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The protein p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies. The carboxyl moiety of p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression. We summarize in this review our current knowledge on the role of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson’s disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies., This work was supported by grants from Fundación Tatiana Pérez de Guzman el Bueno, from Ministerio de Economia y competitividad (MINECO) SAF2015- 64244-R, La Caixa2016-052.407 and from the Instituto de Salud Carlos III CB16/12/00244. AB is supported by grants from the Fondation ARC pour la Reserche sur le Cancer, Ligue National contre le Cancer and Institut National du Cancer. Work in the laboratory of IF is supported by Fundación Botín-Banco Santander.

Different studies have demonstrated multiple effects of arsenite on human physiology. However, there are many open questions concerning the mechanism of response to arsenite. Schizosaccharomyces pombe activates the Sty1 MAPK pathway as a common response to several stress conditions. The specificity of the response is due to the activation of different transcription factors and specific targets such the Cmk2 MAPKAP kinase. We have previously shown that Cmk2 is phosphorylated and activated by the MAPK Sty1 in response to oxidative stress. Here, we report that Cmk2 kinase is specifically necessary to overcome the stress caused by metalloid agents, in particular arsenite. Deletion of cmk2 increases the protein level of various components of the MAPK pathway. Moreover, Cmk2 negatively regulates translation through the Cpc2 kinase: the RACK1 orthologue in fission yeast. RACK1 is a receptor for activated C-kinase. Interestingly, RACK1 is a constituent of the eukaryotic ribosome specifically localized in the head region of the 40 S subunit. Cmk2 controls arsenite response through Cpc2 and it does so through Cpc2 ribosomal function, as observed in genetic analysis using a Cpc2 mutant unable to bind to ribosome. These findings suggest a role for Cmk2 in regulating translation and facilitating adaptation to arsenite stress in the ribosome.

PCAF and GCN5 acetylate cyclin A at specific lysine residues targeting it for degradation at mitosis. We report here that histone deacetylase 3 (HDAC3) directly interacts with and deacetylates cyclin A. HDAC3 interacts with a domain included in the first 171 aa of cyclin A, a region involved in the regulation of its stability. In cells, overexpression of HDAC3 reduced cyclin A acetylation whereas the knocking down of HDAC3 increased its acetylation. Moreover, reduction of HDAC3 levels induced a decrease of cyclin A that can be reversed by proteasome inhibitors. These results indicate that HDAC3 is able to regulate cyclin A degradation during mitosis via proteasome. Interestingly, HDAC3 is abruptly degraded at mitosis also via proteasome thus facilitating cyclin A acetylation by PCAF/GCN5, which will target cyclin A for degradation. Because cyclin A is crucial for S phase progression and mitosis entry, the knock down of HDAC3 affects cell cycle progression specifically at both, S phase and G2/M transition. In summary we propose here that HDAC3 regulates cyclin A stability by counteracting the action of the acetylases PCAF/GCN5., This work was supported by Grants SAF2009-07769 from the Ministerio de Ciencia e Innovación of Spain and Reticc RD06/0020/0010 from the Istituto de Salud Carlos III.

When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.

The administration of the alpha 1-adrenergic antagonist prazosin to hepatectomized rats inhibited DNA synthesis induced in the remaining hepatocytes. This inhibitory effect could be reversed by the simultaneous injection of the agonist phenylephrine. In order to establish how the alpha 1-adrenergic receptors can regulate DNA replication, the effect of prazosin administration on DNA polymerase alpha was examined. At 24 h after partial hepatectomy, the activity of DNA polymerase alpha increased 5, 7 and 9 fold in the homogenates, nuclei and nuclear matrix, respectively. This increase was inhibited by 70%-80% when prazosin was injected at 1, 8 or 11 h after surgery. Kinetic studies revealed that the Km for DNA was 2 fold lower in hepatectomized than in control animals. The administration of prazosin to hepatectomized rats increased the Km to the control values. These results indicate that the alpha 1-adrenergic receptors are involved in the regulation of DNA synthesis through the activation of DNA polymerase alpha and that this activation could be produced by increasing its affinity for DNA.

As has been previously reported one surge in cytosolic calmodulin is produced between 4 and 12 h after a partial hepatectomy. Moreoever, a surge in cytosolic cyclic AMP and another in cyclic AMP-dependent protein kinase activity can be detected during the late period of the prereplicative phase of liver regeneration after a partial hepatectomy. It is known that these three surges are involved in triggering DNA synthesis. By kinetic studies and by injecting transcription (actinomycin D) and translation (cycloheximide) inhibitors into hepatectomized rats we have demonstrated that the cyclic AMP-dependent protein kinase surge is produced by new synthesis of the enzyme. In thyroparathyroidectomized rats subjected to a partial hepatectomy the calmodulin surge was similar to that observed in normal hepatectomized rats whereas the surge in cyclic AMP-dependent protein kinase activity was strongly decreased suggesting that the cyclic AMP-dependent protein kinase surge is not generated by the previous surge in calmodulin.

When rats were infused with a solution containing triiodothyronine, amino acids, glucagon and heparin (solution A) the hepatocytes increased DNA synthesis and decreased plasma membrane sialic acid. In order to study whether the reduced levels of sialic acid in the plasma membrane were associated with hepatocyte proliferation, different mixtures of three components of solution A were infused into rats and the DNA synthetic activity as well as the sialic acid content measured. Results reported here show a correlation between DNA synthetic activity and sialic acid reduction suggesting that the decrease in the plasma membrane sialic acid can be a pre-replicative step associated to cell proliferation.

Enzyme biochemical and histochemical assays during chemical carcinogenesis in rat liver have revealed that several adult enzyme activities are lost and some fetal enzyme activities are re-expressed in the hyperplastic foci as well as in the developed hepatomas. How these enzyme alterations are acquired and to what extent these changes are specifically related to the growth alterations leading to neoplastic development are decisive questions. Using a carcinogenesis protocol that combines a single dose of diethylnitrosamine and phenobarbital given continuously as the promoting agent and by assaying serial liver sections for glucose-6-phosphatase, adenosines-5′-triphosphatase and 5′-nucleotidase, we have identified the three-enzyme pattern of 1,746 islands and measured their section areas. We found a clear trend that clones with more deviated enzyme pattern grow faster than less deviated ones.

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the beta-adrenergic blocker dl-propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occurred between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 mumoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.