Date of Award

6-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

First Advisor

Suraiya Anjum Ansari

Abstract

The nutrient responsive O-GlcNAcylation is a dynamic, posttranslational protein modification present on many nucleocytoplasmic and mitochondrial proteins. Previous research has indicated that hyperglycaemia increases the levels of total O-GlcNAcylation within cells. Transcription factors and histones are among hundreds of proteins that have been reported to be O-GlcNAcylated and have importance in cell fate determination during cell growth, proliferation, and differentiation. However, the role of protein O-GlcNAcylation in epigenome control in response to nutritional perturbations is poorly understood. Hyperglycaemia induced protein O-GlcNAcylation have been linked to several pathologies, including obesity, diabetes, cancer, cardiovascular and neurodegenerative diseases. Given that maternal hyperglycaemia during pregnancy is linked to adverse neurodevelopmental outcomes in the offspring, it is interesting to identify the impact of elevated protein O-GlcNAcylation on embryonic neurogenesis. Herein, we investigate and confirm the implications of protein O-GlcNAcylation by pharmacological induction of O-GlcNAc during embryonic neurogenesis by directed differentiation of human embryonic stem cells (hESCs) into cortical neurons, which precisely mimics early events of human embryonic corticogenesis. The presence of total O-GlcNAc levels during neural differentiation was determined by immunocytochemistry and western blotting techniques. Investigation of several regulatory transcription factor genes and histones needed for stem cell fate during neurogenesis was carried out through different molecular biology techniques including, western blotting, qPCR, and immunocytochemistry. The impact of elevated O-GlcNAc on transcriptional/epigenetic mechanisms was investigated through high-throughput RNA sequencing and ChIP-qPCR. Resulted that increased O-GlcNAcylation is associated with decreased neural progenitor proliferation, premature cortical neurogenesis, reduced AKT signaling, induced apoptosis and defective expression of several genes essential for neural differentiation. This also led to increased expression of key neurogenic transcription factor (TF) genes. Further investigation has shown that de-repression of neurogenic TFs is associated with increased H3K4me3 and decreased H3K27me3 (promoter bi-valency) levels at the promoter of these genes. Increased O-GlcNAc levels also increased Pol IISer5 phosphorylation whereas, levels of H2BS112O-GlcNAc and H2BK120Ub1 were inconsistently affected at different gene promoters. Also studied the effect of elevated O-GlcNAc levels on embryonic neurogenesis in a rat model of maternal hyperglycaemia. We observed similar epigenetic defects including changes in promoter bivalency and induced Pol IISer5p, H2BS112O-GlcNAc and H2BK120Ub1 in the developing embryo brain cortex due to hyperglycaemia. Resulted findings show O-GlcNAc regulated chromatin sensitivity based on maternal nutritional status on neurodevelopment and suggest that metabolic dysregulations can affect stem cell fate decisions via O-GlcNAc mediated epigenetic gene regulating mechanisms during development. These results may have implications in neurodevelopmental disorders associated with metabolically compromised pregnancies.

