School engagement, school climate and youth externalizing behaviors: direct and indirect effects of parenting practices.

This research aimed to explore the direct and indirect effects of students' school engagement, school climate and parenting practices on youth's externalizing behaviors. A quantitative methodology with a sample of 183 Portuguese students, aged between 11 and 16 years old, was used. The main results suggested negative associations between externalizing behaviors and higher levels of school engagement and positive school climate. Poor parental supervision, inconsistent discipline and corporal punishment were positively related with externalizing behaviors, contrary to parental involvement and positive parenting that were associated with lower levels of externalizing behaviors. However, negative parenting practices were associated with lower levels of school engagement. Additionally, the results indicated that parenting practices might influence youth's externalizing behaviors through school engagement.

GSK-3484862 targets DNMT1 for degradation in cells.

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner.

Methyl-lysine readers PHF20 and PHF20L1 define two distinct gene expression-regulating NSL complexes.

The methyl-lysine readers plant homeodomain finger protein 20 (PHF20) and its homolog PHF20-like protein 1 (PHF20L1) are known components of the nonspecific lethal (NSL) complex that regulates gene expression through its histone acetyltransferase activity. In the current model, both PHF homologs coexist in the same NSL complex, although this was not formally tested; nor have the functions of PHF20 and PHF20L1 regarding NSL complex integrity and transcriptional regulation been investigated. Here, we perform an in-depth biochemical and functional characterization of PHF20 and PHF20L1 in the context of the NSL complex. Using mass spectrometry, genome-wide chromatin analysis, and protein-domain mapping, we identify the existence of two distinct NSL complexes that exclusively contain either PHF20 or PHF20L1. We show that the C-terminal domains of PHF20 and PHF20L1 are essential for complex formation with NSL, and the Tudor 2 domains are required for chromatin binding. The genome-wide chromatin landscape of PHF20-PHF20L1 shows that these proteins bind mostly to the same genomic regions, at promoters of highly expressed/housekeeping genes. Yet, deletion of PHF20 and PHF20L1 does not abrogate gene expression or impact the recruitment of the NSL complex to those target gene promoters, suggesting the existence of an alternative mechanism that compensates for the transcription of genes whose sustained expression is important for critical cellular functions. This work shifts the current paradigm and lays the foundation for studies on the differential roles of PHF20 and PHF20L1 in regulating NSL complex activity in physiological and diseases states.

Safety and tolerability of lurbinectedin (PM01183) in patients with acute myeloid leukemia and myelodysplastic syndrome.

Trabectedin is an FDA-approved DNA minor groove binder that has activity against translocation-associated sarcomas. Lurbinectedin is a next-generation minor groove binder with preclinical activity against myeloid leukemia cells. A dose-finding phase 1 clinical trial was performed in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with further assessment of safety and tolerability. Forty-two patients with relapsed/refractory AML/MDS received lurbinectedin administered as a 1-hour intravenous infusion in a 3 + 3 study design. Two dosing schedules were used: 3.5, 5, 7, or 6 mg on days 1 and 8 or 2, 3, 1, or 1.5 mg for 3 consecutive days on days 1 to 3. Three patients experienced dose-limiting toxicities of rhabdomyolysis (grade 4), hyperbilirubinemia (grade 3), and oral herpes (grade 3) with the day 1 and 8 schedule. Otherwise, adverse events mainly consisted of gastrointestinal manifestations (n = 11), febrile neutropenia/infections (n = 4), pulmonary toxicity (n = 2), and renal failure (n = 2). The most common laboratory abnormalities observed were an increase in creatinine (93%) and anemia, neutropenia, and thrombocytopenia (100%). Overall, 33 of 42 patients (79%) had reduction in blasts in peripheral blood or bone marrow. One patient achieved a partial response and 2 patients a morphologic leukemia-free state. Most (n = 30, 71%) were discontinued due to progressive disease. Early deaths occurred from disease-related causes that were not attributable to lurbinectedin. Four patients with a chromosome 11q21-23 abnormality had significantly greater bone marrow blast reduction than those without such abnormality, with decrease of 31 ± 14% (n = 4) vs 8 ± 8% (n = 16), respectively (P = .04). Overall, lurbinectedin was safe and tolerated using the schedules and dose levels tested. While no sustained remissions were observed, single-agent lurbinectedin was transiently leukemia suppressive for some patients.

Histone modifications and the DNA double-strand break response.

The timely and precise repair of DNA damage, or more specifically DNA double-strand breaks (DSBs) - the most deleterious DNA lesions, is crucial for maintaining genome integrity and cellular homeostasis. An appropriate cellular response to DNA DSBs requires the integration of various factors, including the post-translational modifications (PTMs) of chromatin and chromatin-associated proteins. Notably, the PTMs of histones have been shown to play a fundamental role in initiating and regulating cellular responses to DNA DSBs. Here we review the role of the major histone PTMs, including phosphorylation, ubiquitination, methylation and acetylation, and their interactions during DNA DSB-induced responses.

Histone methylation regulator PTIP is required to maintain normal and leukemic bone marrow niches.

The bone is essential for locomotion, calcium storage, and harboring the hematopoietic stem cells (HSCs) that supply the body with mature blood cells throughout life. HSCs reside at the interface of the bone and bone marrow (BM), where active bone remodeling takes place. Although the cellular components of the BM niche have been characterized, little is known about its epigenetic regulation. Here we find that the histone methylation regulator PTIP (Pax interaction with transcription-activation domain protein-1) is required to maintain the integrity of the BM niche by promoting osteoclast differentiation. PTIP directly promotes chromatin changes required for the expression of Pparγ (peroxisome proliferator-activated receptor-γ), a transcription factor essential for osteoclastogenesis. PTIP deletion leads to a drastic reduction of HSCs in the BM and induces extramedullary hematopoiesis. Furthermore, exposure of acute myeloid leukemia cells to a PTIP-deficient BM microenvironment leads to a reduction in leukemia-initiating cells and increased survival upon transplantation. Taken together, our data identify PTIP as an epigenetic regulator of osteoclastogenesis that is required for the integrity of the BM niche to sustain both normal hematopoiesis and leukemia.

Genetic rescue of lineage-balanced blood cell production reveals a crucial role for STAT3 antiinflammatory activity in hematopoiesis.

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage- Sca-1+ c-Kit+ CD150+ CD48- HSPC subset (LSK CD150+ CD48- cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48- cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.