p53 directly represses human LINE1 transposons.

p53 is a potent tumor suppressor and commonly mutated in human cancers. Recently, we demonstrated that p53 genes act to restrict retrotransposons in germline tissues of flies and fish but whether this activity is conserved in somatic human cells is not known. Here we show that p53 constitutively restrains human LINE1s by cooperatively engaging sites in the 5'UTR and stimulating local deposition of repressive histone marks at these transposons. Consistent with this, the elimination of p53 or the removal of corresponding binding sites in LINE1s, prompted these retroelements to become hyperactive. Concurrently, p53 loss instigated chromosomal rearrangements linked to LINE sequences and also provoked inflammatory programs that were dependent on reverse transcriptase produced from LINE1s. Taken together, our observations establish that p53 continuously operates at the LINE1 promoter to restrict autonomous copies of these mobile elements in human cells. Our results further suggest that constitutive restriction of these retroelements may help to explain tumor suppression encoded by p53, since erupting LINE1s produced acute oncogenic threats when p53 was absent.

p53 genes function to restrain mobile elements.

Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53(-) germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility.

Transposons, p53 and Genome Security.

p53, the most commonly mutated tumor suppressor, is a transcription factor known to regulate proliferation, senescence, and apoptosis. Compelling studies have found that p53 may prevent oncogenesis through effectors that are unrelated to these canonical processes and recent findings have uncovered ancient roles for p53 in the containment of mobile elements. Together, these developments raise the possibility that some p53-driven cancers could result from unrestrained transposons. Here, we explore evidence linking conserved features of p53 biology to the control of transposons. We also show how p53-deficient cells can be exploited to probe the behavior of transposons and illustrate how unrestrained transposons incited by p53 loss might contribute to human malignancies.

Tango7 directs cellular remodeling by the Drosophila apoptosome.

It is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, the determinants that specify whether the apoptosome acts to kill or remodel have yet to be identified. Here we report that Tango7 collaborates with the Drosophila apoptosome to drive a caspase-dependent remodeling process needed to resolve individual sperm from a syncytium. In these cells, Tango7 is required for caspase activity and localizes to the active apoptosome compartment via its C terminus. Furthermore, Tango7 directly stimulates the activity of this complex in vitro. We propose that Tango7 specifies the Drosophila apoptosome as an effector of cellular remodeling.

A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans.

We examined how a p53 enhancer transmits regulatory information in vivo. Using genetic ablation together with digital chromosome conformation capture and fluorescent in situ hybridization, we found that a Drosophila p53 enhancer region (referred to as the p53 response element [p53RE]) physically contacts targets in cis and across the centromere to control stress-responsive transcription at these sites. Furthermore, when placed at ectopic genomic positions, fragments spanning this element re-established chromatin contacts and partially restored target gene regulation to mutants lacking the native p53RE. Therefore, a defined p53 enhancer region is sufficient for long-range chromatin interactions that enable multigenic regulation.

An Examination of Prescribing Responsibilities between Psychiatrists and Primary Care Providers.

Patients with comorbid mental health and chronic conditions often receive care from both psychiatrists and primary care physicians (PCPs). The introduction of multiple providers into the care process introduces opportunities for disruptions in care continuity. The purpose of this study was to explore psychiatrists' and PCPs' comfort prescribing, along with their comfort having other physician specialties prescribe medications for cardiometabolic, psychiatric, and neurological/behavioral conditions. This cross-sectional study utilized an online, validated, pilot-tested, anonymous survey to examine prescribing practices of psychiatrists and PCPs. Eligible participants included physicians with medical degrees, U.S. prescribing authority, and active patient care for ≥2 days/week. Outcomes of interest were physicians' self-comfort and cross-specialty comfort (other specialists prescribing mutual patients' medications) prescribing cardiometabolic, psychiatric, and neurological/behavioral medications. Comfort prescribing was measured using 7-point Likert scales. Discrepancies in comfort were analyzed using student's, one-sample, and paired t-tests. Multiple linear regressions examined associations between physician practice characteristics and physicians' comfort-level prescribing cardiometabolic and psychiatric medication categories. Among 50 psychiatrists and 50 PCPs, psychiatrists reported significantly lower self-comfort prescribing cardiometabolic medications (mean ± SD = 2.99 ± 1.63 vs. 6.77 ± 0.39, p < 0.001), but significantly higher self-comfort prescribing psychiatric medications (mean ± SD = 6.79 ± 0.41 vs. 6.00 ± 0.88, p < 0.001) and neurological/behavioral medications (mean ± SD = 6.48 ± 0.74 vs. 5.56 ± 1.68, p < 0.001) than PCPs. After adjusting for covariates, physician specialty was strongly associated with self-comfort prescribing cardiometabolic and psychiatric medication categories (both p < 0.001). Differences between self-comfort and cross-specialty comfort were identified. Because comfort prescribing medications differed by physician type, incorporating psychiatrists through collaborative methods with PCPs could potentially ensure comfort among physicians when initiating medications.

p53 in the game of transposons.

