| dc.description.abstracteng | Histone modifications and gene expression are tightly regulated processes in the brain that has been shown to play crucial role from the beginning of brain development, learning-memory formation and aging. While brain comprises of numerous types of neurons and non-neuronal cells, this regulation is highly cell type specific. To gain more mechanistic insights on cell type specific epigenetic and transcriptomic processes, in this thesis, I demonstrated brain nuclei isolation, cell nuclei specific antibody staining and FACS sorting can be successfully utilized to perform cell type specific genome wide histone mark characterization, gene expression and single nuclei RNA sequencing. I have applied these tools to gain valuable mechanistic insights of the causal epigenetic mechanism for cortical folding, functional role of a histone methyltransferase in memory impairment, and multi omics-based characterization of aged induced cognitive decline model.
In the first manuscript, we found that embryonic mice treated with histone deacetylase inhibitors (therefore, increasing histone acetylation) led to higher amounts of basal progenitor (BP) cells in their cortex. This resulted into higher number of mature neurons, thereby producing cortical gyration phenotypes in lissencephalic rodent brains. To understand causal mechanisms, I established and performed for the first time, BP nuclei specific gene expression and histone 3 lysine 9(H3K9) acetylation dataset from embryonic mice cortex. This cell type specific analysis led to discovering distinct increased H3K9ac induced gene expression signature, that contained key regulatory transcription factor, resulting into higher amount of BP proliferation. Further validation experiments via epigenome editing confirmed the epigenetic basis of cortical gyrification in a lissencephalic brain via increasing histone acetylation.
For the second manuscript, I investigated the molecular role of a histone methyltransferase (HMT), Setd1b in mature neurons. Forebrain excitatory neuron specific Setd1B conditional knockout (cKO) resulted into severe memory impairment which required further characterization of neuron specific epigenetic and transcriptomic perturbation due to this cKO. To understand molecular function of Setd1b cKO in neurons, I isolated neuron specific nuclei from WT vs cKO mice hippocampal CA region and performed 4 different histone modification ChIPseq (H3K4me3, H3K4me1, H3K9ac, H3K27ac) and neuron specific nuclear RNA seq. Bioinformatic data analysis revealed promoter specific alteration of all 4 marks and significant down regulation of memory forming genes. Comparison with other two previously studied HMT revealed Setd1b to be having broadest H3K4me3 peaks and regulating distinct sets of genes, which manifested to the severe most behavioral deficit. To understand expression pattern of those three HMTs, I performed single nuclei RNA sequencing of sorted neurons from wild type mice and found, even though Setd1b is expressed in a small subset of neurons, those neurons had the highest level of neuronal function and memory forming gene expression, compared to other two HMT expressing neurons studied previously by our group. Overall, our work shows neuron specific role of Setd1b and its contribution towards hippocampal memory formation.
In the third manuscript, I applied neuronal and non-neuronal epigenome and transcriptome data generation and analysis of 3 vs 16 months old mice. As it is well known that memory impairment starts during the middle of life, and previous gene expression studies in mice showed very little to no changes while having cognitive deficit, I utilized nuclei based cell sorting method to study two promoter epigenetic marks(H3K4me3, H3K27me3) and RNA expression (including coding and non-coding) in neuronal and non-neuronal cells separately. Due to the novelty of the data, I first characterized the basal activatory H3K4me3, inhibitory H3K27me3, bivalent regions and gene expression in neuronal and non-neuronal nuclei. These epigenomic and transcriptomic datasets would be a valuable resource to the community to compare cell type specific gene expression and epigenomes with their datasets. Moreover, profiling epigenetic marks in old hippocampal CA1 neurons and non-neurons revealed massive decrease of epigenetic marks mostly in the non-neurons, while neurons only had decreased inhibitory H3K27me3 mark. Mechanistically, these epigenome changes correspond to probable non-neuronal dysfunction and neuronal upregulation of aberrant developmental pathways. Surprisingly, nuclear RNAseq revealed significant number of genes deregulated in non-neuronal cells, compared to neurons. By integrating transcriptome and epigenome, I found decreased H3K4me3 leading to decreased gene expression in non-neuronal cells, that resulted into probably downregulated neuronal support function and downregulated important glial metabolic pathways related to extra cellular matrix.
Therefore, in this thesis, I have described cell type specific neurodevelopmental, neuronal and cognitive decline related epigenetic and transcriptional pathways that would add valuable knowledge and resources to the neuroscientific community. | de |