FOXD1 regulates cell division in clear cell renal cell carcinoma

Background Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported. Methods Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database. Results FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation. Conclusions We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.

DIPG-76. HISTONE H3 PHOSPHORYLATION IN H3K27M MIDLINE GLIOMAS

Diffuse midline gliomas (DMG) patients have a dire prognosis despite radiation therapy and there is an urgent need to develop more effective treatments. DMG are characterized by heterozygous mutations in select H3 genes resulting in the replacement of lysine 27 by methionine (K27M) that leads to global epigenetic reprogramming and drives tumorigenesis. We previously reported that pharmacological inhibition of aurora kinase (AKI) may represent a targeted approach for treating tumors with this mutation. Our analysis with both published dataset and patient samples showed that patients with higher aurora kinase A (AKA) expression were associated with worse survival. AKA phosphorylates H3S10 and H3S28 during mitosis. Intriguingly, phosphorylation of the H3S28 (H3S28ph) by AKA blocks PRC2 methyltransferase activity and decreases global H3K27me3 in certain stem cells. We propose that a similar mechanism occurs in H3K27M DMG tumors, where there is a reciprocal relationship between H3S28ph and H3K27me3. We found that AKI significantly decreases H3S28ph while increasing H3K27me3 specifically in H3K27M tumors. To further evaluate the link between the H3K27M mutation and H3 serine phosphorylation, we used CRISPR/Cas9-directed gene editing to silence H3S28ph by replacing serine with alanine (H3S28A) in DIPG cell lines. Ectopic expression of histone H3S28A leads to a prominent epigenetic changes in H3K27M tumors and is similar to AKA inhibition. Overall, this study highlights H3S28ph, one of the targets of AK, is a key driver of epigenetic changes in H3K27M tumors through both direct and indirect changes to H3K27me3 and H3K27ac across the genome.

EFFECT OF LONG-TERM EMOTIONALPAINFUL STRESS ON THE HISTONE H3 PHOSPHORYLATION IN THE MEDIAL PREFRONTAL CORTEX AND BASOLATERAL AMYGDALA OF RATS WITH GENETIC DIFFERENCES IN EXCITABILITY OF THE NERVOUS SYSTEM

Цель - исследование влияния длительного эмоционально-болевого стрессорного воздействия (ДЭБС) в разные сроки после его окончания (24 ч, 2 нед, 2 мес) на фосфорилирование гистона Н3 (по серину 10) (phH3-Ser10) в клетках медиальной префронтальной коры (мПК) и базолатеральной области амигдалы (блА) у крыс двух линий с разным порогом возбудимости нервной системы к электрическому току (генетическая модель постстрессорных тревожно-депрессивных расстройств). Материал и методы. С использованием иммуногистохимического метода исследована иммунореактивность клеток мПК и блА к phH3-Ser10 у крыс 2 селекционных линий: низковозбудимых с высоким порогом возбудимости нервной системы (линия ВП) и высоковозбудимых с низким порогом возбудимости (линия НП). В качестве стрессора применяли длительное (15 сут) эмоционально-болевое воздействие по схеме К. Гехта. Результаты. У низковозбудимых крыс линии ВП в блА обнаружен более высокий базовый уровень phH3-Ser10 по сравнению с высоковозбудимыми крысами линии НП. В мПК межлинейных различий в базовом уровне phH3-Ser10 не обнаружено. Выявлено влияние ДЭБС на уровень phH3-Ser10 у крыс обеих линий. Показано кратковременное (через 24 ч) повышение phH3-Ser10 в мПК у крыс линии НП и устойчивое (до 2 мес после ДЭБС) у животных линии ВП. В блА только у высоковозбудимых крыс линии НП обнаружено индуцируемое ДЭБС возрастание и устойчивое до 2 мес сохранение уровня phH3-Ser10. Выводы. Выявлены долговременные изменения фосфорилирования гистона Н3, имеющие структурную специфичность и зависящие от генетически детерминированного функционального состояния нервной системы крыс. Objective - to study the effect of the long-term emotional-painful stress on the level of histone H3 phosphorylation at Ser10 (phH3-Ser10) in the medial prefrontal cortex (mPC) and basolateral amygdala (BLA) in the rats of two strains characterized by different excitability of the nervous system in normal conditions and at various intervals (24 hours, 2 weeks, 2 months) after the long-term emotional-painful stress (LEPS). Material and methods. The immunoreactivity of mPC and BLA cells to phH3-Ser10 was studied using the immunohistochemical method. The objects of investigation were selected rat strains: НТ (high threshold, low excitability of the nervous system) and LT (low threshold, high excitability of the nervous system). A long-term (15 days) exposure to emotional-painful stress according to K. Hecht’s scheme was used. Results. Intact rats with low nervous excitability (HT strain - high threshold) demonstrated more high basal level of phH3Ser10 in BLA cells than rats with high excitability (LT strain - low threshold). No differences in basal level of phH3-Ser10 between two rat strains were found. The exposure to emotional- painful stress caused alterations in the level of phH3-Ser10 in rats from both strains. Increase of phH3-Ser10 level in the mPC was short-term (24h after LEPS) in LT rats and long-term (up to 2 months) in HT rats. The long-term (up to 2 months) increase of phH3-Ser10 level after stress in the BLA was discovered in LT rats only. Conclusions. Long-term changes in histone H3 phosphorylation, which have structural specificity and depend on genetically determined functional state of rats nervous system, were revealed.