Arabic Abstract

يعتبر O-GlcNAcylation اﻟﻤﺴﺘﺠﯿﺐ ﻟﻠﻤﻐﺬﯾﺎت ﺗﻌﺪﯾﻼً دﯾﻨﺎﻣﯿﻜﯿًﺎ ﻟﻠﺒﺮوﺗﯿﻦ ﺑﻌﺪ ﻋﻤﻠﯿﺔ ﺗﻜﻮن اﻟﺒﺮوﺗﯿن (اﻟﺘﺮﺟﻤﺔ) وﻣﻮﺟﻮد ﻋﻠﻰ اﻟﻌﺪﯾﺪ ﻣﻦ ﺑﺮوﺗﯿﻨﺎت nucleocytoplasmic واﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ. ﻟﻘﺪ أﺷﺎرت اﻷﺑﺤﺎث اﻟﺴﺎﺑﻘﺔ إﻟﻰ أن ارﺗﻔﺎع اﻟﺴﻜﺮ ﻓﻲ اﻟﺪم ﯾﺰﯾﺪ ﻣﻦ ﻣﺴﺘﻮﯾﺎت O-GlcNAcylation اﻟﻜﻠﻲ داﺧﻞ اﻟﺨﻼﯾﺎ. ﺗﻌﺪ ﻋﻮاﻣﻞ اﻟﻨﺴﺦ واﻟﮭﯿﺴﺘﻮﻧﺎت ﻣﻦ ﺑﯿﻦ ﻣﺌﺎت اﻟﺒﺮوﺗﯿﻨﺎت اﻟﺘﻲ ﺗﻢ اﻹﺑﻼغ ﻋﻦ أﻧﮭﺎ O-GlcNAcylated وﻟﮭﺎ أھﻤﯿﺔ ﻓﻲ ﺗﺤﺪﯾﺪ ﻣﺼﯿﺮ اﻟﺨﻠﯿﺔ أﺛﻨﺎء ﻧﻤﻮ اﻟﺨﻼﯾﺎ وﺗﻜﺎﺛﺮھﺎ وﺗﻤﺎﯾﺰھﺎ. وﻣﻊ ذﻟﻚ، ﻓﺈن دور اﻟﺒﺮوﺗﯿﻦ O-GlcNAcylation ﻓﻲ اﻟﺘﺤﻜﻢ ﻓﻲ اﻹﯾﺒﯿﺠﯿﻨﻮم اﺳﺘﺠﺎﺑﺔً ﻻﺿﻄﺮاﺑﺎت اﻟﺘﻐﺬﯾﺔ ﻏﯿﺮ ﻣﻔﮭﻮم ﺟﯿﺪًا. ﺗﻢ رﺑﻂ ﺑﺮوﺗﯿﻦ O-GlcNAcylation اﻟﻤﺴﺘﺤﺚ ﺑﻔﺮط ﺳﻜﺮ اﻟﺪم ﺑﺎﻟﻌﺪﯾﺪ ﻣﻦ اﻷﻣﺮاض، ﺑﻤﺎ ﻓﻲ ذﻟﻚ اﻟﺴﻤﻨﺔ واﻟﺴﻜﺮي واﻟﺴﺮطﺎن وأﻣﺮاض اﻟﻘﻠﺐ واﻷوﻋﯿﺔ اﻟﺪﻣﻮﯾﺔ واﻷﻣﺮاض اﻟﺘﻨﻜﺴﯿﺔ اﻟﻌﺼﺒﯿﺔ. ﺑﺎﻟﻨﻈﺮ إﻟﻰ أن ﻓﺮط ﺳﻜﺮ اﻟﺪم ﻟﺪى اﻷم أﺛﻨﺎء اﻟﺤﻤﻞ ﻣﺮﺗﺒﻂ ﺑﻨﺘﺎﺋﺞ ﻧﻤﻮ ﻋﺼﺒﻲ ﻋﻜﺴﯿﺔ ﻓﻲ اﻟﻨﺴﻞ، ﻓﻤﻦ اﻟﻤﺜﯿﺮ ﻟﻼھﺘﻤﺎم ﺗﺤﺪﯾﺪ ﺗﺄﺛﯿﺮ اﻟﺒﺮوﺗﯿﻦ اﻟﻤﺮﺗﻔﻊ O-GlcNAcylation ﻋﻠﻰ ﺗﻜﻮﯾﻦ اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ اﻟﺠﻨﯿﻨﯿﺔ. ھﻨﺎ، ﻗﻤﻨﺎ ﺑﺎﻟﺘﺤﻘﯿﻖ واﻟﺘﺄﻛﺪ ﻣﻦ آﺛﺎر ﺑﺮوﺗﯿﻦ O-GlcNAc ﻋﻦ طﺮﯾﻖ اﻟﺤﺚ اﻟﺪواﺋﻲ لـO-GlcNAc أﺛﻨﺎء ﺗﻜﻮﯾﻦ اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ اﻟﺠﻨﯿﻨﯿﺔ ﻋﻦ طﺮﯾﻖ اﻟﺘﻤﺎﯾﺰ اﻟﻤﻮﺟﮫ ﻟﻠﺨﻼﯾﺎ اﻟﺠﺬﻋﯿﺔ اﻟﺠﻨﯿﻨﯿﺔ اﻟﺒﺸﺮﯾﺔ (hESCs) ﻓﻲ اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ اﻟﻘﺸﺮﯾﺔ، واﻟﺘﻲ ﺗﺤﺎﻛﻲ ﺑﺪﻗﺔ اﻷﺣﺪاث اﻟﻤﺒﻜﺮة ﻟﺘﻜﻮﯾﻦ اﻟﻘﺸﺮة اﻟﺠﻨﯿﻨﯿﺔ اﻟﺒﺸﺮﯾﺔ. ﺗﻢ ﺗﺤﺪﯾﺪ وﺟﻮد ﻣﺴﺘﻮﯾﺎت O-GlcNAc اﻟﻜﻠﯿﺔ أﺛﻨﺎء اﻟﺘﻤﺎﯾﺰ اﻟﻌﺼﺒﻲ ﻋﻦ طﺮﯾﻖ اﻟﻜﯿﻤﯿﺎء اﻟﻤﻨﺎﻋﯿﺔ وﺗﻘﻨﯿﺎت اﻟﺒﺮوﺗﯿﻦ. ﺗﻢ إﺟﺮاء اﻟﺘﺤﻘﯿﻖ ﻓﻲ اﻟﻌﺪﯾﺪ ﻣﻦ ﺟﯿﻨﺎت ﻋﺎﻣﻞ اﻟﻨﺴﺦ اﻟﺘﻨﻈﯿﻤﻲ واﻟﮭﯿﺴﺘﻮﻧﺎت اﻟﻼزﻣﺔ ﻟﻤﺼﯿﺮ اﻟﺨﻼﯾﺎ اﻟﺠﺬﻋﯿﺔ أﺛﻨﺎء ﺗﻜﻮﯾﻦ اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ ﻣﻦ ﺧﻼل ﺗﻘﻨﯿﺎت اﻟﺒﯿﻮﻟﻮﺟﯿﺎ اﻟﺠﺰﯾﺌﯿﺔ اﻟﻤﺨﺘﻠﻔﺔ ﺑﻤﺎ ﻓﻲ ذﻟﻚ، ﺗﻘﻨﯿﺎت اﻟﺒﺮوﺗﯿﻦ، و qPCR، واﻟﻜﯿﻤﯿﺎء اﻟﺨﻠﻮﯾﺔ اﻟﻤﻨﺎﻋﯿﺔ. ﺗﻢ اﻟﺘﺤﻘﯿﻖ ﻓﻲ ﺗﺄﺛﯿﺮ O-GlcNAc اﻟﻤﺮﺗﻔﻊ ﻋﻠﻰ آﻟﯿﺎت اﻟﻨﺴﺦ / اﻟﻮراﺛﺔ اﻟﻼﺟﯿﻨﯿﺔ ﻣﻦ ﺧﻼل ﺗﺴﻠﺴﻞ اﻟﺤﻤﺾ اﻟﻨﻮوي اﻟﺮﯾﺒﻲ ﻋﺎﻟﻲ اﻹﻧﺘﺎﺟﯿﺔ و ChIP-qPCR. ﻟﻘﺪ وﺟﺪﻧﺎ أن زﯾﺎدة O-GlcNAcylation ﻣﺮﺗﺒﻄﺔ ﺑﺎﻧﺨﻔﺎض ﺗﻜﺎﺛﺮ اﻟﺴﻼﻻت اﻟﻌﺼﺒﯿﺔ، وﺗﻜﻮﯾﻦ اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ اﻟﻘﺸﺮﯾﺔ اﻟﻤﺒﻜﺮة، واﻧﺨﻔﺎض إﺷﺎرات AKT، واﻻﺳﺘﻤﺎﺗﺔ اﻟﻤﺴﺘﺤﺜﺔ واﻟﺘﻌﺒﯿﺮ اﻟﻤﻌﯿﺐ ﻟﻠﻌﺪﯾﺪ ﻣﻦ اﻟﺠﯿﻨﺎت اﻟﻀﺮورﯾﺔ ﻟﻠﺘﻤﺎﯾﺰ اﻟﻌﺼﺒﻲ.

