Final Report Summary - CHAPERONES (The role of histone chaperones during epigenetic reprogramming)
➢ description of the project objectives
During the life of an individual, large scale epigenetic reprogramming occurs twice: in zygote, shortly after fertilization and during the development of primordial germ cells. We have previously shown that both reprogramming events entail large scale chromatin remodelling connected with dynamic changes in the abundance of histone variants. During the epigenetic reprogramming in zygote, histone variant H2A.Z disappears and there is a selective early incorporation of histone variant H3.3 into paternal chromatin. Histone chaperones are responsible for the incorporation and removal of histones, and Nap1l1 and Hira have been found enriched during epigenetic reprogramming in vivo. In this project, we are going to generate Nap1l1 and Hira conditional knockout mouse models to investigate the exact role of these factors in epigenetic reprogramming in the developing germ line and in zygotes, respectively. Especially and based on the data obtained during the first period of this project, we will address the role of histone chaperone Hira during oocyte as well as pre-implantation embryonic development. Meanwhile, we will try to understand how Hira mediated histone dynamics regulates efficient transcription and de novo DNA methylation during oogenesis.
➢ description of the work performed since the beginning of the project
First of all, we generated Nap1l1f/f mouse line in the host laboratory and obtained Hiraf/f mouse strain through direct collaboration with Prof Richard Festenstein (Imperial College London). These two mouse strains were cross-bred with different promoter driven Cre mice to achieve tissue specific genetic ablation during early germ cells and oocyte development. For example, Hiraf/f mice were cross bred with Prdm1-Cre and Gdf9-Cre (or Zp3-Cre) strains to achieve genetic ablation of Hira during early germ cell (primordial germ cells) development and oocyte development, respectively. Among these attempts, Gdf9-Cre or Zp3-Ce mediated genetic ablation of histone chaperone Hira during oocyte development had very clear and promising phenotypes and our subsequent studies further revealed the underlying molecular mechanisms. The mRNA microinjection experiments in oocytes showed that Hira is required for continuous histone replacement during oogenesis. This result was further confirmed using H3.3-EGFP knock-in mouse strain which was generated in the host laboratory in collaboration with Prof Richard Festenstein and the MRC Clinical Sciences Centre Transgenic Facility. Next, the detrimental effect of Hira depletion on oocyte and early embryonic development was analysed systematically using a range of molecular and cellular methods. Then, we used single oocyte RNA-sequencing experiments to elucidate the cross-talk between Hira mediated histone turn over and fine-tuned transcriptional regulation during oogenesis. Finally, single oocyte whole genome bisulphite sequencing experiment was performed in collaboration with Dr Gavin Kelsey (Cambridge University) group to address how Hira mediated histone dynamics affect de novo DNA methylation during oocyte development.
As a backup plan for the in vivo experiments and also as an alternative, unbiased approach, we set up a SILAC (Stable isotope labeling by amino acids in cell culture) based cell culture system to identify novel H2A.Z or H2A.X histone chaperone and interacting proteins by immuno-affinity purification followed by mass spectrometric analysis.
➢ description of the main results achieved so far
o Using our genetic models we found that Hira is responsible for continuous H3.3/H4 turnover in postnatal oocytes
o Hira depletion results in a severe ovarian phenotype, associated with extensive oocyte death and the failure to support zygotic reprogramming or embryonic development
o Hira-depleted oocytes show increased DNA accessibility and accumulation of DNA damage
o Continuing histone replacement mediated by Hira is required to maintain the full dynamic range of gene expression
o Hira-mediated H3.3/H4 deposition is essential for transcriptional transitions associated with the oocyte developmental programme
o Hira depletion leads to aberrant transcription from regions not normally transcribed within the genome
o Continuing histone replacement is essential for efficient de novo methylation during oogenesis.
o Identified several H2A.X specific proteins including Dnmt3l, Parp1 and Parp2.
