The role of lysine specific demethylase 1 (LSD1) and the functional consequences during development 公开
Scott, Alyssa (Fall 2021)
Published
Abstract
The importance of lysine specific demethylase 1 (LSD1) during cell fate transitions has been established as a crucial step for developmental processes. We add to the accumulating body of evidence that LSD1 is critical in erasing epigenetic memory during these cell fate transitions. In neural stem cells in mice, we have shown that LSD1 is required for survival and appropriate neurodevelopment. We also examined the role of LSD1 during the maternal-to-zygotic transition. In C. elegans, LSD1/KDM1A (lysine specific demethylase 1) acts as part of the CoREST repressor complex to enable this transition by removing H3K4me1/2 and preventing the transgenerational inheritance of transcription patterns. In mouse, the loss of maternal LSD1 results in embryonic arrest at the 1-2 cell stage, with arrested embryos similarly failing to undergo the maternal-to zygotic transition. This suggests that LSD1 maternal reprogramming is conserved. Moreover, partial loss of maternal LSD1 results in striking phenotypes weeks after fertilization, including perinatal lethality and abnormal behavior in surviving adults. To explore the mechanism underlying these heritable defects further, we developed a new maternally hypomorphic LSD1 allele that predominantly affects the binding of LSD1 to CoREST. This new hypomorphic allele phenocopies our mouse model with reduced LSD1, suggesting that the maternal reprogramming function of LSD1 is CoREST dependent. In addition, we find that the incidence of perinatal lethality in our new model is higher in a mother’s first litter, as well as with advanced maternal age (>9 months). This modulation of the phenotype by maternal age is reminiscent of the epidemiological data in autism, raising the possibility that defective maternal epigenetic reprogramming can contribute to neurodevelopmental disorders.
Table of Contents
Chapter 1: An introduction to epigenetic mechanisms and cellular reprogramming
Epigenetic modifications influence gene expression………………………………..Page 12
H3K4 methylation is an active mark…………………………………………………..Page 15
LSD1 is an amine oxidase demethylase…………………………………….……….Page 16
Figure 1: LSD1 protein domains……………………………………………………....Page 16
Binding partners affect LSD1 function………………………………………………..Page 17
Figure 2: LSD1 demethylase specificity changes depending on complex composition...……………………………………………………………………………Page 18
LSD1 decommissions enhancers in stem cell populations……………………...…Page 18
LSD1 plays a role in neural stem cell development…………………………………Page 19
The maternal to zygotic transition requires extensive epigenetic regulation……..Page 20
DNA methylation is inherited between generations and must be reprogrammed.Page 22
LSD1 is required for the maternal to zygotic transition……………………………..Page 24
Figure 3: Maternally-provided LSD1 is required for epigenetic reprogramming at fertilization……………………………………………………………………………….Page 26
A partial loss of LSD1 maternally results in defects that manifest postnatally…...Page 27
Mutations in epigenetic proteins cause human disease…………………………....Page 28
Outstanding questions………………………………………………………………….Page 29
Chapter 2: Maternally provided LSD1 prevents defects that manifest postnatally
Generation of a novel hypomorphic LSD1 allele…………………………………….Page 31
Figure 1: Generation of M448V hypomorphic allele………………………………...Page 33
Maternally hypomorphic LSD1 leads to perinatal lethality which may be modified by maternal age…………………………………………………………………………….Page 33
Figure 2: Genetic crosses to obtain mutant and control progeny………………….Page 35
Figure 3: Maternally hypomorphic LSD1 leads to higher rates of perinatal lethality…………………………………………………………………………………...Page 38
Reasons underlying perinatal lethality are unclear……………………...……….…Page 39
Figure 4: Maternal mutants do not have craniofacial defects……………………...Page 40
Figure 5: RNA sequencing analysis of blastocysts………………………………….Page 44
Lsd1M448V progeny may have imprinting defects…………………………………….Page 44
Figure 6: Maternal mutants do not display developmental delay………………….Page 45
Figure 7: Zac1 imprinting is defective in maternal mutants………………………...Page 48
Maternal mutant behavioral phenotypes are modified by genetic background…..Page 48
Figure 8: There are no detectable behavioral differences in maternal mutants in the B6 background………………………………………………………………………………Page 50
Figure 9: Maternal mutants in the CAST background display looping behavior…Page 52
Chapter 3: Materials and methods
Solutions and buffers…………………………………………………………………...Page 53
Mouse lines……………………………………………………………………………...Page 54
Mouse genotyping by PCR…………………………………………………………….Page 55
Morris water maze………………………………………………………………………Page 56
Skeletal preps…………………………………………………………………………...Page 57
Developmental delay…………………………………………………………………...Page 57
Flushing blastocysts for RNA seq……………………………………………..…...…Page 57
RNA sequencing analysis……………………………………………………………...Page 58
