Cell Cycle Control Research Articles

472 Nicotinamide improves cutaneous field cancerization on an ex-vivo study

Cutaneous field cancerization (FC) is a tissue area chronically exposed to sun light, in which cells present altered cell cycle, proliferation, differentiation, and death despite their normal appearance. On normal sun-exposed skin the most mutated gene is p53 which regulates the expression of p21 which controls cell cycle progression and cellular differentiation. Treatment of cutaneous FC with safe and low-cost molecules could reduce the probability of skin cancer recurrence. Nicotinamide (NAM) is the amide form of vitamin B3, a water-soluble molecule assumed by daily diet.

Differential dynamics of cullin deneddylation via COP9 signalosome subunit 5 interaction

Cullin-RING E3 ubiquitin ligases (CRLs) spatiotemporally regulate the proteasomal degradation of numerous cellular proteins involved in cell cycle control, DNA replication, and maintenance of genome stability. Activation of CRLs is controlled via neddylation by NEDD8-activating, -conjugating, and -attaching enzymes to the C-terminus of scaffold cullins (CULs), whereas the COP9 signalosome (CSN) inactivates CRLs via deneddylation. Here, we show that the deneddylation rate of each CUL is differentially modulated. Dose- or time-dependent treatment with pevonedistat, a small molecule inhibitor of NEDD8-activating enzyme (NAE), rapidly inhibits neddylation in most CULs, including CUL1, CUL3, CUL4A/B, and CUL5, whereas the deneddylation of CUL2 is slowly increased. We revealed that the different deneddylation speeds of each CUL depend on its binding strength with CSN5, the catalytic core of the CSN complex. Immunoprecipitation analysis revealed that CUL2 has a lower binding affinity for CSN5 than other CULs. Consistently, released cells treated with CSN5 inhibitor showed that CUL2 was slowly converted to the deneddylated form compared to the rapid deneddylation of other CULs. These findings provide mechanistic insights into the different dynamics of CULs in neddylation–deneddylation conversion.

Regulators specifying cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues.

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programmes. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. Notochord and muscle cells stop dividing after eight or nine rounds of cell division depending on their lineages. In the present study, we showed that a Cdk inhibitor, Cdkn1.b, is responsible for stopping cell cycle progression in these lineages. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells continue to divide even after hatching, and Myc is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by transcription factors that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm is under control of transcription factors that specify the developmental fate of mesenchyme and endoderm. Thus, cell fate specification and cell cycle control are linked by these transcription factors.

NPM1 Mutation Reprograms Leukemic Transcription Network Via Reshaping CTCF-Defined TAD Topology

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, it remains poorly understood how NPM1C+ mutation results in altered myeloid master transcription program and HOX expression profile leading to AML disease and whether NPM1 mutant associated transcription signature can be targeted to prevent pathogenesis of acute myeloid leukemia. Here, we report that NPM1C+ reprograms MIZ-1/MYC regulatory axis by altering NPM1-associated CTCF-driven topologically associated domains (TADs) that switches the balance of MIZ1 interaction with coactivator NPM1/p300 and corepressors MYC/G9A complexes to control cell cycle progression and myeloid lineage-specific PU.1/CEBPa transcription networks leading to impairment of myeloid differentiation and leukemic transformation. To test the hypothesis that mislocalization of the NPM1C+ mutant to cytoplasm may alter CTCF mediated three-dimensional (3D) TADs, we carried out Hi-C to examine whether NPM1C+ mutation altered hematopoietic TAD integrity by comparing mouse bone marrow (BM) Lin-cKit+ (LK) stem- and progenitor-enriched cells purified from WT and NPM1C+ knock-in animals, which develop a myeloproliferative neoplasm/myelodsplasia-like condition with a long latency characterized by enlarged spleens, leukocytosis, and thrombocytopenia. Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to altered chromatin accessibility and NPM1 mutant associated gene expression signature, which results in disrupted regulation of the cell cycle and myeloid master transcription factors, and eventually myeloid differentiation block. In contract, Retention of NPM1 in OCI-AML3 nucleus, a NPM1C+ mutant driven AML cells, reverses NPM1C+-driven signature TAD formation and gene transcription leading to proper cell cycle control and myeloid differentiation. Mechanistically, we demonstrated that cytoplasmic mislocalization of NPM1C+ mutant exerts two coordinated effects: (a) alteration of CTCF dependent TAD structure, and (b) MYC-MIZ-1 transcription axis. Both of these regulate target genes critical for cell cycle control and myeloid master transcription factors, Pu.1 and CEBPA, mediated myeloid differentiation. Re-activation of Pu.1 or CEBPa within the nucleus by CRISPR-dCas9 mediated endogenous promoter activation restores myeloid differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators, and prevents NPM1C+-driven gene expression. FinalIy, when we transplanted NPM1OE, activated Pu.1, or activated CEBPA OCI-AML3 cell line or primary NPM1C+ AML patient cells into NSG mice along with control cells, NPM1 overexpression or re-activation of PU.1 or CEBPA in OCI-AML3 cells or NPM1C+ primary AML patient cells prolonged transplanted or PDX mice survival and mitigate AML leukemic blast in transplant or PDX mouse models. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation. Restoration of myeloid transcription program in nucleus reversed NPM1C+-driven transcription signature and promotes myeloid differentiation leading to mitigation of AML.

Combined Inhibition of CDK2 and BCL2 Overrides Resistance to Targeting BTK and CDK4/6 in Mantle Cell Lymphoma Therapy

Mantle cell lymphoma (MCL) remains incurable due to the development of drug resistance, despite the plethora of therapies available. Each successive treatment failure is associated with a more rapidly proliferating disease and fewer treatment options. Understanding the drug-resistance mechanism and developing effective, well-tolerated, new therapies for MCL are urgently needed. Unrestrained proliferation of MCL cells is mainly driven by aberrant cyclin D1 expression and dysregulated CDK4 activity that promotes cell cycle progression from G1 to S. In our phase 1 clinical trial inhibiting CDK4/6 with palbociclib and BTK with ibrutinib (PALIBR) in recurrent MCL, the complete response rate (CR) was 42% compared to 21% in response to ibrutinib alone, despite a comparable 67% overall response rate. Moreover, 5 patients remain on therapy (2 CR and 3 PR) for ~ 8 years, suggesting that CDK4/6 inhibition deepens and prolongs the clinical response to BTK inhibition in MCL. However, resistance emerges in some patients. To elucidate the underpinnings, we undertook integrative longitudinal genomic analysis of sequential tissue and blood specimens (n=53) of 27 MCL patients before, during therapy and on progression, and 4 treatment-naïve MCL patients and 4 normal subjects as controls. Whole exome and whole transcriptome sequencing of freshly isolated MCL cells and B cells revealed that resistance to PALIBR is rooted in homozygous deletion of RB1 (direct target of CDK4/6) or compound copy number variation (cCNV) - concurrent hemizygous loss of RB1 and CDKN2A (p16, CDK4/6 inhibitor) and gain of CDK4. In other resistant patients, the RB protein is silenced by translational termination mutation. Single-cell RNA-sequencing (scRNA-seq, 210,000 cells) of corresponding PBMCs using a unique in-house MCL RNA reference library reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative, expanding with disease progression. RNA velocity analysis further uncovered that C2 fuels the transition to C4 with disease progression, whereas C3 is a terminal node (Fig 1). In C2 and C3 MCL cells, CDK2 is highly elevated, indicating evasion of CDK4/6 inhibition, and PIK3IP1 (negative PI3K regulator) is silenced, suggesting that compensatory PI3K activation in BTKi resistance (Chiron et al, Cancer Discovery 2014) is secondary to loss of cell cycle control. BCL2 is markedly elevated in C2, greater in C3 MCL cells along with increases in anti-apoptotic BCL2L1(BCL-XL) and BCL-2A1 and decreases in pro-apoptotic PMAIP1 (NOXA) expression. This suggests combined CDK2 and BCL2 inhibition as a rational approach to override CDK4/6i and BTKi resistance. Indeed, combined inhibition of CDK2 with PF-07104091 and BCL2 with venetoclax (PF-VEN) cooperatively impaired growth and killed ibrutinib-resistant MAVER-1 cells, more effectively in the isogenic derivative MAVER-1R cells which we generated to recapitulate cCNV in PALIBR resistance - depletion of RB protein through translational termination mutation (W99, AAF 75%) in RB1, loss of CDKN2A and gain of CDK4. Mechanistically, MAVER-1R cells are accelerated in G1 to S progression, based on elevation of CDK2 and TK1 expression and cell cycle profiling. They are intrinsically apoptotic and vulnerable to BCL2 inhibition due to a marked reduction in cells expressing anti-apoptotic BCL2L1 and IRF4, and universal expression of pro-apoptotic PMAIP1 (NOXA). Importantly, combined CDK2 and BCL2 inhibition synergistically kills MCL cells of a patient resistant to PALIBR ex vivo. In summary, by longitudinal genomic analysis, we have provided the first evidence that 1) resistance to CDK4/6i and BTKi stems from MCL intrinsic cCNV; 2) MCL cells comprise 4 major transcriptomically distinct clusters; cluster 2 expressing copious CDK2 and BCL2 is pivotal in fueling proliferation of MCL cells in resistance; 3) cluster 3 cells also accumulate with resistance due to longevity; 4) combined inhibition of CDK2 and BCL2 overrides resistance to CDK4i and BTKi, in MCL cell lines and primary MCL cells from PALIBR resistant patient. Combined inhibition of CDK2 and BCL2, therefore, represents a mechanism-based strategy to overcome CDK4/6i and BTKi resistance in MCL therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PHF6 Represses Leukemia Stem Cell Self-Renewal By Dysregulating Cell Cycle Progression