Helicase LSH/Hells regulates kinetochore function, histone H3/Thr3 phosphorylation and centromere transcription during oocyte meiosis

Centromeres are epigenetically determined nuclear domains strictly required for chromosome segregation and genome stability. However, the mechanisms regulating centromere and kinetochore chromatin modifications are not known. Here, we demonstrate that LSH is enriched at meiotic kinetochores and its targeted deletion induces centromere instability and abnormal chromosome segregation. Superresolution chromatin analysis resolves LSH at the inner centromere and kinetochores during oocyte meiosis. LSH knockout pachytene oocytes exhibit reduced HDAC2 and DNMT-1. Notably, mutant oocytes show a striking increase in histone H3 phosphorylation at threonine 3 (H3T3ph) and accumulation of major satellite transcripts in both prophase-I and metaphase-I chromosomes. Moreover, knockout oocytes exhibit centromere fusions, ectopic kinetochore formation and abnormal exchange of chromatin fibers between paired bivalents and asynapsed chromosomes. Our results indicate that loss of LSH affects the levels and chromosomal localization of H3T3ph and provide evidence that, by maintaining transcriptionally repressive heterochromatin, LSH may be essential to prevent deleterious meiotic recombination events at repetitive centromeric sequences.

Sangivamycin and its derivatives inhibit Haspin-Histone H3-survivin signaling and induce pancreatic cancer cell death

Current treatment options for patients with pancreatic cancer are suboptimal, resulting in a five year survival rate of about 9%. Difficulties with treatment are due to an immunosuppressive, fibrotic tumor microenvironment that prevents drugs from reaching tumor cells, but also to the limited efficacy of existing FDA-approved chemotherapeutic compounds. We here show that the nucleoside analog Sangivamycin and its closely-related compound Toyocamycin target PDA cell lines, and are significantly more efficient than Gemcitabine. Using KINOMEscan screening, we identified the kinase Haspin, which is overexpressed in PDA cell lines and human PDA samples, as a main target for both compounds. Inhibition of Haspin leads to a decrease in Histone H3 phosphorylation and prevents Histone H3 binding to survivin, thus providing mechanistic insight of how Sangivamycin targets cell proliferation, mitosis and induces apoptotic cell death. In orthotopically implanted tumors in mice, Sangivamycin was efficient in decreasing the growth of established tumors. In summary, we show that Sangivamycin and derivatives can be an efficient new option for treatment of PDA.