Chapter 4: SPR-5/LSD1 functions through CoREST to maternally reprogram histone methylation
Abstract…………………………………………………………………………………..Page 61
Introduction………………………………………………………………………………Page 61
Results…………………………………………………………………………………...Page 66
spr-1 mutants have reduced fertility but do not exhibit germline mortality..Page 66
met-2; spr-1 double mutants exhibit germline mortality…………………….Page 67
The sterility of met-2;spr-1 mutants resembles spr-5;met-2 mutants……..Page 68
Transcriptional misregulation in met-2;spr-1 progeny resembles that observed in
spr-5;met-2 progeny but is less effected……………………………………..Page 68
MES-4 germline genes are enriched in met-2;spr-1 mutants, but less affected
compared to spr-5;met-2 mutants…………………………………………….Page 71
LSD1 and CoREST are expressed during each stage of mouse oocyte
development………………………………….…………………………………Page 72
Reducing the function of maternally-provided LSD1 causes perinatal
lethality…………………………………………………………………...………Page 72
Discussion……………………………………………………………………………….Page 76
CoREST regulated LSD1 maternal reprog of histone methylation………..Page 76
Evidence from diverse developmental processes across multiple phyla support a
role for CoREST in LSD1 function…………………………………………….Page 79
Potential roles for CoREST in regulating LSD1 activity…………………….Page 81
Potential implications for CoREST function in humans…………………….Page 82
Acknowledgements…..…………………………………………………………………Page 82
Materials and methods…………………………….………………………………...…Page 83
C. elegans strains………………………………………………………………Page 83
Generation of M448V hypomorphic allele……………………………………Page 84
Mouse husbandry and genotyping……………………………………………Page 85
Perinatal lethality………………………………………………………………..Page 86
Germline mortality assay………………………………………………………Page 86
RNA sequencing and analysis……………………………………………...…Page 87
Differential interference contrast (DIC) microscopy…………………………Page 88
Figure 1: Germline mortality in spr-1 and met-2;spr-1 mutants……………………Page 89
Figure 2: Transcriptional misregulation in met-2;spr-1 progeny resembles that observed in spr-5;met-2 progeny but is less affected…………………………………………..Page 90
Figure 3: MES-4 germline genes are enriched in met-2;spr-1 mutants, but less affected compared to spr-5;met-2 mutants…………………………………………………….Page 91
Figure 4: LSD1 and CoREST are expressed during each stage of mouse oocyte development…………………………………………………………………………….Page 92
Figure 5: Hypomorphic maternal LSD1 results in perinatal lethality………….…..Page 94
Supplementary material……………………………………………………………….Page 95
Figure S1: Germline mortality in spr-1 and met-2;spr-1 mutants replicate
experiment………………………………………………………………………Page 95
Figure S2: Sterile spr-5;met-2 and met-2;spr-1 double mutant gonads….Page 96
Figure S3: Differential gene expression spr-1, met-2, and met-2;spr-1 progeny
compared to wild type………………………………………………………….Page 98
Figure S4: Differential expression and replicate comparison of RNA seq
experiments performed on wild type, spr-1, met-2, and met-2;spr-1progeny………………………………………………………………………….Page 99
Figure S5: Generation of M448V hypomorphic allele……………………..Page 100
Figure S6: Percent survival by genotype per experimental condition…...Page 101
Chapter 5: LSD1 is required for neural stem cell development in vivo in mice
Introduction……………………………………………………………………………Page 102
Materials and methods………………………………………………………………Page 104
Mouse husbandry and genotyping………………………………………….Page 104
Histological methods…………………………………………………………Page 104
Primary motor neuron culture and transfection……………………………Page 105
Results…………………………………………………………………………………Page 106
LSD1 is expressed in neural stem cells in vivo……………………………Page 106
Figure 1: LSD1 is expressed in neural stem cells…………………………Page 107
Lsd1NSC animals die postnatally with motor defects……………………….Page 107
Figure 2: Lsd1NSC animals do not survive past weaning and show stunted growth……..……………………………………………………………………Page 108
Figure 3: Differentiation of motor neurons in Lsd1NSC animals do not appear to be
affected…………………………………………………………………………Page 110
Motor neurons differentiated from LSD1-deficient NSCs inappropriately express
stem cell genes……………………………………………………………….Page 110
Figure 4: Lsd1NSC-derived motor neurons inappropriately express stem cell genes.. …………………………………………………………………………Page 111
Postnatal Lsd1NSC mutants show abnormal brain morphology in vivo…..Page 112
Figure 5: Lsd1NSC animals show brain morphology defects in vivo……..Page 113
Discussion……………………………………………………………………………..Page 114
Chapter 6: A discussion on LSD1 and its role during developmental processes
Major findings…………………………………………………………………………Page 116
Future experiments…………………………………………………………………..Page 116
Figure 1: Schematic of GFP imprinting reporter mice……………………………Page 121
Implications in humans………………………………..…………………………….Page 122
A model for maternal LSD1 and maternal age……………………………………Page 124
Figure 2: A model for the relationship between LSD1 and maternal age………Page 125
References……………………………………………………………………………Page 126
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