أدى ھﺬا أﯾﻀًﺎ إﻟﻰ زﯾﺎدة اﻟﺘﻌﺒﯿﺮ ﻋﻦ ﺟﯿﻨﺎت ﻋﺎﻣﻞ اﻟﻨﺴﺦ اﻟﻌﺼﺒﻲ اﻟﺮﺋﯿﺴﻲ (TF). أظهر مزيد ﻣﻦ اﻟﺘﺤﻘﯿﻖ أن إﻟﻐﺎء ﻗﻤﻊ TFs اﻟﻌﺼﺒﯿﺔ ﯾﺮﺗﺒﻂ ﺑﺰﯾﺎدة H3K4me3 واﻧﺨﻔﺎض ﻣﺴﺘﻮﯾﺎت H3K27me3 (ﻣﺤﻔﺰ ﺛﻨﺎﺋﯿﺔ اﻟﺘﻜﺎﻓﺆ) ﻋﻨﺪ ﻣﺤﻔﺰ ھﺬه اﻟﺠﯿﻨﺎت. أدت زﯾﺎدة ﻣﺴﺘﻮﯾﺎت O-GlcNAc أﯾﻀًﺎ إﻟﻰ زﯾﺎدة ﻓﺴﻔﺮة Pol IISer5، ﺑﯿﻨﻤﺎ ﺗﺄﺛﺮت ﻣﺴﺘﻮﯾﺎت H2BS112O-GlcNAc و H2BK120Ub1 ﺑﺸﻜﻞ ﻏﯿﺮ ﻣﺘﺴﻖ ﻓﻲ ﻣﺤﻔﺰات ﺟﯿﻨﯿﺔ ﻣﺨﺘﻠﻔﺔ. درﺳﻨﺎ أﯾﻀﺎ ﺗﺄﺛﯿﺮ ﻣﺴﺘﻮﯾﺎت O-GlcNAc اﻟﻤﺮﺗﻔﻌﺔ ﻋﻠﻰ ﺗﻜﻮﯾﻦ اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ اﻟﺠﻨﯿﻨﯿﺔ ﻓﻲ ﻧﻤﻮذج اﻟﻔﺌﺮان ﻟﻔﺮط ﺳﻜﺮ اﻟﺪم ﻟﺪى اﻷﻣﮭﺎت.

