Dissertation - Atiyeh Eghbali

Abstract:

Spermatogenesis is a highly coordinated developmental process that relies on the precise temporal regulation of gene expression. During this process, spermatogonial stem cells give rise to differentiating germ cells that undergo
mitotic proliferation, meiosis, and spermiogenesis to ultimately generate mature spermatozoa. Proper execution of these developmental transitions requires transcription factors and chromatin regulators that establish and activate stage-specific gene-expression programs. Disruption of these regulatory mechanisms can impair spermatogenesis and lead to male
infertility. In this thesis, I investigated the molecular mechanisms controlling the temporal regulation of gene expression during mouse spermatogenesis, with a particular focus on how transcription factors and chromatin-remodeling complexes contribute to the establishment and activation of stage-specific transcriptional programs.

In paper 1, we identified the transcription factor NFYA as a regulator of meiotic entry. We found that promoters of genes expressed later during spermatogenesis were already accessible in pre-meiotic spermatogonia and investigated how this permissive chromatin state is regulated. NFYA occupied a subset of developmentally poised meiotic promoters before
meiosis, and germ-cell-specific deletion of Nfya reduced chromatin accessibility at NFYA-bound promoters and caused arrest at meiotic entry. Together, these findings support NFYA as a candidate pioneer transcription factor that primes meiotic gene promoters for timely activation.

In paper 2, we investigated the role of the ATP-dependent chromatin remodeler SMARCA5 in regulating accessible chromatin at developmentally poised meiotic promoters. We found that SMARCA5 interacts with NFYA and that NFYA is required for SMARCA5 occupancy at these promoters, where SMARCA5 co-occupies chromatin with NFYA and the NURF subunit BPTF. Germ-cell-specific deletion of Smarca5 disrupted the mitosis-to-meiosis transition and impaired meiotic progression, resulting in spermatogenic arrest during early meiotic prophase I. Together, these findings support a model in which NFYA cooperates with SMARCA5 to maintain accessible chromatin at poised meiotic promoters, with
BPTF co-occupancy suggesting the involvement of the NURF chromatin-remodeling complex.

In papers 3 and 4, we demonstrated that the testis-specific transcription factor TCFL5 cooperates with A-MYB through an interlocking positive feedback loop to regulate meiotic and spermiogenic transcriptional programs and ensure the production of pachytene piRNAs. We found a similar A-MYB/TCFL5 regulatory network in mouse and rhesus macaque,
suggesting that this mechanism evolved before rodents and primates diverged. Loss of TCFL5 in male mice caused spermatogenic arrest at the mid- to late-pachytene stage of meiosis I, resulting in infertility.

Together, these papers reveal how transcription factors and chromatin regulators coordinate the timely activation of developmental gene-expression programs during spermatogenesis. Disruption of these regulatory mechanisms can impair germ-cell development and male fertility.

Keywords: Spermatogenesis, transcription factor network, nucleosome remodeler, meiotic initiation, meiosis, chromatin accessibility, pioneer transcription factor.