o Successfully generated Nap1l1f/f mouse strain and achieved tissue specific depletion of Nap1l1 in oocytes
➢ final results
Our findings regarding the roles of histone chaperone Hira during oocyte development has resulted in a high impact factor paper. This paper is going to be published on line by the peer reviewed journal “Molecular Cell” on the 5th November 2015, under the title “Continuous histone replacement by Hira is essential for normal transcriptional regulation and de novo DNA methylation during mouse oogenesis”. Based on the findings we developed a hypothesis: In wild type oocyte, normal chromatin structure and nucleosome density is maintained by Hira mediated continuous H3.3/H4 replacement. This is required for fine-tuned transcriptional regulation and efficient de novo DNA methylation. In Hira mutant oocytes, nucleosome density is reduced due to the lack of H3.3/H4 replacement. This not only leads to aberrant transcriptional regulation (inability to fully activate genes as well as defect in gene silencing and appearance in spurious transcripts) but also compromises Dnmt3a activity on chromatin leading to reduced global DNA methylation.
➢ potential impact
We have systematically elucidated the critical roles of histone chaperone Hira during oogenesis and showed that this factor is essential for normal oocyte development. Mutation of Hira leads to various oocyte defects and results in extensive oocyte loss which is reminiscent of a common human disease- Premature Ovarian Failure (POF). Premature ovarian failure is the loss of function of the ovaries before age 40. POF is a devastating diagnosis for reproductive-aged women and about 1%-3% of women experience POF before age 40. This condition not only results in infertility but also other serious health consequences including psychological distress, osteoporosis, autoimmune disorders, ischemic heart disease, and increased risk for mortality. The cause of this condition has not been fully understood. However, there is an association between the failure of germ cell development and complete ovarian failure. Hira mutation in female mice ovaries resembles this human condition. The ovarian size is significantly smaller in Hira mutant female mice and mature oocytes are lost from ovaries prematurely (as early as 5 weeks age). As the functions of histone chaperone Hira is well conserved in mammals, it is likely that this factor also plays a vital role in human oogenesis. Thus, ovarian mutation of histone chaperone Hira might be one of the reasons leading to Premature Ovarian Failure in women. Our findings regarding the molecular mechanism underlying Hira mutation in mice contributes to better understanding of the cause of Premature Ovarian Failure and could provide clinical indication for the treatment of the Premature Ovarian Failure or infertility treatment.
During the life of an individual, large scale epigenetic reprogramming occurs twice: in zygote, shortly after fertilization and during the development of primordial germ cells. We have previously shown that both reprogramming events entail large scale chromatin remodelling connected with dynamic changes in the abundance of histone variants. During the epigenetic reprogramming in zygote, histone variant H2A.Z disappears and there is a selective early incorporation of histone variant H3.3 into paternal chromatin. Histone chaperones are responsible for the incorporation and removal of histones, and Nap1l1 and Hira have been found enriched during epigenetic reprogramming in vivo. In this project, we are going to generate Nap1l1 and Hira conditional knockout mouse models to investigate the exact role of these factors in epigenetic reprogramming in the developing germ line and in zygotes, respectively. Especially and based on the data obtained during the first period of this project, we will address the role of histone chaperone Hira during oocyte as well as pre-implantation embryonic development. Meanwhile, we will try to understand how Hira mediated histone dynamics regulates efficient transcription and de novo DNA methylation during oogenesis.
➢ description of the work performed since the beginning of the project
First of all, we generated Nap1l1f/f mouse line in the host laboratory and obtained Hiraf/f mouse strain through direct collaboration with Prof Richard Festenstein (Imperial College London). These two mouse strains were cross-bred with different promoter driven Cre mice to achieve tissue specific genetic ablation during early germ cells and oocyte development. For example, Hiraf/f mice were cross bred with Prdm1-Cre and Gdf9-Cre (or Zp3-Cre) strains to achieve genetic ablation of Hira during early germ cell (primordial germ cells) development and oocyte development, respectively. Among these attempts, Gdf9-Cre or Zp3-Ce mediated genetic ablation of histone chaperone Hira during oocyte development had very clear and promising phenotypes and our subsequent studies further revealed the underlying molecular mechanisms. The mRNA microinjection experiments in oocytes showed that Hira is required for continuous histone replacement during oogenesis. This result was further confirmed using H3.3-EGFP knock-in mouse strain which was generated in the host laboratory in collaboration with Prof Richard Festenstein and the MRC Clinical Sciences Centre Transgenic Facility. Next, the detrimental effect of Hira depletion on oocyte and early embryonic development was analysed systematically using a range of molecular and cellular methods. Then, we used single oocyte RNA-sequencing experiments to elucidate the cross-talk between Hira mediated histone turn over and fine-tuned transcriptional regulation during oogenesis. Finally, single oocyte whole genome bisulphite sequencing experiment was performed in collaboration with Dr Gavin Kelsey (Cambridge University) group to address how Hira mediated histone dynamics affect de novo DNA methylation during oocyte development.