ﻻﺣﻈﻨﺎ ﻋﯿﻮﺑًﺎ ﺟﯿﻨﯿﺔ ﻣﻤﺎﺛﻠﺔ ﺑﻤﺎ ﻓﻲ ذﻟﻚ اﻟﺘﻐﯿﺮات ﻓﻲ ﺛﻨﺎﺋﯿﺔ اﻟﺘﻜﺎﻓﺆ اﻟﻤﺤﻔﺰ و Pol IISer5p وH2BS112O-GlcNAc و H2BK120Ub1 ﻓﻲ ﻗﺸﺮة دﻣﺎغ اﻟﺠﻨﯿﻦ اﻟﻨﺎﻣﯿﺔ ﺑﺴﺒﺐ ارﺗﻔﺎع اﻟﺴﻜﺮ ﻓﻲ اﻟﺪم. ﺗﻈﮭﺮ اﻟﻨﺘﺎﺋﺞ اﻟﺘﻲ ﺗﻮﺻﻠﻨﺎ إﻟﯿﮭﺎ ﺣﺴﺎﺳﯿﺔ اﻟﻜﺮوﻣﺎﺗﯿﻦ اﻟﻤﻨﻈﻤﺔ O-GlcNAc ﺑﻨﺎءً ﻋﻠﻰ اﻟﺤﺎﻟﺔ اﻟﺘﻐﺬوﯾﺔ ﻟﻸم ﻋﻠﻰ اﻟﺘﻄﻮر اﻟﻌﺼﺒﻲ وﺗﺸﯿﺮ إﻟﻰ أن اﺧﺘﻼﻻت اﻟﺘﻤﺜﯿﻞ اﻟﻐﺬاﺋﻲ ﯾﻤﻜﻦ أن ﺗﺆﺛﺮ ﻋﻠﻰ ﻗﺮارات ﻣﺼﯿﺮ اﻟﺨﻼﯾﺎ اﻟﺠﺬﻋﯿﺔ ﻋﺒﺮ آﻟﯿﺎت ﺗﻨﻈﯿﻢ اﻟﺠﯿﻨﺎت اﻟﻼﺟﯿﻨﯿﺔ ﺑﻮﺳﺎطﺔ O-GlcNAc أﺛﻨﺎء اﻟﺘﻄﻮر. ﻗﺪ ﯾﻜﻮن ﻟﮭﺬه اﻟﻨﺘﺎﺋﺞ آﺛﺎر ﻓﻲ اﻻﺿﻄﺮاﺑﺎت اﻟﻨﻤﺎﺋﯿﺔ اﻟﻌﺼﺒﯿﺔ اﻟﻤﺮﺗﺒﻄﺔ ﺑﺤﻤﻞ ﺿﻌﯿﻒ اﻟﺘﻤﺜﯿﻞ اﻟﻐﺬاﺋﻲ

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