Cell fate decision and proliferation of hematopoietic stem cells (HSCs) are tightly regulated to maintain a lifelong stem cell pool size while meeting the continuous demand for mature blood cells. In Acute Myeloid Leukemia (AML), mutations in HSC-regulatory genes are thought to disrupt cell cycle control and differentiation, leading to hyperproliferation of immature myeloid cells. The X-chromosome gene PHF6 (PHD-finger protein 6) shows loss-of-function mutations in 3% of AML patients, indicating its role as a leukemia suppressor. PHF6 is a ubiquitously expressed nucleolar and chromatin binding protein with two ePHD domains. Its knockout in mice is known to cause increased HSC self-renewal, but the functional and molecular consequence of its loss in the context of AML are poorly understood. In this study, we characterize the leukemia suppressing function of PHF6 in AML using an in vivo mouse model. Hematopoietic knockout of Phf6 alone is insufficient to cause hematopoietic malignancy, and we therefore used the retroviral HoxA9 mouse model of AML. We transduced cKO (Vav-Cre; Phf6 flox) or Ctrl (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HoxA9 (cKO+HoxA9 and Ctrl+HoxA9) and performed both in vitro and in vivo studies. cKO+HoxA9 cells showed persistent replating ability in vitro compared to Ctrl+HoxA9, with larger colonies on serial replating indicating increased proliferation. On serial transplantation into irradiated recipient mice, we found higher engraftment ability of cKO+HoxA9 leukemic cells, and reduced survival of secondary recipients (Fig 1A). Limiting dilution transplantation showed that Phf6 cKO increased the frequency of Leukemic Stem Cells (LSCs). Thus, loss of PHF6 increases self-renewal of LSCs, and increased rapidity of AML progression with serial transplantation. To further understand the role of PHF6 in LSC self -renewal, we performed RNA-Seq on an LSC-enriched cell population that we phenotypically characterized with C-Kit+, Ly6C-. Gene Set Enrichment Analysis (GSEA) showed that the cKO LSC transcriptome (compared to Ctrl) positively correlated with leukemic stem cell gene sets, and negatively correlated with gene sets for neutrophil and monocytic differentiation (Fig 1B). On in vitro culture of LSCs, we found that Phf6 cKO cells maintained higher self-renewal rate in vitro, with a shorter G1 phase accompanied by prolonged persistence of an undifferentiated LSC population in culture. In summary, we have found that PHF6 represses LSC self-renewal and inhibits AML progression. Deletion of PHF6 leads to shortening of G1 phase of cell cycle, and maintains undifferentiated leukemic cells. Our future work will involve establishing the molecular mechanism by which PHF6 control self-renewal associated cell cycle progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Patterns of interdivision time correlations reveal hidden cell cycle factors.

