Meiotic Synapsis: a Novel Role for Dynein Light Chain 1 in Synapsis Regulation and Sex-specific Processes in Meiotic Prophase I Público
Fielder, Sara (Summer 2020)
Abstract
Meiotic synapsis is an essential process of connecting homologous chromosomes and holding them in close proximity for recombination. As C. elegans chromosomes search for their homologous partner, one end of each chromosome attaches to nuclear membrane proteins and movements at the periphery are driven by cytoplasmic dynein motors to aid the search. The dynein motor has been proposed to generate tension between homolog pairs that tests the matches and signals for synapsis initiation. The central element of the synaptonemal complex is composed of six synapsis proteins (SYPs 1-6) that form a physical, phase-separated, zipper-like bridge between homologs. Chromosomes that fail to synapse are targeted for enrichment of the heterochromatin mark Histone H3 Lysine 9 dimethylation (H3K9me2).
We have discovered that the dynein light chain 1 (DLC-1) has a potential additional chaperone-like role in aiding SYP protein folding at slightly elevated temperatures. Knockdown of dlc-1 results in aggregation of SYP proteins, and this phenotype increases in frequency at higher growth temperatures. DLC-1 interacts with SYP-2 through a binding motif, and elimination of the binding motif partially, but not completely, eliminates the interaction. This work adds to the understanding of the roles of DLC-1 in meiosis, and motor-independent roles of the light chain.
Additionally, we have identified several sex-specific differences in meiotic regulation, including a new sex-specific mechanism that senses the initiation of synapsis. If synapsis is unable to initiate, H3K9me2 enrichment in hermaphrodite chromatin is not observed. However, mutants that are able to partially synapse their chromosomes display widespread H3K9me2 enrichment. In contrast, widespread H3K9me2 enrichment is observed in males when synapsis is prevented. This indicates that there may be a hermaphrodite/female-specific checkpoint-like mechanism that stalls meiosis when synapsis does not initiate, and this arrest precedes H3K9me2 targeting. Additionally, whereas hermaphrodite synapsis is temperature-sensitive and will form un-recoverable SYP protein aggregates at temperatures above 26.5°, males still synapse their chromosomes even when raised at 27.8°. Furthermore, the SYP aggregates that occur when DLC-1 is depleted in hermaphrodites are not observed in males. These differences indicate that studying male meiosis could reveal as yet undiscovered mechanisms regulating meiosis.
Table of Contents
CHAPTER 1: INTRODUCTION..................................................................................................... 1
1.1 Chromosome movement and homolog pairing............................................................ 2
1.2 Synaptonemal complex axis formation......................................................................... 4
1.3 Central element assembly and synapsis....................................................................... 6
1.4 Meiotic silencing of unsynapsed chromatin................................................................. 8
1.5 Meiotic checkpoints............................................................................................................ 9
1.6 Differences in male meiosis........................................................................................... 10
1.7 Dynein light chain............................................................................................................... 11
1.8 Scope of this dissertation............................................................................................... 12
1.9 Figures.................................................................................................................................. 15
CHAPTER 2: A MOTOR-INDEPENDENT REQUIREMENT FOR DYNEIN LIGHT CHAIN IN C. ELEGANS MEIOTIC SYNAPSIS 18
2.1 Abstract............................................................................................................................... 18
2.2 Introduction......................................................................................................................... 19
2.3 Methods............................................................................................................................... 21
2.4 Results................................................................................................................................. 26
2.4.1 Knockout of dynein heavy chain and/or dynactin does not induce SYP foci formation 26
2.4.2 SYP proteins form foci in a temperature sensitive manner with knockdown of DLC-1 27
2.4.3 SYPs form insoluble foci with knockdown of DLC-1............................................. 27
2.4.4 SYP-2 interacts with DLC-1 in vivo through a conserved binding motif............ 28
2.4.5 Mutation of the putative DLC-1 binding site in SYP-2 results in temperature sensitive meiotic defects 30
2.5 Discussion........................................................................................................................... 32
2.6 Acknowledgements.......................................................................................................... 37
2.7 Figures.................................................................................................................................. 38
2.8 Tables................................................................................................................................... 51
2.9 Supplemental Material..................................................................................................... 55
CHAPTER 3: MULTIPLE SEX-SPECIFIC DIFFERENCES EXIST IN THE REGULATION OF SYNAPSIS IN C. ELEGANS MEIOSIS 59
3.1 Abstract............................................................................................................................... 59
3.2 Introduction......................................................................................................................... 60
3.3 Methods............................................................................................................................... 65
3.4 Results................................................................................................................................. 68
3.4.1 H3K9me2 targeting to unsynapsed chromosomes differs between sexes... 68
3.4.2 PCH-2 is not required for adult hermaphrodite- specific prevention of H3K9me2 enrichment with disrupted synapsis 70
3.4.3 Initiation of synapsis is required for H3K9me2 enrichment of unsynapsed chromosomes in adult hermaphrodite meiosis 71
3.4.4 Dynein is not required for males to correctly pair and synapse chromosomes............ 72
3.4.5 Male synapsis is not as sensitive to heat as hermaphrodite synapsis............ 74
3.4.6 Spindle assembly checkpoint proteins in males................................................... 75
3.5 Discussion........................................................................................................................... 76
3.6 Figures.................................................................................................................................. 85
3.7 Tables................................................................................................................................... 97
3.8 Supplemental Figures....................................................................................................... 98
Chapter 4. Discussion and Future Directions................................................................. 101
4.1 Summary of Dissertation.............................................................................................. 101
4.2 A screen for components in the synapsis initiation “checkpoint”..................... 103
4.3 Post-translational modification of axial elements and the synapsis initiation failure response 104
4.4 Male C. elegans evolution favors less stringent meiosis..................................... 105
4.5 Dynein light chain’s myriad of functions.................................................................... 107
4.6 Ability of males to complete synapsis without DLC-1............................................ 107
4.7 Future directions for study of interactions of DLC-1 with SYP proteins............ 108
4.8 Future study required into CRL4 and DLC-1 similarities........................................ 109
4.9 MPK-1 as a potential player in early meiosis........................................................... 110
4.10 Future direction of study of SYP-2 in late meiosis................................................ 111
4.11 Final Conclusions.......................................................................................................... 111
REFERENCES........................................................................................................................... 113
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