Functionality of placental 5-hydroxymethylcytosine and associations with birthweight Open Access
Mortillo, Michael (Fall 2023)
Published
Abstract
Genetic and environmental influences during in utero development may contribute to the etiology of adverse health outcomes in both early life and adulthood. As the main regulator of early development, the placenta acts as an interface in the exchange of maternal physiological and environmental cues with the fetus. It also functions to ensure the success of the pregnancy and allow for proper fetal growth and development. Together, these roles position the placenta as a unique window to understanding molecular mechanisms underlying these potentially deleterious genetic and environmental impacts. One mechanism that can regulate placental function through control of gene expression is the DNA modification 5-hydroxymethylcytosine (5hmC). 5hmC is an epigenetic modification found on the cytosine base of a cytosine-phosphate- guanine (CpG) dinucleotide. It is formed through oxidation of 5-methylcytosine (5mC), an epigenetic mark known to play a complicated role in gene expression control. Though evidence has suggested 5hmC to be a transient intermediate on the demethylation pathway, it may also play a functional role similar to that of 5mC. In this study, placentae from the Rhode Island Child Health Study (RICHS) were selected for CpG 5hmC and 5mC profiling, as well as total mRNA sequencing. The RICHS cohort also collected anthropomorphic and medical data from mother-infant pairs, including data related to newborn birthweight, an early life predictor of long-term health. Here, we characterize associations between placental 5hmC and gene expression, as well as associations between 5hmC, 5mC, and birthweight. We show that although 5hmC proportions are generally low across placenta, it is positively associated with expression of specific genes based on genomic context. We also identify few relationships between 5hmC and birthweight, but show that the characterization of 5mC is not overtly biased by 5hmC content. The characterization of placental 5hmC and its associations with expression suggests a role for this mark in the placenta, with as of yet unknown phenotypic consequence. Future work should consider potentially other roles for 5hmC and explore how environmental and physiologic factors impact this functional mark.
Table of Contents
Table of Contents
Chapter 1 – Introduction........................................................................................1
Birthweight outcomes are an early life predictor of chronic disease risk through the developmental origins of health and disease .............................................................................. 1
The placenta is an important mediator of the maternal-fetal environment and can be used to assess the DOHaD hypothesis ....................................................................... 2
Placental methylation is a potential biomarker for fetal birthweight.......................................... 3
5hmC is formed through oxidation of 5mC and is a stable epigenetic modification that regulates expression.................................................................................................................... 5
Dissertation Overview ................................................................................................................ 6
Figures ........................................................................................................................................ 9
Figure 1-1: Developmental programming of chronic disease..............................................................9
Figure 1-2: Overview of the critical functions of the placenta...........................................................10
Figure 1-3: Association between DNA methylation and infant birthweight.......................................11
Figure 1-4: Overview of the active demethylation pathway ...............................................................12
Figure 1-5: Dissertation Overview .....................................................................................................13
References................................................................................................................................. 14
Chapter 2 - Select early-life environmental exposures and DNA methylation in
the placenta ............................................................................................................19
Abstract..................................................................................................................................... 20 Introduction............................................................................................................................... 21
Methods .................................................................................................................................... 23 Results....................................................................................................................................... 23
Air pollution ........................................................................................................................................24
Maternal smoking ...............................................................................................................................27
Environmental chemicals....................................................................................................................29
Trace metals........................................................................................................................................30
Socioeconomic adversity.....................................................................................................................32
Circadian Disruption (CD)....................................................................................... 33
Conclusions and Future Perspectives......................................................................................33
Statements and Declarations..................................................................................................... 35
Funding ...............................................................................................................................................35
Conflicts of Interest.............................................................................................................................35
Human and animal rights informed consent.......................................................................................35
Tables........................................................................................................................................ 36
Table 2-1: Summary of recent publications focusing on various environmental exposures in relation
to placental DNA methylation.............................................................................................................40
Figures ...................................................................................................................................... 41
Figure 2-1: Early life environmental exposures and their impact on genome-wide DNA methylation and disease risk...................................................................................................................................41
Figure 2-2: Human placental structure and function.........................................................................42
Figure 2-3: Flow chart illustrating paper selection process for conducting literature search..........43
References................................................................................................................................. 44
Chapter 3 - Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta..........................................................................50
Abstract..................................................................................................................................... 51 Introduction............................................................................................................................... 52
Materials and Methods.............................................................................................................. 53
Study population .................................................................................................................................53
Placental sample collection ................................................................................................................53
CpG methylation and hydroxymethylation profiling ..........................................................................54
RNA-sequencing, Quality Control and Read Filtering.......................................................................55
Annotation ........................................................................................................................................... 56