Throughout the animal kingdom, p53 genes function to restrain mobile elements and recent observations indicate that transposons become derepressed in human cancers. Together, these emerging lines of evidence suggest that cancers driven by p53 mutations could represent "transpospoathies," i.e. disease states linked to eruptions of mobile elements. The transposopathy hypothesis predicts that p53 acts through conserved mechanisms to contain transposon movement, and in this way, prevents tumor formation. How transposon eruptions provoke neoplasias is not well understood but, from a broader perspective, this hypothesis also provides an attractive framework to explore unrestrained mobile elements as inciters of late-onset idiopathic disease. Also see the video abstract here.

Genome-wide silencing in Drosophila captures conserved apoptotic effectors.

Apoptosis is a conserved form of programmed cell death firmly established in the aetiology, pathogenesis and treatment of many human diseases. Central to the core machinery of apoptosis are the caspases and their proximal regulators. Current models for caspase control involve a balance of opposing elements, with variable contributions from positive and negative regulators among different cell types and species. To advance a comprehensive view of components that support caspase-dependent cell death, we conducted a genome-wide silencing screen in the Drosophila model. Our strategy used a library of double-stranded RNAs together with a chemical antagonist of Inhibitor of apoptosis proteins (IAPs) that simulates the action of native regulators in the Reaper and Smac (also known as Diablo) families. Here we present a highly validated set of targets that is necessary for death provoked by several stimuli. Among these, Tango7 is identified as a new effector. Cells depleted for this gene resisted apoptosis at a step before the induction of effector caspase activity, and the directed silencing of Tango7 in Drosophila prevented caspase-dependent programmed cell death. Unlike known apoptosis regulators in this model system, Tango7 activity did not influence stimulus-dependent loss of Drosophila DIAP1 (also known as th and IAP1), but instead regulated levels of the apical caspase Dronc (Nc). Similarly, the human Tango7 counterpart, PCID1 (also known as EIF3M), impinged on caspase 9, revealing a new regulatory axis affecting the apoptosome.

RNA Sensing and Innate Immunity Constitutes a Barrier for Interspecies Chimerism.

Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs. To date, however, it remains unexplored if human chimerism can be enhanced in animals through modifying the host embryos. Leveraging the interspecies PSC competition model, here we discovered retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, an RNA sensor, in "winner" cells plays an important role in the competitive interactions between co-cultured mouse and human PSCs. We found that genetic inactivation of Ddx58/Ifih1-Mavs-Irf7 axis compromised the "winner" status of mouse PSCs and their ability to outcompete PSCs from evolutionarily distant species during co-culture. Furthermore, by using Mavs-deficient mouse embryos we substantially improved unmodified donor human cell survival. Comparative transcriptome analyses based on species-specific sequences suggest contact-dependent human-to-mouse transfer of RNAs likely plays a part in mediating the cross-species interactions. Taken together, these findings establish a previously unrecognized role of RNA sensing and innate immunity in "winner" cells during cell competition and provides a proof-of-concept for modifying host embryos, rather than donor PSCs, to enhance interspecies chimerism.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

A collective form of cell death requires homeodomain interacting protein kinase.

We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective "suicide waves" promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.

Generalized nuclear localization of retroelement transcripts.

BACKGROUND: LINE-1s, Alus and SVAs are the only retrotransposition competent elements in humans. Their mobilization followed by insertional mutagenesis is often linked to disease. Apart from these rare integration events, accumulation of retrotransposition intermediates in the cytoplasm is potentially pathogenic due to induction of inflammatory response pathways. Although the retrotransposition of LINE-1 and Alu retroelements has been studied in considerable detail, there are mixed observations about the localization of their RNAs. RESULTS: We undertook a comprehensive and unbiased approach to analyze retroelement RNA localization using common cell lines and publicly available datasets containing RNA-sequencing data from subcellular fractions. Using our customized analytic pipeline, we compared localization patterns of RNAs transcribed from retroelements and single-copy protein coding genes. Our results demonstrate a generalized characteristic pattern of retroelement RNA nuclear localization that is conserved across retroelement classes as well as evolutionarily young and ancient elements. Preferential nuclear enrichment of retroelement transcripts was consistently observed in cell lines, in vivo and across species. Moreover, retroelement RNA localization patterns were dynamic and subject to change during development, as seen in zebrafish embryos. CONCLUSION: The pronounced nuclear localization of transcripts arising from ancient as well as de novo transcribed retroelements suggests that these transcripts are retained in the nucleus as opposed to being re-imported to the nucleus or degraded in the cytoplasm. This raises the possibility that there is adaptive value associated with this localization pattern to the host, the retroelements or possibly both.

Distinct p53 isoforms code for opposing transcriptional outcomes.

p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. Here, we investigate synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control in vivo. We show that transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

Coordination of cell death and the cell cycle: linking proliferation to death through private and communal couplers.

In development and in the adult, complex signaling pathways operate within and between cells to coordinate proliferation and cell death. These networks can be viewed as coupling devices that link engines driving the cell cycle and the initiation of apoptosis. We propose three simple frameworks for modeling the effects of proliferative drive on apoptotic propensity. This perspective offers a potentially useful foundation for predicting group behaviors of cells in normal and pathological settings.

Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia.

Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.

Drosophila GFAT1 and GFAT2 enzymes encode obligate developmental functions.

Glutamine: fructose-6-phosphate amidotransferase (GFAT) enzymes catalyse the first committed step of the hexosamine biosynthesis pathway (HBP) using glutamine and fructose-6-phosphate to form glucosamine-6-phosphate (GlcN6P). Numerous species (e.g. mouse, rat, zebrafish, chicken) including humans and Drosophila encode two broadly expressed copies of this enzyme but whether these perform redundant, partially overlapping or distinct functions is not known. To address this question, we produced single gene null mutations in the fly counterparts of gfat1 and gfat2. Deletions for either enzyme were fully lethal and homozygotes lacking either GFAT1 or GFAT2 died at or prior to the first instar larval stage. Therefore, when genetically eliminated, neither isoform was able to compensate for the other. Importantly, dietary supplementation with D-glucosamine-6-phosphate rescued GFAT2 deficiency and restored viability to gfat2-/- mutants. In contrast, glucosamine-6-phosphate did not rescue gfat1-/- animals.

Colorectal Neoplasia among Patients with and without Human Immunodeficiency Virus.

BACKGROUND: Increasing availability of highly active antiretroviral therapy (HAART) for human immunodeficiency virus (HIV) has led to prolonged survival and rising incidence of non-HIV-defining cancers among patients with HIV. Compared with the general population, risk of colorectal cancer may differ among those with HIV due to immunosuppression, oncogenic viral coinfections, and higher prevalence of risk factors. METHODS: We identified patients (age ≥50 years) diagnosed with HIV, prescribed HAART for ≥6 months, and receiving care in two large health care systems in Dallas, TX. Patients received a first colonoscopy between January 2009 and December 2017. We calculated a standardized prevalence ratio as the ratio of observed to expected number of advanced neoplasia (high-risk adenoma or colorectal cancer) using an age- and sex-matched cohort of patients without HIV (n = 10,250). RESULTS: Among patients with HIV (n = 839), about two thirds (60.1%) had normal findings at colonoscopy; 6.8% had hyperplastic polyps only, 20.4% had low-risk adenomas, 11.7% had high-risk adenomas, and 1.1% had colorectal cancer. Prevalence of advanced neoplasia was similar between patients with and without HIV, with a standardized prevalence ratio of 0.99 (95% confidence interval, 0.81-1.19). CONCLUSIONS: There was no difference in the prevalence of colorectal neoplasia between patients with and without HIV. IMPACT: Patients with HIV appear to have similar risk of colorectal neoplasia compared to those without HIV and can therefore follow average-risk colorectal cancer screening guidelines.

The apical caspase dronc governs programmed and unprogrammed cell death in Drosophila.

Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, we produced a null mutation in dronc. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis.

Drosophila p53 directs nonapoptotic programs in postmitotic tissue.

TP53 is the most frequently mutated gene in human cancers, and despite intensive research efforts, genome-scale studies of p53 function in whole animal models are rare. The need for such in vivo studies is underscored by recent challenges to established paradigms, indicating that unappreciated p53 functions contribute to cancer prevention. Here we leveraged the Drosophila system to interrogate p53 function in a postmitotic context. In the developing embryo, p53 robustly activates important apoptotic genes in response to radiation-induced DNA damage. We recently showed that a p53 enhancer (p53RErpr) near the cell death gene reaper forms chromatin contacts and enables p53 target activation across long genomic distances. Interestingly, we found that this canonical p53 apoptotic program fails to activate in adult heads. Moreover, this failure to exhibit apoptotic responses was not associated with altered chromatin contacts. Instead, we determined that p53 does not occupy the p53RErpr enhancer in this postmitotic tissue as it does in embryos. Through comparative RNA-seq and chromatin immunoprecipitation-seq studies of developing and postmitotic tissues, we further determined that p53 regulates distinct transcriptional programs in adult heads, including DNA repair, metabolism, and proteolysis genes. Strikingly, in the postmitotic context, p53-binding landscapes were poorly correlated with nearby transcriptional effects, raising the possibility that p53 enhancers could be generally acting through long distances.

Neuronal expression of p53 dominant-negative proteins in adult Drosophila melanogaster extends life span.

Hyperactivation of p53 leads to a reduction in tumor formation and an unexpected shortening of life span in two different model systems . The decreased life span occurs with signs of accelerated aging, such as osteoporosis, reduction in body weight, atrophy of organs, decreased stress resistance, and depletion of hematopoietic stem cells. These observations suggest a role for p53 in the determination of life span and the speculation that decreasing p53 activity may result in positive effects on some aging phenotypes . In this report, we show that expression of dominant-negative versions of Drosophila melanogaster p53 in adult neurons extends life span and increases genotoxic stress resistance in the fly. Consistent with this, a naturally occurring allele with decreased p53 activity has been associated with extended survival in humans . Expression of the dominant-negative Drosophila melanogaster p53 constructs does not further increase the extended life span of flies that are calorie restricted, suggesting that a decrease in p53 activity may mediate a component of the calorie-restriction life span-extending pathway in flies.