alterations observed in freely cycling ES cells. Accordingly, we are undertaking global analysis of H3.3 deposition at 12 hrs (using the H3.3 HA knockin fusion created using zinc finger nucleases) to test how this correlates with expression alteration at specific loci. We have preliminary evidence for some effects on specific imprinted genes, and are therefore also undertaking global methylated analysis. In vitro findings will then be validated in vivo. Finally, given independent evidence for a role of H3.3 in cardiovascular system development, Dr. Chapgier was awarded a BHF Intermediate Fellowship to investigate this in greater detail. We plan to publish our ES cell work, and then return with a research council bid to follow these aspects further, once we have peer reviewed recognition of their importance and validity. |
Exploitation Route | Not applicable The research will be put to use in follow on work. As mentioned, we have British Heart Foundation support to examine Hira during cardiovascular development, and we collaborate will colleagues at UCL who look at histone chaperones and H3.3 deposition in cancer (brain tumours). |
Sectors | Healthcare |
URL | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885838/ |
Description | The have been used to inform work on chromatin regulation by the histone variant H3.3 |
First Year Of Impact | 2010 |
Sector | Healthcare |
Impact Types | Cultural |
Description | BHF Intermediate Fellowship |
Amount | £361,713 (GBP) |
Funding ID | FS/10/031/28395 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 12/2010 |
End | 11/2014 |
Title | Conditional allele for Hira |
Description | Floxed exon of Hira, proved to result in null allele upon cre mediated recombination. Created from WTSI ES line. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | Paper in prep |
Title | Hira null cell lines |
Description | ES line null for Hira and its wild type control |
Type Of Material | Cell line |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | Several papers |
Title | Transcriptome analysis of Hira-/- versus Hira +/+ cells |
Description | Affymetric mouse array. Primary data submitted to Array Express and the reference to this dataset given in the publication Goldberg et al (see publication attached to report) |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
Description | Collaboration with Allis lab. |
Organisation | Rockefeller University |
Country | United States |
Sector | Academic/University |
PI Contribution | Dr Chapgier obtained an EMBO short term fellowship to cement this collaboration, which is ongoing towards another paper |
Collaborator Contribution | Provision of expertise and critical comment |
Impact | Paper see publication |
Start Year | 2009 |