BIOL 3211 Group Presentation Leah Broger Tristan Chan

BIOL 3211 Group Presentation Leah Broger Tristan Chan HAO Zijing

Index ● ● ● ● ● Why this paper was chosen Research Aims Background and relevant terminology ○ Beta Cells and Diabetes ○ Vit D in HK (general) ○ BAF & BRD 7/9 (novel) Preliminary methods ○ Mechanisms regulating β cell survival ○ Functional significance of VDR expression Continued experimentation and results ○ VDR mechanism in protecting cells against inflammation ○ Molecular mechanism underlying VDR function ○ Nutritional experimentation (dietary stress mouse model) Proposed Pathway and Discussion Strengths and Weaknesses Limitations with comparisons from other journals Future considerations/Conclusion 2

Why was this paper chosen? 1. Strong Impact Factor (31. 4) 2. Relevance to Diabetes 3. Nutritional Impacts of Vitamin D on a Transcriptional level 3

Research Aims/Hypothesis Investigate the modulatory significance of Vitamin D Receptor (VDR) expression on inflammation and β cell survival by: 1. Exploring the significance of VDR expression in β cells 2. Observe whether VDR activation can protect β cells against inflammatory damage 3. Examine the transcriptional expression of Vitamin D on BAF complexes 4

Background and Relevant Terminology β Cells and Diabetes Type II: 1. Diabetes Type II is characterized by insulin resistance of β cells called β cell dysfunction 1. cells are a predominant type of islets of Langerhans found in the pancreas β 5

Background cont’d. Vitamin D and VDR: 1. Vitamin D is a fat-soluble secosteroid found in fatty fish. Putatively known for its contribution to bone growth, and calcium absorption. 1. Vitamin D receptor is a transcription factor part of the nuclear receptor family, Vitamin D binds to it. 6

Diabetes Relevance in HK Mortality rates for diabetes is on a decline, but remains within the top 10 causes of death accounting for 1. 1% in 2015. Previous research discovered that DM 2 caused by increased apoptosis of β-cells Literature gap in diabetes “disease treatments that target β-cell pathogenesis” (Wei et al. ) (Source: Centre for Health Protection, Department of Health, Government of the Hong Kong SAR) 7

Novel Terminology BAF Complex 1. Polymorphic assemblies containing upwards of 15 subunits 2. Bromodomain-containing proteins a. SWI/SNF b. BAF & PBAF in humans 3. Epigenetic regulation a. Chromatin remodeling b. Proliferation & differentiation of cell types c. Recognizes acetylated lysines 4. Perceived to be upstream modulators of β cell stress Source: http: //dev. biologists. org/content/143/16/2882 8

Novel Terminology Bromodomain-containing factor (BRD) ◉ Components of BAF complex BAF-BRD 9 1. Absence of ligand 2. BRD 9 recruits BAF 3. Docks onto VDR (suppressive) PBAF-BRD 7 1. 2. 3. 4. 5. Source: BPS Bioscience, http: //bpsbioscience. com/bromodomain Ligand activation Inhibits BAF-BRD 9 action PTM of VDR leads to conformational changes Allow PBAF-BRD 7 binding for transcriptional response Genes up/down regulated (Insulin produced) 9

Preliminary Methods Explore Mechanisms Regulating β cell Survival Use a CRISPR knockout screen into β-like pluripotent stem cells, with human insulin promoter- driven GFP reporter (INS-GFP) Explore the Functional Significance of VDR Expression i. PS cell lines with RNA knockdowns of VDR were differentiated into β like cells Results 1. Gene ontology analysis revealed that VDR was the one of the most enriched gene targets. 2. Increase in islet staining for pro-insulin in the VDR knockdown 3. β cell function is compromised upon loss of VDR 10

Results of Preliminary Methods 11

Experimentation and Results Exploring VDR activation in protecting β cells against inflammatory damage Method Mouse islet cells cultured in IL 1 B +/- Cal (Synthetic D 2) Relative to VDR @ 25 (RED) Results 1. IL 1 B suppressed β cell function 2. Presence of Cal a. Decrease in BRD 9 interaction b. Increase in BRD 7 interaction c. Partially restored repressed genes Figure. Heatmap of VDR interacting proteins in human β-like cells in the absence or presence of ligand Cal. Spectral counts normalized to VDR. 12

Experimentation Cont’d. Exploring molecular underpinning of VDR function ● ● Given the established roles of the BAF and PBAF complexes in chromatin remodeling, they examined the impact of this VDR-shifted equilibrium on genome-wide chromatin accessibility. Added VDR ligand (Cal) to favor BRD 7 - PBAF association, i. BRD 9 to block re-association to BAF, as well as both compounds simultaneously Results: ● These findings support a model in which dissociating BRD 9 from VDR by ligand or inhibitor results in selected chromatin accessibility 13

Nutritional Experimentation and Results B 6 mice ◉ Treatment started at 6 weeks of age ◉ Cal 60 ug/kg + i. BRD 9 10 mg/kg injected intraperitoneally ◉ IPGTT & Fasting serum obtained after 9 week treatment Increase in serum insulin levels Decrease in blood glucose levels 14

