SUMMARY The importance of the p53 protein in the cellular response to DNA damage is well known, but its function during steady-state hematopoiesis has not been established. We have defined a critical role of p53 in regulating hematopoietic stem cell quiescence, especially in promoting the enhanced quiescence seen in HSCs that lack the MEF/ELF4 transcription factor. Transcription profiling of HSCs isolated from wild type and p53 null mice identified Gfi-1 and Necdin as p53 target genes and using lentiviral vectors to upregulate or knockdown the expression of these genes, we show their importance in regulating HSC quiescence. Establishing the role of p53 (and its target genes) in controlling the cell cycle entry of HSCs may lead to therapeutic strategies capable of eliminating quiescent cancer (stem) cells.

Background: Accumulating evidence proposed Janus-associated kinase (JAK) inhibitors as therapeutic targets warranting rapid investigation. Objective: This study evaluated the efficacy and safety of ruxolitinib, a JAK1/2 inhibitor, for coronavirus disease 2019. Methods: We conducted a prospective, multicenter, single-blind, randomized controlled phase II trial involving patients with severe coronavirus disease 2019. Results: Forty-three patients were randomly assigned (1:1) to receive ruxolitinib plus standard-of-care treatment (22 patients) or placebo based on standard-of-care treatment (21 patients). After exclusion of 2 patients (1 ineligible, 1 consent withdrawn) from the ruxolitinib group, 20 patients in the intervention group and 21 patients in the control group were included in the study. Treatment with ruxolitinib plus standard-of-care was not associated with significantly accelerated clinical improvement in severe patients with coronavirus disease 2019, although ruxolitinib recipients had a numerically faster clinical improvement. Eighteen (90%) patients from the ruxolitinib group showed computed tomography improvement at day 14 compared with 13 (61.9%) patients from the control group (P 5 .0495). Three patients in the control group died of respiratory failure, with 14.3% overall mortality at day 28; no patients died in the ruxolitinib group. Ruxolitinib was well tolerated with low toxicities and no new safety signals. Levels of 7 cytokines were significantly decreased in the ruxolitinib group in comparison to the control group. Conclusions: Although no statistical difference was observed, ruxolitinib recipients had a numerically faster clinical improvement. Significant chest computed tomography improvement, a faster recovery from lymphopenia, and favorable side-effect profile in the ruxolitinib group were encouraging and informative to future trials to test efficacy of ruxolitinib in a larger population. (

DNA and histone modifications exhibit noticeable impacts on gene expression 1 . Being the most prevalent internal modification in mRNA, N 6 -Methyladenosine (m 6 A) mRNA modification emerges as an important post-transcriptional mechanism of gene regulation 2 - 4 and plays critical roles in various normal and pathological bioprocesses 5 - 12 . However, how m 6 A is precisely and dynamically deposited in the transcriptome remains elusive. Here we report that H3K36me3 histone modification, a marker for transcription elongation, globally guides m 6 A modification. We found that m 6 A modifications enrich in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a critical component of the m 6 A methyltransferase complex (MTC), which in turn facilitates the binding of the m 6 A MTC to adjacent RNA polymerase II, and thereby delivering the m 6 A MTC to actively transcribed nascent RNAs to deposit m 6 A co-transcriptionally. In mouse embryonic stem cells, phenocopying Mettl14 silencing, H3K36me3 depletion also induces m 6 A reduction transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the critical roles of H3K36me3 and METTL14 in determining precise and dynamic m 6 A deposition in mRNA, and uncover another layer of gene expression regulation involving crosstalk between histone modification and RNA methylation.

Cleidocranial dysplasia (CCD), an autosomal-dominant human bone disease, is thought to be caused by heterozygous mutations in runt-related gene 2 (RUNX2)͞polyomavirus enhancer binding protein 2␣A (PEBP2␣A)͞core-binding factor A1 (CBFA1). To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of RUNX2, CCD␣A376, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We show that signal transducers of transforming growth factor ␤ superfamily receptors, Smads, interact with RUNX2 in vivo and in vitro and enhance the transactivation ability of this factor. The truncated RUNX2 protein failed to interact with and respond to Smads and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts on stimulation by bone morphogenetic protein. Therefore, the pathogenesis of CCD may be related to the impaired Smad signaling of transforming growth factor ␤͞bone morphogenetic protein pathways that target the activity of RUNX2 during bone formation.

