CDK2 regulates the NRF1/Ehmt1 axis during meiotic prophase I

Abstract

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1. We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

A novel function for CDK2 activity at meiotic crossover sites

“…This work illustrates the importance for the normal regulation of CDK2 activity during meiotic prophase and adds yet further novel, to our knowledge, functions of CDK2 in addition to those currently established. These include promoting homolog synapsis through the regulation of telomere–nuclear envelope interactions [ 10 , 23 ], regulating meiotic transcription [ 18 ], and maintaining homeostasis and differentiation status of spermatogonial stem cells [ 17 ]. We conclude that CDK2-associated activity is required at multiple stages during the development of germ cells and that this is enabled by the association of CDK2 with multiple activating partner proteins beyond only the currently known Speedy A.…”

“…For histological analyses, testes were fixed for 16 hours at 4°C in modified Davidson’s fixative (8.2% formalin, 33% ethanol, 11% glacial acetic acid in water). Fixed tissues were paraffin-embedded, sectioned at 5 μm, and then stained with the ApopTag Plus Peroxidase In Situ Apoptosis Kit (Sigma-Aldrich: S7101; St. Louis, MO, USA) or HE were used as previously described [ 18 ]. For immunohistochemical staining of testis sections or immunofluorescent staining of meiotic chromosome spreads, slides were blocked in PBS containing 0.15% Triton X-100, 10% BSA, 3% skim milk powder for 1 hour at RT, followed by incubation with primary antibodies for 16 hours at 4°C and detection by secondary antibodies (see antibody details in S1 Table ).…”

“…CDK2 is a multifunctional kinase that is recognized to play multiple roles during germ cell development. These include regulating cell fate in spermatogonial stem cells [ 17 ], regulation of transcription in spermatocytes [ 18 ], and promoting homolog synapsis during meiotic prophase I ([ 10 ]; for reviews, also see [ 19 – 22 ]). During zygonema of meiotic prophase, CDK2 and its noncanonical activating partner Speedy A (see below) interact to promote stable interactions between telomeres and the inner nuclear envelope.…”

Loss of hepatocyte cell division leads to liver inflammation and fibrosis

“…For antigen retrieval in Proteinase K solution, slides were incubated with 20μg/mL Proteinase K in Tris-EDTA buffer (10mM Tris-HCl pH8.0, 1mM EDTA) at 37°C for 20 minutes. Antigen retrieval in trisodium citrate solution has been described previously [ 82 ]. Following antigen retrieval, slides were washed in PBS and blocked in 1% BSA in PBS supplemented with 0.1% Tween (0.1% PBS-T).…”

Diverse roles for CDK‐associated activity during spermatogenesis

“…Remarkably, through ChIPseq and ChIP-reChIP approaches, we detected interactions of CDK2 with chromatin-bound NRF1 at its target gene promoters. Despite the fact that this transcription factor is canonically associated with the regulation of mitochondrial respiration, many of the genes regulated by NRF1 in male germ cells were found to function within meiotic processes, a finding also noted by a prior study [232,233]. Although a thorough analysis of this relationship in meiotic cells was precluded by the early arrest stage of Cdk2 À/À spermatocytes and the lethality associated with Nrf1 deletion [234], we were able to observe that CDK2 was a negative regulator of NRF1 transcriptional activity.…”