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Principal Investigator: Johan Girgenrath
Organization: UNIV OF MASSACHUSETTS MED SCH WORCESTER
Fiscal Year: 2024
Award: $33,780
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
PROJECT SUMMARY
Germline immortality is essential for species survival. Evidence from plants to animals suggests germline
immortality depends on small RNA pathways that promote genome stability. In Caenorhabditis elegans germline,
small RNAs that interact with the PIWI Argonaute protein PRG-1—called piRNAs—initiate silencing of foreign
sequences that is then maintained by Worm Argonautes (WAGOs). Nuclear WAGO-9 binds the nucleosome
remodeling and deacetylase (NuRD) complex via histone deacetylase (HDA-1), which initiates heterochromatin
formation on piRNA targets. piRNA pathway defects activate expression of foreign sequences and disrupt germ
cell development and fertility. How collaboration between the piRNA pathway and heterochromatin machinery is
regulated to promote germline immortality is unclear.
In the C. elegans germline, assembly of HDA-1 into the NuRD complex depends on the small ubiquitin-like
modifier (SUMO). In worms expressing mutant HDA-1 that cannot be SUMOylated, piRNA-mediated silencing,
germline heterochromatin formation, and fertility are lost. Interestingly, HDA-1 SUMOylation is not needed for its
assembly into the NuRD complex in somatic embryo. Together, these findings suggest that the piRNA pathway
relies on SUMO to co-opt NuRD complex assembly in the germline for silencing of piRNA targets.
This proposal seeks to explore how germline NuRD complex assembly and its recruitment by the piRNA pathway
are regulated. Aim 1 will explore the role of SUMOylated and germline-specific factors in preventing default
(SUMO independent) assembly of the NuRD complex in germline. The highly-conserved MRG-1 chromodomain
protein is essential for piRNA-mediated silencing and is SUMOylated. Preliminary data suggest SUMOylated
MRG-1 in germline renders HDA-1 function to be SUMO-dependent. Co-immunoprecipitation assays will be
used to determine whether SUMOylated MRG-1 prevents unmodified HDA-1 from assembling into the germline
NuRD complex. Because SUMOylated MRG-1 is abundant in embryonic tissue, this aim will also use mass
spectrometry and immunofluorescence assays to identify potential germline-specific factors interacting with
SUMOylated MRG-1 to render germline HDA-1 function SUMO dependent. Aim 2 will determine whether HDA-
1 SUMOylation promotes interaction with WAGO-9. Putative SUMO-interacting motifs of WAGO-9 will be
mutated and the effect on piRNA-mediated silencing and HDA-1interaction will be determined. This aim will also
computationally analyze published data characterizing strength of piRNA-target interactions to explore how the
piRNA pathway may utilize its targeting of transcripts to enact downstream silencing via heterochromatin. This
work will reveal how highly conserved small RNA and chromatin factors coordinate to promote germline
immortality, and provide the fellow with training in genetics, microscopy, biochemistry, and bioinformatics to
prepare them for a future career as an independent investigator.
Terms: <APF-1><ATP-Dependent Proteolysis Factor 1><Affect><Animals><Assay><Basic Mechanisms of SUMOylation><Binding><Bio-Informatics><Bioassay><Biochemistry><Bioinformatics><Biological Assay><Biological Chemistry><Biological Function><Biological Process><Body Tissues><C elegans><C. elegans><C.elegans><CRISPR><CRISPR/Cas system><Caenorhabditis elegans><Chromatin><Clustered Regularly Interspaced Short Palindromic Repeats><Co-Immunoprecipitations><Collaborations><Complex><Data><Deacetylase><Defect><Development><Developmental Biology><Embryo><Embryonic><Embryonic Tissue><Ensure><Fecundability><Fecundity><Fertility><Future><Gametes><Gene Expression><Gene Inactivation><Gene Silencing><Generations><Genetic><Genome Stability><Genomic Stability><Germ Cells><Germ Lines><Germ-Line Cells><Goals><Gonadal structure><HDAC><HDAC Proteins><HMG-20><Heterochromatin><High Mobility Protein 20><Histone Deacetylase><Human><Hybrids><Immunofluorescence><Immunofluorescence Immunologic><Investigators><Knock-out><Knockout><Lab Findings><Laboratory Finding><Maintenance><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Messenger RNA><Mice><Mice Mammals><Microscopy><Modern Man><Molecular Interaction><Murine><Mus><Mutate><Nuclear><Nucleosomes><Pathway interactions><Plants><Proteins><Publishing><Regulation><Reproductive Cells><Research Personnel><Researchers><Role><SUMOylation><Sex Cell><Site><Small RNA><Sumoylation Pathway><Testing><Tissues><Training><Transcript><Transmission><Ubiquitin><Work><career><developmental><embryo tissue><genetic information><gonad><gonads><iPS><iPSC><iPSCs><induced pluripotent cell><induced pluripotent stem cell><inducible pluripotent stem cell><initial cell><insight><mRNA><mRNA seq><mRNA sequencing><mRNA-seq><mRNAseq><mutant><pathway><piRNA><piwi RNA><prevent><preventing><recruit><self-renew><self-renewal><sexual cell><social role><transcriptional silencing><transmission process>