(336) Chromatin Accessibility Dynamics and Transcriptional Regulation in Human Granulosa Cells in Response to Follicle Stimulating Hormone Stimulation.

Navya Prabhu Basrur¹; Dipanwita Das^1,2; Ailenn C. Castillo¹; Sankarasubramanian Jagadesan³; Chittibabu Guda^3,4; John S. Davis^1,2,5.

1. Olson Center for Women’s Health, Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

2. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Centre, Omaha, NE 68198, USA.

3. Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, USA.

4. Center for Biomedical Informatics Research and Innovation, University of Nebraska Medical Center, Omaha, NE 68198, USA.

5. VA Nebraska-Western Iowa Health care System, Omaha, NE 68198, USA.

Abstract Text:

The adenylyl cyclase and hippo signaling pathway are vital for female fertility, regulating granulosa cell gene expression and follicle maturation to ensure successful ovulation and conception. A key player in this cascade is the cAMP response element-binding protein (CREB), which drives gene expressions essential for ovarian function. Disruptions in CREB activity are known to be associated with infertility, emphasizing its pivotal role in reproductive health. The formation and maturation of follicles in the ovary primarily depend on Follicle-Stimulating Hormone (FSH). FSH plays a significant role in granulosa cell function, compelling processes such as proliferation, differentiation, and steroidogenesis during follicular development. FSH signaling, relying on the cAMP/PKA/CREB pathway, undergoes age-related alterations that can disrupt granulosa cell function. Upon phosphorylation, CREB binds to cAMP response elements (CREs) to regulate target gene transcription. To date, there is a lack of knowledge on how CREB contributes to FSH action and its impact on chromatin accessibility in human granulosa cells remain largely unexplored. To address this gap, we employed ATAC sequencing as an unbiased approach to examine the putative transcription factors (TF) that may mediate the FSH-responsive gene regulation in human granulosa cells. To assess early and long-term responses, the granulosa cells isolated from in vitro fertilization patients were cultured and treated with 30 ng/ml of FSH for 30 minutes and 2 hours and performed immunofluorescence to confirm CREB activation and localization. Additionally, we analyzed chromatin accessibility dynamics in granulosa cells (n=2/group) using ATAC-sequencing with nf-core/atacseq to identify regulatory elements controlling gene expression. Immunofluorescence revealed nuclear translocation of phosphorylated CREB (p-CREB) upon FSH stimulation, confirming its activation. This finding was validated by Western blotting in our prior studies. Furthermore, sequencing reads underwent quality control, alignment to the reference genome, and peak calling to identify open chromatin regions. The differential footprinting analysis revealed high binding activity of the KLF15, SP1, SP2 (p< 0.001) TFs in control granulosa cells, suggesting their role in maintaining basal gene expression and cellular homeostasis. In contrast, FSH-treated cells showed increased accessibility for CREB, RUNX, GATA, NR5A2 and AP-1 family of TFs, reflecting their activation in granulosa cell differentiation and steroidogenesis. Functional enrichment analysis identified Transcription factor AP-1 complex (FDR=0.0004), ATF4-CREB1 TF complex (FDR=0.01), and TEAD-YAP complex (FDR=0.006) in accessible chromatin regions, revealing key transcriptional regulators of FSH signaling. Motif enrichment analysis also highlighted a progressive increase in SF-1 (NR5A1), in 2-hour FSH treated granulosa cells compared to 30 minutes treatment and control granulosa cells suggests increasing enrichment of SF-1 binding motifs over time. Interestingly, our foot printing and motif analysis findings indicate that NR5A1 and NR5A2 play a prominent role in FSH-treated cells compared to controls. These findings demonstrate that FSH acutely actives multiple TF complexes in human granulosa cells, consistent with its ability to promote, proliferation, differentiation, and steroidogenesis during follicular development. Our findings provide novel insights into the transcription factors, regulatory elements, and key signaling pathways underlying the response of ovarian granulosa cells to FSH stimulation, offering a deeper understanding of ovarian function and potential targets for improving fertility treatments.
Statement of financial support: This study is supported by National Institutes of Health (NIH) (HD11261801, P01AG02953111A1, R01HD113570A1) and Veterans Affairs (VA) (IK6BX005797).