COVID-19 is a disease with heterogeneous clinical appearances. Most patients are asymptomatic or exhibit mild to moderate symptoms; approximately 15% progress to severe pneumonia and about 5% are eventually admitted to the intensive care unit (ICU) due to acute respiratory distress syndrome (ARDS), septic shock and/ or multiple organ failure. ICU patients respond poorly to currently available treatments and exhibit a high mortality rate. 1-3 Inadequate identification of the determinants of fatal outcomes is one of the major obstacles to the improvement of the outcomes in severe COVID-19 patients. A previous study reported a scoring system (COVID-GRAM) which accurately predicted the occurrence of critical illness in hospitalized COVID-19 patients. 4 Damage-associated molecular patterns (DAMPs), or alarmins, are a number of molecules, released by stressed cells undergoing microbial infection or sterile injury, that act as danger signals to promote and exacerbate the inflammatory response. 5,6 Of note, the serum level of S100A8/A9 and HMGB1 was found to be correlated with both the severity of pathogen-associated tissue damage and excessive cytokine storm. 7 Despite the hypothesis that S100A8/A9 and HMGB1 are significantly involved in COVID-19, so far, no study has yet tried to substantiate the hypothesis. In this study, we aimed to define the role of S100A8/ A9 and HMGB1 in progression to a fatal outcome and develop clinically relevant risk strata for COVID-19 patients. A total of 121 patients were enrolled in this retrospective study, of which 40 patients were in ICU and 81 patients in general wards at enrollment (Table S1). ICU Patients had much higher COVID-GRAM risk scores in comparison to those in general wards. Complications, including ARDS, sepsis, septic shock, secondary infection, acute renal injury, acute cardiac injury or failure, were more frequent in CCOVID-19 patients admitted to ICU. As of the cutoff date of April 30, 2020, most of non-ICU patients (96.3%) had been discharged alive, while 82.5% of ICU patients had died in ICU.

The chromosomal translocations found in acute myelogenous leukemia (AML) generate oncogenic fusion transcription factors with aberrant transcriptional regulatory properties. Although therapeutic targeting of most leukemia fusion proteins remains elusive, the posttranslational modifications that control their function could be targetable. We found that AML1-ETO, the fusion protein generated by the t(8;21) translocation, is acetylated by the transcriptional coactivator p300 in leukemia cells isolated from t(8;21) AML patients, and that this acetylation is essential for its self-renewal–promoting effects in human cord blood CD34+ cells and its leukemogenicity in mouse models. Inhibition of p300 abrogates the acetylation of AML1-ETO and impairs its ability to promote leukemic transformation. Thus, lysine acetyltransferases represent a potential therapeutic target in AML.

Both PU.1 (also called SFPI1), an Ets-family transcription factor, and AML1 (also called RUNX1), a DNA-binding subunit of the CBF transcription factor family, are crucial for the generation of all hematopoietic lineages, and both act as tumor suppressors in leukemia. An upstream regulatory element (URE) of PU.1 has both enhancer and repressor activity and tightly regulates PU.1 expression. Here we show that AML1 binds to functionally important sites within the PU.1 upstream regulatory element and regulates PU.1 expression at both embryonic and adult stages of development. Analysis of mice carrying conditional AML1 knockout alleles and knock-in mice carrying mutations in all three AML1 sites of the URE proximal region demonstrated that AML1 regulates PU.1 both positively and negatively in a lineage dependent manner. Dysregulation of PU.1 expression contributed to each of the phenotypes observed in these mice, and restoration of proper PU.1 expression rescued or partially rescued each phenotype. Thus, our data demonstrate that PU.1 is a major downstream target gene of AML1.

The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of de®nitive hematopoiesis and osteogenesis. The RUNX genes encode the a subunit of the transcription factor PEBP2/CBF. The b subunit consists of the non-RUNX protein PEBP2b. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin±proteasome pathway. When PEBP2b is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2b and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2b ±/± mouse. CBF(PEBP2)b± SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more ef®ciently than PEBP2b. These results reveal a hitherto unknown and major role of PEBP2b, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFb±SMMHC. Keywords: AML1/PEBP2b/proteolytic degradation/ Runx1/ubiquitylation IntroductionThe Runt domain transcription factor, PEBP2/CBF, is a heterodimeric transcription factor which is one of the major targets of transforming growth factor-b (TGF-b) and bone morphogenetic protein (BMP) (Hanai et al., 1999;Zhang et al., 2000). It is involved in the regulation of gene expression in a variety of biological activities, most notably hematopoiesis and osteogenesis (Ito, 1997(Ito, , 1999. Its b subunit, denoted as PEBP2b/CBFb, is homologous to the Drosophila Runt-binding proteins, Brother and Big brother. It does not bind to DNA, but the a subunit of PEBP2, which is homologous to the Drosophila gene products Runt and Lozenge, does have DNA-binding properties.The a subunit is encoded by RUNX, the mammalian homolog of the runt gene from Drosophila. RUNX contains an evolutionarily conserved 128 amino acid region termed the Runt domain, which is required both for DNA binding and heterodimerization with the b subunit (Kagoshima et al., 1993). In mammals, three a subunit genes exist, namely RUNX1, RUNX2 and RUNX3 (also referred to, respectively, as PEBP2aB, PEBP2aA and PEBP2aC, or CBFA2, CBFA1 and CBFA3, or AML1, AML3 and AML2) (Ito, 1999). RUNX1, which is frequently altered by the chromosome translocations associated with human leukemia (Look, 1997), is essential for inducing de®nitive hematopoiesis. It is also critical in regulating hematopoietic cell-speci®c genes in a variety of blood cells (Ito, 1997(Ito, , 1999. RUNX2 is essential for the generation and maturation of osteoblasts (Komori et al., 1997;Otto et al., 1997) and it plays pivotal roles in regulating the expression of bone-speci®c genes such as osteocalcin and osteopontin (Ducy et al., 1996). Haploinsuf®ciency of RUNX2 causes cleidocranial dysplasia, a human autosom...