The time taken for cells to complete a round of cell division is a stochastic process controlled, in part, by intracellular factors. These factors can be inherited across cellular generations which gives rise to, often non-intuitive, correlation patterns in cell cycle timing between cells of different family relationships on lineage trees. Here, we formulate a framework of hidden inherited factors affecting the cell cycle that unifies known cell cycle control models and reveals three distinct interdivision time correlation patterns: aperiodic, alternator, and oscillator. We use Bayesian inference with single-cell datasets of cell division in bacteria, mammalian and cancer cells, to identify the inheritance motifs that underlie these datasets. From our inference, we find that interdivision time correlation patterns do not identify a single cell cycle model but generally admit a broad posterior distribution of possible mechanisms. Despite this unidentifiability, we observe that the inferred patterns reveal interpretable inheritance dynamics and hidden rhythmicity of cell cycle factors. This reveals that cell cycle factors are commonly driven by circadian rhythms, but their period may differ in cancer. Our quantitative analysis thus reveals that correlation patterns are an emergent phenomenon that impact cell proliferation and these patterns may be altered in disease.

High Expression of POGK Predicts Poor Prognosis in Patients with Hepatocellular Carcinoma.

Kruppel-associated box (KRAB) proteins reportedly play a dual role in neoplastic transformation. At present, little is known about the function of the proteins encoded by the human pogo transposable element derived with KRAB domain (POGK) gene. Herein, we evaluated the prognostic significance of POGK expression in patients with hepatocellular carcinoma (HCC). The data of HCC patients was downloaded from The Cancer Genome Atlas (TCGA) database. To determine the relationship between POGK and clinical features, logistic regression was applied. Cox regression and Kaplan-Meier analyses were used to evaluate the correlation between POGK and survival rates. Gene ontology (GO) analysis and Gene set enrichment analysis (GSEA) were conducted to identify the enriched pathways and functions associated with POGK. A total of 374 HCC patients were identified in TCGA. POGK was significantly upregulated in HCC and correlated with tumor status (p = 0.036), race (p = 0.025), weight (p = 0.002), body mass index (p = 0.033), histologic grade (p < 0.001), and alpha-fetoprotein (p < 0.001). High POGK expression in HCC patients correlated with a poor outcome in terms of overall survival (p = 0.0018), progression-free survival (p = 0.0087), relapse-free survival (p = 0.045), and disease-specific survival (p = 0.014), according to Kaplan-Meier analysis. Receiver operating characteristic curve analysis showed that the area under the curve of POGK expression for HCC diagnosis was 0.891. GSEA showed that high POGK expression might activate mitotic prometaphase, kinesins, homologous DNA pairing and strand exchange, MET activates PTK2 signaling pathway, G1 to S cell cycle control, Aurora B pathway, ncRNAs involved in WNT signaling pathway, hepatitis C, and ncRNAs involved in the STAT3 signaling pathway. POGK expression correlated with the abundance of adaptive and innate immunocytes in HCC. High expression of POGK has high diagnostic and prognostic values in patients with HCC. Moreover, POGK expression is correlated with immune infiltration in HCC.

EXTH-95. UNCOVERING THERAPEUTIC VULNERABILITIES IN MISMATCH REPAIR-DEFICIENT GLIOMAS

Abstract Approximately 25% of recurrent gliomas exhibit hypermutation, which is most often acquired in chemotherapy sensitive gliomas post-treatment with standard of care alkylating agent temozolomide, Intriguingly, nearly all of these recurrent hypermutant tumors harbor deficiencies in mismatch repair (MMR) genes. Unlike other MMR-deficient cancers, hypermutant gliomas do not exhibit detectable microsatellite instability (MSI) at the population level, and do not share similar dependencies. Thus, strategies to therapeutically target MMR-deficient and hypermutant gliomas are urgently needed, and likely to impact many patients. To evaluate vulnerabilities associated with MMR-deficient gliomas, we have generated isogenic MMR-deficient models by systematic ablation of core MMR genes MSH2, MSH6, MLH1, and PMS2, in multiple patient-derived glioma cell lines using a robust all-in-one CRISPR-Cas9 system. We characterized these isogenic MMR-deficient glioma lines in comparison to respective MMR-proficient control line by performing differential gene expression analysis and subsequent gene set enrichment analyses. Our analyses reveal in an unbiased fashion an enrichment of several hallmark gene sets including cell cycle control and DNA repair in the MMR-deficient cell lines. We performed a CRISPR-Cas9 knockout screen in these MMR deficient and hypermutant models and identified candidate genes involved in DNA repair, cell cycle, RNA metabolism and other pathways as preferential dependencies in the MMR-deficient glioma cells. We also screened multiple MMR-deficient isogenic models against the high-throughput drug repurposing (REPO) chemical library and our preliminary results reveal a number of compounds from a variety of drug classes that selectively kill the MMR-deficient glioma cells, indicating that the loss of MMR confers differential dependencies to these small molecule inhibitors. The candidate drugs and genes identified from these high-throughput assays are currently being subjected to further validation, and they possibly will help us identify novel therapeutic strategies or improve existing therapeutic strategies to target recurrent MMR-deficient gliomas.

Quantifying and mathematical modelling of the influence of soluble adenylate cyclase on cell cycle in human endothelial cells with Bayesian inference.

Adenosine-3', 5'-cyclic monophosphate (cAMP) produced by adenylate cyclases (ADCYs) is an established key regulator of cell homoeostasis. However, its role in cell cycle control is still controversially discussed. This study focussed on the impact of soluble HCO3 - -activated ADCY10 on cell cycle progression. Effects are quantified with Bayesian inference integrating a mathematical model and experimental data. The activity of ADCY10 in human umbilical vein endothelial cells (HUVECs) was either pharmacologically inhibited by KH7 or endogenously activated by HCO3 - . Cell numbers of individual cell cycle phases were assessed over time using flow cytometry. Based on these numbers, cell cycle dynamics were analysed using a mathematical model. This allowed precise quantification of cell cycle dynamics with model parameters that describe the durations of individual cell cycle phases. Endogenous inactivation of ADCY10 resulted in prolongation of mean cell cycle times (38.7 ± 8.3h at 0 mM HCO3 - vs 30.3 ± 2.7h at 24 mM HCO3 - ), while pharmacological inhibition resulted in functional arrest of cell cycle by increasing mean cell cycle time after G0 /G1 synchronization to 221.0 ± 96.3h. All cell cycle phases progressed slower due to ADCY10 inactivation. In particular, the G1 -S transition was quantitatively the most influenced by ADCY10. In conclusion, the data of the present study show that ADCY10 is a key regulator in cell cycle progression linked specifically to the G1 -S transition.

Molecular phenotyping uncovers differences in basic housekeeping functions among closely related species of hares (Lepus spp., Lagomorpha: Leporidae).

Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.

Arabidopsis thaliana sirtuins control proliferation and glutamate dehydrogenase activity

Sirtuins are part of a gene family of NAD-dependent deacylases that act on histone and non-histone proteins and control a variety of activities in all living organisms. Their roles are mainly related to energy metabolism and include lifetime regulation, DNA repair, stress resistance, and proliferation. A large amount of knowledge concerning animal sirtuins is available, but data about their plant counterparts are scarce. Plants possess few sirtuins that have, like in animals, a recognized role in stress defense and metabolism regulation. However, engagement in proliferation control, which has been demonstrated for mammalian sirtuins, has not been reported for plant sirtuins so far. In this work, srt1 and srt2 Arabidopsis mutant seedlings have been used to evaluate in vivo the role of sirtuins in cell proliferation and regulation of glutamate dehydrogenase, an enzyme demonstrated to be involved in the control of cell cycle in SIRT4-defective human cells. Moreover, bioinformatic analyses have been performed to elucidate sequence, structure, and function relationships between Arabidopsis sirtuins and between each of them and the closest mammalian homolog. We found that cell proliferation and GDH activity are higher in mutant seedlings, suggesting that both sirtuins exert a physiological inhibitory role in these processes. In addition, mutant seedlings show plant growth and root system improvement, in line with metabolic data. Our data also indicate that utilization of an easy to manipulate organism, such as Arabidopsis plant, can help to shed light on the molecular mechanisms underlying the function of genes present in interkingdom species.

Simulated Microgravity Modulates Focal Adhesion Gene Expression in Human Neural Stem Progenitor Cells.