As a backup plan for the in vivo experiments and also as an alternative, unbiased approach, we set up a SILAC (Stable isotope labeling by amino acids in cell culture) based cell culture system to identify novel H2A.Z or H2A.X histone chaperone and interacting proteins by immuno-affinity purification followed by mass spectrometric analysis.
➢ description of the main results achieved so far
o Using our genetic models we found that Hira is responsible for continuous H3.3/H4 turnover in postnatal oocytes
o Hira depletion results in a severe ovarian phenotype, associated with extensive oocyte death and the failure to support zygotic reprogramming or embryonic development
o Hira-depleted oocytes show increased DNA accessibility and accumulation of DNA damage
o Continuing histone replacement mediated by Hira is required to maintain the full dynamic range of gene expression
o Hira-mediated H3.3/H4 deposition is essential for transcriptional transitions associated with the oocyte developmental programme
o Hira depletion leads to aberrant transcription from regions not normally transcribed within the genome
o Continuing histone replacement is essential for efficient de novo methylation during oogenesis.
o Identified several H2A.X specific proteins including Dnmt3l, Parp1 and Parp2.
o Successfully generated Nap1l1f/f mouse strain and achieved tissue specific depletion of Nap1l1 in oocytes
➢ final results
Our findings regarding the roles of histone chaperone Hira during oocyte development has resulted in a high impact factor paper. This paper is going to be published on line by the peer reviewed journal “Molecular Cell” on the 5th November 2015, under the title “Continuous histone replacement by Hira is essential for normal transcriptional regulation and de novo DNA methylation during mouse oogenesis”. Based on the findings we developed a hypothesis: In wild type oocyte, normal chromatin structure and nucleosome density is maintained by Hira mediated continuous H3.3/H4 replacement. This is required for fine-tuned transcriptional regulation and efficient de novo DNA methylation. In Hira mutant oocytes, nucleosome density is reduced due to the lack of H3.3/H4 replacement. This not only leads to aberrant transcriptional regulation (inability to fully activate genes as well as defect in gene silencing and appearance in spurious transcripts) but also compromises Dnmt3a activity on chromatin leading to reduced global DNA methylation.
➢ potential impact
We have systematically elucidated the critical roles of histone chaperone Hira during oogenesis and showed that this factor is essential for normal oocyte development. Mutation of Hira leads to various oocyte defects and results in extensive oocyte loss which is reminiscent of a common human disease- Premature Ovarian Failure (POF). Premature ovarian failure is the loss of function of the ovaries before age 40. POF is a devastating diagnosis for reproductive-aged women and about 1%-3% of women experience POF before age 40. This condition not only results in infertility but also other serious health consequences including psychological distress, osteoporosis, autoimmune disorders, ischemic heart disease, and increased risk for mortality. The cause of this condition has not been fully understood. However, there is an association between the failure of germ cell development and complete ovarian failure. Hira mutation in female mice ovaries resembles this human condition. The ovarian size is significantly smaller in Hira mutant female mice and mature oocytes are lost from ovaries prematurely (as early as 5 weeks age). As the functions of histone chaperone Hira is well conserved in mammals, it is likely that this factor also plays a vital role in human oogenesis. Thus, ovarian mutation of histone chaperone Hira might be one of the reasons leading to Premature Ovarian Failure in women. Our findings regarding the molecular mechanism underlying Hira mutation in mice contributes to better understanding of the cause of Premature Ovarian Failure and could provide clinical indication for the treatment of the Premature Ovarian Failure or infertility treatment.