Systematic 5hmC distribution .............................................................................................................57
eQTHM Identification.........................................................................................................................57
Differentially-hydroxymethylated regions (DHMRs)..........................................................................59
Enrichment tests..................................................................................................................................59
Results....................................................................................................................................... 60
Sample cohort .....................................................................................................................................60
Placental 5hmC distribution ...............................................................................................................60
eQTHM analysis .................................................................................................................................61
DHMR analysis...................................................................................................................................63
Discussion................................................................................................................................. 64
Statements and Declarations..................................................................................................... 70
Data Availability .................................................................................................................................70
Funding ...............................................................................................................................................70
Conflicts of Interest.............................................................................................................................70
Ethical Standards................................................................................................................................70
Tables........................................................................................................................................ 71
Table 3-1: RICHS participant demographics .....................................................................................71
Table 3-2. eQTHM summary results...................................................................................................72
Figures ...................................................................................................................................... 73
Figure 3-1: Distribution of systematic 5hmC across the placental epigenome..................................73
Figure 3-2: Placental 5hmC distribution across ChromHMM states ................................................74
Figure 3-3: Placental 5hmC distribution across gene compartments of genes with varying expression levels .................................................................................................................................75 Figure 3-4: Characterization and distribution of genomic location of eQTHM signals....................77
Figure 3-5: Characterization and distribution of transcription-associated DHMRs.........................78
Supplementary Tables............................................................................................................... 79
Table S3-1: Significant CpG-gene pairs from eQTHM analysis ........................................................79
Table S3-2: Significant DHMRs .........................................................................................................93
References................................................................................................................................. 98
Chapter 4 - Associations between placental hydroxymethylation and birthweight ...........................................................................................................101
Abstract................................................................................................................................... 102 Introduction............................................................................................................................. 103
Methods .................................................................................................................................. 104
Study cohort ......................................................................................................................................104
Placental sample collection ..............................................................................................................105
CpG methylation and hydroxymethylation profiling and RNA-sequencing of transcripts ...............105
Cell type estimations .........................................................................................................................106
Robust linear modeling of 5hmC and 5mC data (EWAS).................................................................106
Statistical tests ..................................................................................................................................107
CpG annotation.................................................................................................................................107
Results..................................................................................................................................... 108
Sample cohort ...................................................................................................................................108
Hydroxymethylation and methylation associations with birthweight...............................................108
Discussion............................................................................................................................... 109
Conclusion .............................................................................................................................. 112
Statements and Declarations................................................................................................... 112
Data Availability ...............................................................................................................................112
Funding .............................................................................................................................................113
Conflicts of Interest...........................................................................................................................113
Ethical Standards..............................................................................................................................113
Tables...................................................................................................................................... 114
Table 4-1: RICHS participant demographics ...................................................................................114
Figures .................................................................................................................................... 115
Figure 4-1: Comparison of placental hydroxymethylation and methylation β-values and resulting EWAS effect estimates.......................................................................................................................115
Figure 4-2: Association between placental CUBN expression and infant birthweight Z-score.......116
Supplemental Figures ............................................................................................................. 117
Figure S4-1: EWAS’s between placental A) 5hmC, B) 5mC+5hmC, and C) 5mC with birthweight Z-score ..................................................................................................................................................117
References............................................................................................................................... 118
Chapter 5 – Summary, Limitations, Future Directions, and Conclusions.....122
Summary ................................................................................................................................. 122
Overall Limitations ................................................................................................................. 126
Future Directions .................................................................................................................... 127 Conclusions............................................................................................................................. 128 References............................................................................................................................... 129
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