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

Histone deacetylases 1 and 2 target gene regulatory networks of nephron progenitors to control nephrogenesis

Our studies demonstrated the critical role of Histone deacetylases (HDACs) in the regulation of nephrogenesis. To better understand the key pathways regulated by HDAC1/2 in early nephrogenesis, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) of HDAC1/2 on isolated nephron progenitor cells (NPCs) from mouse E16.5 kidneys. Our analysis revealed that 11,802 (40.4%) of HDAC1 peaks overlap with HDAC2 peaks, further demonstrates the redundant role of HDAC1 and HDAC2 during nephrogenesis. Common HDAC1/2 peaks are densely concentrated close to the transcriptional start site (TSS). GREAT Gene Ontology analysis of overlapping HDAC1/2 peaks reveals that HDAC1/2 are associated with metanephric nephron morphogenesis, chromatin assembly or disassembly, as well as other DNA checkpoints. Pathway analysis shows that negative regulation of Wnt signaling pathway is one of HDAC1/2′s most significant function in NPCs. Known motif analysis indicated that Hdac1 is enriched in motifs for Six2, Hox family, and Tcf family members, which are essential for self-renewal and differentiation of nephron progenitors. Interestingly, we found the enrichment of HDAC1/2 at the enhancer and promoter regions of actively transcribed genes, especially those concerned with NPC self-renewal. HDAC1/2 simultaneously activate or repress the expression of different genes to maintain the cellular state of nephron progenitors. We used the Integrative Genomics Viewer to visualize these target genes associated with each function and found that HDAC1/2 co-bound to the enhancers or/and promoters of genes associated with nephron morphogenesis, differentiation, and cell cycle control. Taken together, our ChIP-Seq analysis demonstrates that HDAC1/2 directly regulate the molecular cascades essential for nephrogenesis.

ERH: a plug-and-play protein important for gene silencing and cell cycle progression.

In metazoans, most proteins have pleiotropic cellular functions and have the ability to interact with several factors to accomplish these different functions. This is the case of eukaryotic ERH proteins, a family of homodimeric proteins involved in DNA replication and cell cycle control as well as in gene silencing by contributing either to the biogenesis of small interference RNAs (miRNAs, piRNAs) or to the recruitment of RNA decay machineries. Very recently, several crystal structures describing complexes formed by eukaryotic ERH proteins and several small peptides from various partners have highlighted the existence of different binding sites on the surface of ERH proteins. In this issue of The FEBS Journal, Wang etal. present the crystal structure of the complex formed between the human ERH protein and a short peptide of the CIZ1 protein, one of its partners. Altogether, this information will be particularly important for future studies aimed at dissecting the different biological functions governed by this family of highly conserved proteins. Comment on: https://doi.org/10.1111/febs.16611.

Age-related SUMOylation of PLK1 is essential to meiosis progression in mouse oocytes.

Polo like kinase 1 (PLK1) is a protein kinase involved in regulating the spindle assembly and cell cycle control in mammalian oocytes. SUMOylation, one way of post-translational modification, regulates oocyte meiosis by controlling several substrates. However, the relation between PLK1 and SUMOylation in oocytes is still unknown. In this study, we investigated that whether PLK1 was modified by SUMOylation in oocytes and its potential relationship with age-related meiotic abnormalities. We showed that PLK1 had colocalization and protein interaction with Small Ubiquitin-Like Modifier (SUMO)-1 and SUMO-2/3 in mouse oocytes, indicating that PLK1 could be modified by SUMO-1 and SUMO-2/3. Overexpression of PLK1 SUMOylation site mutants PLK1K178R and PLK1K191R caused the increase of the abnormal spindle rate of oocytes and the decline of the first polar body extrusion rate with the abnormal localization of PLK1, suggesting that the SUMOylation modification of PLK1 is essential for normal meiosis in oocytes. Compared with young mice, the expression of PLK1 protein increased and the expression of SUMO-1 and SUMO-2/3 protein decreased in the oocytes of aged mice, indicating that the SUMOylation of PLK1 might be related to the mouse aging. Therefore, our data suggested that PLK1 could be SUMOylated by SUMO-1 and SUMO-2/3 in mouse oocytes and SUMOylation of PLK1 regulated the meiosis progression of oocytes which was related with aging.

