Functional analysis of the Cdk7/cyclin H/Mat1 complex in mouse embryonic stem cells and embryos

The trimeric Cdk7/cyclin H/Mat1 complex functions in cell cycle regulation, as the Cdk-activating kinase (CAK), and in transcription, as a module of the general transcription factor TFIIH. As a component of TFIIH, Cdk7 phosphorylates serine-5 and serine-7 of the carboxy-terminal domain (CTD) of RNA polymerase II and can also directly phosphorylate transcription factors to regulate gene expression. Here we have investigated the function of the Cdk7/cyclin H/Mat1 complex in murine embryonic stem (ES) cells and preimplantation embryos to determine whether it regulates the unique cell cycle structure and transcriptional network of pluripotent cells. We demonstrate that depletion of cyclin H leads to differentiation of ES cells independent of changes in cell cycle progression. In contrast, we observed that developmental genes are acutely upregulated after cyclin H downregulation, likely perturbing normal ES self-renewal pathways. Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass (ICM) of blastocysts, a transient pluripotent stem cell population in vivo. Thus, cyclin H has an essential function in promoting the self-renewal of pluripotent stem cells from blastocyst-stage embryos. Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development

PARP Inhibitors in Small-Cell Lung Cancer: Rational Combinations to Improve Responses

Despite recent advances in first-line treatment for small-cell lung cancer (SCLC), durable responses remain rare. The DNA repair enzyme poly-(ADP)-ribose polymerase (PARP) was identified as a therapeutic target in SCLC using unbiased preclinical screens and confirmed in human and mouse models. Early trials of PARP inhibitors, either alone or in combination with chemotherapy, showed promising but limited responses, suggesting that selecting patient subsets and treatment combinations will prove critical to further clinical development. Expression of SLFN11 and other components of the DNA damage response (DDR) pathway appears to select for improved responses. Combining PARP inhibitors with agents that damage DNA and inhibit DDR appears particularly effective in preclinical and early trial data, as well as strategies that enhance antitumor immunity downstream of DNA damage. A robust understanding of the mechanisms of DDR in SCLC, which exhibits intrinsic replication stress, will improve selection of agents and predictive biomarkers. The most effective combinations will target multiple nodes in the DNA damage/DDR/immune activation cascade to minimize toxicity from synthetic lethality

Mammalian ASH1L Is a Histone Methyltransferase That Occupies the Transcribed Region of Active Genes▿ †

Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, we examined ASH1L, the mammalian homolog of the Drosophila melanogaster Trithorax group (TrxG) protein Ash1. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. Here we demonstrate that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines we found that their recruitments occur independently of each other. Collectively, our data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes

Psychiatric disorders and risk of transition to chronicity in men with first onset low back pain

OBJECTIVE: To assess whether pre-existing psychiatric diagnoses increase the likelihood of transitioning from sub-acute to chronic back pain. DESIGN: Prospective cohort study. METHODS: Men (N = 140) experiencing a first onset of low back pain (LBP) were examined for lifetime psychiatric disorders approximately 8 weeks post pain-onset using the Diagnostic Interview Schedule (DIS-III-R), then re-evaluated at 6 months after pain onset to determine who did or did not progress to pain chronicity. OUTCOME MEASURE: Transition to chronic pain and disability was based on 6-month self-report measures of pain intensity and perceived disability. RESULTS: Men with a pre-pain lifetime diagnosis of major depressive disorder had 5 times greater risk of transitioning to chronic LBP (odds ratio [OR] = 4.99; 95% confidence interval [CI] 1.49-16.76). Increased risk was also associated with a pre-pain lifetime diagnosis of generalized anxiety (OR = 2.45; 95% CI 1.06-5.68), post-traumatic stress (OR = 3.23; 95% CI 1.11-9.44), and with current nicotine dependence (OR = 2.49; 95% CI 1.15-5.40). There were no statistically significant effects for abuse or dependence of alcohol or other psychoactive substances. DISCUSSION: Lifetime history of major depression or a major anxiety disorder may represent potential psychosocial yellow flags for the transition to chronicity in men with first-onset LBP. Screening for lifetime depressive or anxiety disorders may identify individuals at higher risk, who may benefit from referral for more intensive rehabilitation

Hypoxia-inducible factor 2α regulates macrophage function in mouse models of acute and tumor inflammation

Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1α and HIF-2α, and overexpression of HIF-2α in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2α during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1α versus HIF-2α must be elucidated. We demonstrate here that mice lacking HIF-2α in myeloid cells (Hif2aΔ/Δ mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2α directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aΔ/Δ mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2α modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2α as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer