Circadian Genes, Stress Axis and Fetal Alcohol Spectrum Disorder PI: Dipak K. Sarkar

Goal: to determine changes in levels of Per1 and Per2 gene methylation and expression and cortisol and testosterone from biological samples of FASD and non-FASD children from longitudinal studies conducted by CIFASD in order to identify a stable epigenetic signature of a specific group or all FASD patients.

Fetal alcohol exposed offspring often shows health problems Fetal Alcohol Syndrome, partial FAS or physical expression Sleep-wake disturbance Mental diseases such as anxiety and depression Abnormality in coping stress Metabolic disease Immune dysfunction & Cancer

Studies in laboratory rodents Fetal ethanol exposure alter the daily rhythm of clock genes and POMC levels in the adult hypothalamus of rats Fetal alcohol exposed adult rats show abnormality in clock gene expression in the arcuate nucleus and in the SCN and in POMC expression in the arcuate nucleus. (Chen CP, et al. 2006 J Neurochem. 97:1026-1033).

Studies in laboratory rodents Fetal alcohol exposures induce hyper-response of stress hormones following stress in adult mice Logan and Sarkar, unpublished

Studies in laboratory rodents Per2 knocking down reduces beta-endorphin levels and prevents fetal alcohol-induced increase in stress-induced corticosterone release in mice Agapito and Sarkar, Endocrinology, in revision

Does fetal alcohol exposure program the POMC system to control the neuroendocrine-immune axis functions? Reward Amygdala N. Accumb. SCN (-) CRH, ANS PVN -EP (-) Arcuate POMC PIT ACTH -EP (+) (+) (-) Lymphoid organs CORT Adrenal NK cells

Why fetal alcohol exposures alter Per2 and POMC gene expression Why fetal alcohol exposures alter Per2 and POMC gene expression? Is there any role of the epigenetic mechanism? Adopted from S. Pandey

Fetal alcohol’s epigenetic programming of POMC neurons Microarray analysis revealed differential significant expression of 20 genes with a P<0.001 with a 2 fold change and around 2,000 genes with a P<0.01 and a P<0.05 with less than 2 fold change in linear scale. We linked particular sets of these 20 genes to biological functions including methylation, transcriptional and translational regulation, alcohol metabolism, folate metabolism and immunity (Table 2). Bekdash, Agapito and Sarkar, unpublished

Fetal alcohol’s epigenetic programming of POMC neurons Fetal Alcohol Increases Methylation States of Individual CpG Sites in the Hypothalamic POMC Promoter Govorko et al., 2012, Biol Psychiatry

5’-Aza or TSA FAE Pomc gene repression Pomc gene activation SAM HDACs Dnmts Ac Hypermethylation MBDs Deacetylation CH3 CH3 CH3 CH3 CH3 CH3 CH3 Inaccessible chromatin Hypermethylated CpG island SAM Acetylation HATs Fig. 1. Conceptual diagram of the epigenetic effects of fetal alcohol exposure on POMC gene. Dnmt=DNA methyltransferase, MBD (methyl-binding domain proteins), HDAC=histone deacetylase, HAT= Histone acetyltransferase, Ac=acetylation, FAE=fetal alcohol exposure, TSA=trichostatin, 5’-Aza=5’-azacytidine, CH3=methyl group, SAM=S-adenosylmethionine. Dnmts Ac Normal methylation CH3 CH3 CH3 CH3 Accessible chromatin CpG island Bekdash et al, in communication FAE Pomc gene activation

Fetal alcohol’s epigenetic programming of POMC neurons Choline supplementation reversed ethanol effects on POMC gene promoter methylation, POMC gene expression, β-EP production, and normalized the corticosterone hyper-response to LPS challenge in alcohol-exposed rats. Bekdash et al, ACER i2013

Fetal alcohol’s epigenetic programming of POMC neurons Govorko et al.,2012, Biological Psychiatry Rachdaoui and Sarkar, in press

Interrelationship between the epigenetic programming of POMC and Per2 genes: using fetal alcohol exposed rats Logan and Sarkar, unpublished

Interrelationship between the epigenetic programming of Per2 genes and stress axis: using human alcoholic patients 30 light social drinking controls (HC) matched to 70 treatment engaged alcohol dependent (AD) individuals were used. Methylation of the Per2 DNA expression as assayed by MSRP in AD vs HC (P<0.002). Increased methylation levels were significantly associated with stress-induced cortisol (r=.39, P<0.03) and ACTH levels (r=.41, P<0.02). Methylation/unmethylation Ratio in AD vs HC (p<.002). Sinha and Sarkar, unpublished

FAS and fetal alcohol exposed children show persistent hypermethylation of POMC gene Collaborative work with Dr. Foroud and CIASD

FAS and fetal alcohol exposed children show persistent hypermethylation of Per2 gene Collaborative work with Dr. Foroud and CIASD

Interrelation with Aims of the Consortium and Other Projects We are working with other Consortium projects conducted by Drs. Tatiana Foroud, Christina Chamber, Elizabeth R. Sowell, Sara Mattson, Jeff Wozniak and Claire Coles.

Questions we are addressing and our expected outcome Question 1. Do methylation and expression levels of Per genes correlate with the stress abnormality (high cortisol)? Question 2. Can these methylation sites and patterns be used as biomarkers that predict future endophenotypes of stress abnormalities (e.g., anxiety, hyperactivity)? Question 3. If Per genes hypermethylation is involved in stress abnormalities, whether this is manifested in patients with FASD? Expected outcome: These studies may allow identification of a stable epigenetic marker for earlier detection of alcohol-related birth outcomes.