1. Microdeletion 13q12.2 in B cell acute lymphoblastic leukemia: Little but important!

Chromosomal microarray analysis (CMA) is instrumental for identification of recurrent focal deletions in patients with B cell acute lymphoblastic leukemia (B-ALL). These microdeletions include genes involved in B cell differentiation, cell cycle control and transcriptional regulation often with prognostic or therapeutic significance. Recently, a 13q12.2 microdeletion with breakpoints upstream of FLT3 and within intronic regions of the PAN3 gene was reported. Functionally, this microdeletion resulted in enhancer hijacking and constitutive activation of FLT3 in pediatric B-ALL. Seven institutions performed internal database searches to identify similar deletions. In total, 10 patients were identified, all with B-ALL, confirming the specificity for this diagnosis. This cohort included seven pediatric cases (3 male, 4 female; ages 2-14 yo, mean 5) and 3 adult cases (1 male, 2 female; ages 22, 39, 42). Subtypes included two high hyperdiploid, one hypodiploid, one iAMP21, one P2RY8::CLRF, one unbalanced rearrangement involving TFE3, one PAX5::PML, three B-ALL NOS.? Deletions ranged in size from?6.7 kb ? 136 kb (mean 76 kb); each with proximal breakpoints between FLT3 and PAN3 and 9/10 with distal breakpoints within intron 5 of the PAN3 gene. Our study is the first to report the microdeletion in adults, and unlike the discovery cohorts, our patients showed no sex bias. Our data does not confirm a strong association of the deletion with high hyperdiploidy, since 8/10 had other B-ALL subtypes. Constitutive activation of FLT3 may have significant clinical implications for prognosis and treatment; thus, detection of 13q12.2 microdeletion at diagnosis or disease relapse is paramount.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

Heat-Shock Proteins.

Heat-shock proteins (HSPs), or stress proteins, are abundant and highly conserved, present in all organisms and in all cells. Selected HSPs, also known as chaperones, play crucial roles in folding and unfolding of proteins, assembly of multiprotein complexes, transport and sorting of proteins into correct subcellular compartments, cell-cycle control and signaling, and protection of cells against stress and apoptosis. More recently, HSPs have been shown to be key players in immune responses: during antigen presentation as well as cross-priming, they chaperone and transfer antigenic peptides to class I and class II molecules of the major histocompatibility complexes. In addition, extracellular HSPs can stimulate and cause maturation of professional antigen-presenting cells of the immune system, such as macrophages and dendritic cells. They also chaperone several toll-like receptors, which play a central role in innate immune responses. HSPs constitute a large family of proteins that are often classified based on their molecular weight as Hsp10, Hsp40, Hsp60, Hsp70, Hsp90, etc. This unit contains a table that lists common HSPs and summarizes their characteristics including (a) name, (b) subcellular localization, (c) known function, (d) chromosome assignment, (e) brief comments, and (f) references. © 2022 Wiley Periodicals LLC.

Cyclin D1 mediates pain behaviour in a rat model of breast cancer-induced bone pain by a mechanism involving regulation of the proliferation of spinal microglia.

The involvement of cyclin D1 in the proliferation of microglia, and the generation and maintenance of bone cancer pain (BCP), have not yet been clarified. We investigated the expression of microglia and cyclin D1, and the influences of cyclin D1 on pain threshold. Female Sprague Dawley (SD) rats were used to establish a rat model of BCP, and the messenger RNA (mRNA) and protein expression of ionized calcium binding adaptor molecule 1 (IBA1) and cyclin D1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. The proliferation of spinal microglia was detected by immunohistochemistry. The pain behaviour test was assessed by quantification of spontaneous flinches, limb use, and guarding during forced ambulation, mechanical paw withdrawal threshold, and thermal paw withdrawal latency. IBA1 and cyclin D1 in the ipsilateral spinal horn increased in a time-dependent fashion. Spinal microglia proliferated in BCP rats. The microglia inhibitor minocycline attenuated the pain behaviour in BCP rats. The cyclin-dependent kinase inhibitor flavopiridol inhibited the proliferation of spinal microglia, and was associated with an improvement in pain behaviour in BCP rats. Our results revealed that the inhibition of spinal microglial proliferation was associated with a decrease in pain behaviour in a rat model of BCP. Cyclin D1 acts as a key regulator of the proliferation of spinal microglia in a rat model of BCP. Disruption of cyclin D1, the restriction-point control of cell cycle, inhibited the proliferation of microglia and attenuated the pain behaviours in BCP rats. Cyclin D1 and the proliferation of spinal microglia may be potential targets for the clinical treatment of BCP.Cite this article: Bone Joint Res2022;11(11):803-813.