The NUCKS1-SKP2-p21/p27 axis controls S phase entry

Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.

M-Phase oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis

Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-Phase transitions (e.g., G2/M). Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in activity driving mitotic M-Phase entry, progression, and exit, with evidence from a variety of experimental systems pointing to roles in meiosis as well. Here we report that PP1 is important for M-Phase transitions through mouse oocyte meiosis. Employing a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis, we found that aberrations in normal cyclical PP1 activity leads to meiotic abnormalities. We report here that temporal control of PP1 activity is essential for G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase I is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in oocytes and female fertility, and more broadly, M-Phase regulation.

The NUCKS1-SKP2-p21/p27 axis controls S phase entry

Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. TCGA data reveal that this mechanism is hijacked in cancer, potentially allowing cancer cells to sustain uncontrolled proliferation.

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

Replication of genomic DNA is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase on DNA replication origins during the G1 phase is essential in initiating DNA replication. Here, we show that stepwise loading of two hexamer complexes of MCM occurs during G1 progression in human cells. This transition from the single-to-double hexamer was associated with levels of methylation at lysine 20 of histone H4 (H4K20). A single hexamer of MCM complexes was loaded at the replication origins with the presence of H4K20 monomethylation (H4K20me1) in the early G1 phase, then another single hexamer was recruited to form a double hexamer later in G1 as H4K20me1 was converted to di-/tri-methylation (H4K20me2/me3). Under non-proliferating conditions, cells stay halted at the single-hexamer state in the presence of H4K20me1. We propose that the single-hexamer state on chromatin is a limiting step in making the proliferation-quiescence decision.

The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.

Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.

The Influence of Anti-Hiv-1 Specific IgY In Inhibiting HIV-1 Infection in Binding Phase with Syncytium Examination of CD4 Receptor Density Using the Flowcytometry Method

HIV/ AIDS infections have increased and spread very quickly in the world, including in Indonesia. The absence of an effective vaccine and the fact that antiretroviral drugs can only suppress the progression of infection but cannot eradicate it lead to the efforts to find materials containing immunoglobulins that can replace the immune system which greatly declines in HIV/ AIDS patients. The successful use of specific IgY in other studies opens up opportunities for the use of anti-HIV-1 specific IgY as passive immunotherapy. This type of research is true experimental research design with post-test only control group design. IgY was obtained from Lohmann Laying hens chicken eggs immunized with the inactivated HIV-1 virus. The concentration of IgY was determined using the Bradford method and then the characterization test was continued using the AGPT, ELISA, SDS-PAGE and Western blot tests which showed anti-HIV-1 specific IgY. The results of the test showed specific anti-HIV-1 IgY was effective in inhibiting the formation of syncytium in HIV-1 infection against CD4+ T lymphocytes in the binding phase (entry stage) in the treatment group p-value 0.000 (p <0.05). The results of CD4 receptor density tests using the Flowcytometry method showed that specific anti-HIV-1 IgY was effective in inhibiting HIV-1 infection against CD4+ T lymphocytes in the binding phase (entry stage) in the treatment group p-value 0.047 (p <0.05).HIV/ AIDS infections have increased and spread very quickly in the world, including in Indonesia. The absence of an effective vaccine and the fact that antiretroviral drugs can only suppress the progression of infection but cannot eradicate it lead to the efforts to find materials containing immunoglobulins that can replace the immune system which greatly declines in HIV/ AIDS patients. The successful use of specific IgY in other studies opens up opportunities for the use of anti-HIV-1 specific IgY as passive immunotherapy. This type of research is true experimental research design with post-test only control group design. IgY was obtained from Lohmann Laying hens chicken eggs immunized with the inactivated HIV-1 virus. The concentration of IgY was determined using the Bradford method and then the characterization test was continued using the AGPT, ELISA, SDS-PAGE and Western blot tests which showed anti-HIV-1 specific IgY. The results of the test showed specific anti-HIV-1 IgY was effective in inhibiting the formation of syncytium in HIV-1 infection against CD4+ T lymphocytes in the binding phase (entry stage) in the treatment group p-value 0.000 (p <0.05). The results of CD4 receptor density tests using the Flowcytometry method showed that specific anti-HIV-1 IgY was effective in inhibiting HIV-1 infection against CD4+ T lymphocytes in the binding phase (entry stage) in the treatment group p-value 0.047 (p <0.05).