Discussion Established Mechanism 1. BRD 9 and BRD 7 bromodomains recognize VDR K 91 Ac 2. VDR ligand switches association from BRD 9/BAF (inactive) to BRD 7/PBAF (active) 3. Inhibiting BRD 9 enhances vitamin D response 4. Enhanced VDR signaling reduces β cell failure and curbs DT 2 progression 15

Strengths of the Study 1. The advantages of research on Vitamin D and VDR a. Vitamin D has been proven to have systemic anti-inflammatory effects (Krishnan & Feldman, 2011) b. Vitamin D modulates the human epigenome and enhances the accessible chromatin for VDR binding (Veijo Nurminen et al. ) 2. Very cohesive and structured, terminology well explained 3. Methodologies used were contemporary and efficient (CRISPR screening) 16

Weaknesses of the Study 1. Reduced adiposity of VDR KO mice that they used limited their utility in evaluating the contribution of β cell specific defects to altered glucose homeostasis 1. Mice studies may not translate well when scaled up to humans 17

Limitations in relation to Diabetes Clinical Viability: Dual effect of hypercalcemia “Unfortunately, the doses needed for disease prevention in NOD mice lead to hypercalcemia and bone decalcification” (Decallonne et al. 2005) “

Limitations in relation to Diabetes Are there enough β Cells to make a Quantifiable difference in Mitigating Diabetes? “It is possible that at this point the number of remaining βcell mass is simply not sufficient to restore insulin needs. ” (Keymeulen B et al. 2005) “

Limitations in relation to Diabetes Only focused on Acetylation of K 91 on VDR. Other PTMs may also occurs at same site “In a preliminary analysis we identify lysine 91, a residue known to be critical formation and DNA binding of the VDR-RXR heterodimer, as a minor SUMO acceptor site within VDR. ” (Lee et al. , 2014) “ Source: (Zenata & Vrzal, 2017)

Further Considerations and Conclusion: ◉ Overall, there is insufficient evidence to claim that Vitamin D can clinically prevent or mitigate Diabetes Type II because: ○ Only mice trials have been conducted ○ There are other mechanisms which Vitamin D would be inadvertently affecting ○ Best option would be a combination of pharmacological and nutritional intervention (Vit D + i. BRD 9) Further Considerations: ◉ ◉ ◉ Conduct nutritional trials on diabetic humans looking at different Vit D intakes More research into the enzymatic reaction of Vit D and VDR and the enzymatic substrate interaction (saturation point, efficacy of enzyme) Study showed that this dual ligand mechanism can be pharmacologically manipulated which is very promising with regards to possible genomic reprogramming therapies for DT 2 21

Thank you! 22

References Butler, A. , Janson, J. , Bonner-Weir, S. , Ritzel, R. , Rizza, R. , & Butler, P. (2003). -Cell Deficit and Increased Cell Apoptosis in Humans With Type 2 Diabetes, 52(1), 102 -110. doi: 10. 2337/diabetes. 52. 1. 102 Krishnan, A. , & Feldman, D. (2011). Mechanisms of the Anti-Cancer and Anti-Inflammatory Actions of Vitamin D. Annual Review Of Pharmacology And Toxicology, 51(1), 311 -336. doi: 10. 1146/annurev-pharmtox-010510100611 Lee SC, Pu YB, Chow CC, Yeung VT, Ko GT, So WY, Li JK, Chan WB, Ma RC, Critchley JA, Cockram CS, Chan JC. Diabetes in Hong Kong Chinese: evidence for familial clustering and parental effects. Diabetes Care 23: 1365– 1368, 2000. Xu, C. , Perera, R. , Chan, Y. , Fang, V. , Ng, S. , & Ip, D. et al. (2015). Determinants of serum 25 -hydroxyvitamin D in Hong Kong. British Journal Of Nutrition, 114(01), 144 -151. doi: 10. 1017/s 0007114515001683 Zenata, O. , & Vrzal, R. (2017). Fine tuning of vitamin D receptor (VDR) activity by post-transcriptional and posttranslational modifications. Oncotarget, 8(21). doi: 10. 18632/oncotarget. 15697 23

References Lee, W. , Jena, S. , Doherty, D. , Ventakesh, J. , Schimdt, J. , & Furmick, J. et al. (2014). Sentrin/SUMO Specific Proteases as Novel Tissue-Selective Modulators of Vitamin D Receptor. Mediated Signaling. Plos ONE, 9(2), e 89506. doi: 10. 1371/journal. pone. 0089506 Mark D. Long, Moray J. Campbell(2017). Integrative genomic approaches to dissect clinicallysignificant relationships between the VDR cistrome and gene expression in primary colon cancer. The Journal of Steroid Biochemistry and Molecular Biology 173: 130 -138 Veijo Nurminen, Antonio Neme, Sabine Seuter, Carsten Carlberg(2018). The impact of the vitamin D-modulated epigenome on VDR target gene regulation. Biochimica et Biophysica Acta (BBA) Gene Regulatory Mechanisms, 1861(8): 1874 -9399 24