Context dependent activity of p63-bound gene regulatory elements.

The p53 family of transcription factors regulate numerous organismal processes including the development of skin and limbs, ciliogenesis, and preservation of genetic integrity and tumor suppression. p53 family members control these processes and gene expression networks through engagement with DNA sequences within gene regulatory elements. Whereas p53 binding to its cognate recognition sequence is strongly associated with transcriptional activation, p63 can mediate both activation and repression. How the DNA sequence of p63-bound gene regulatory elements is linked to these varied activities is not yet understood. Here, we use massively parallel reporter assays (MPRA) in a range of cellular and genetic contexts to investigate the influence of DNA sequence on p63-mediated transcription. Most regulatory elements with a p63 response element motif (p63RE) activate transcription, with those sites bound by p63 more frequently or adhering closer to canonical p53 family response element sequences driving higher transcriptional output. The most active regulatory elements are those also capable of binding p53. Elements uniquely bound by p63 have varied activity, with p63RE-mediated repression associated with lower overall GC content in flanking sequences. Comparison of activity across cell lines suggests differential activity of elements may be regulated by a combination of p63 abundance or context-specific cofactors. Finally, changes in p63 isoform expression dramatically alters regulatory element activity, primarily shifting inactive elements towards a strong p63-dependent activity. Our analysis of p63-bound gene regulatory elements provides new insight into how sequence, cellular context, and other transcription factors influence p63-dependent transcription. These studies provide a framework for understanding how p63 genomic binding locally regulates transcription. Additionally, these results can be extended to investigate the influence of sequence content, genomic context, chromatin structure on the interplay between p63 isoforms and p53 family paralogs.

A feedback loop between heterochromatin and the nucleopore complex controls germ-cell-to-oocyte transition during Drosophila oogenesis.

Germ cells differentiate into oocytes that launch the next generation upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrently with oocyte specification. Here, we discovered that nuclear SETDB1 is required for silencing a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for upregulating 18 of the ∼30 nucleoporins (Nups) that compose the nucleopore complex (NPC), promoting NPC formation. NPCs anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to the loss of SETDB1 or Nups results in the loss of oocyte identity, cell death, and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification, which is critical for establishing oocyte identity.

Shared Gene Targets of the ATF4 and p53 Transcriptional Networks.

The master tumor suppressor p53 regulates multiple cell fate decisions, such as cell cycle arrest and apoptosis, via transcriptional control of a broad gene network. Dysfunction in the p53 network is common in cancer, often through mutations that inactivate p53 or other members of the pathway. Induction of tumor-specific cell death by restoration of p53 activity without off-target effects has gained significant interest in the field. In this study, we explore the gene regulatory mechanisms underlying a putative anticancer strategy involving stimulation of the p53-independent integrated stress response (ISR). Our data demonstrate the p53 and ISR pathways converge to independently regulate common metabolic and proapoptotic genes. We investigated the architecture of multiple gene regulatory elements bound by p53 and the ISR effector ATF4 controlling this shared regulation. We identified additional key transcription factors that control basal and stress-induced regulation of these shared p53 and ATF4 target genes. Thus, our results provide significant new molecular and genetic insight into gene regulatory networks and transcription factors that are the target of numerous antitumor therapies.

Shared gene targets of the ATF4 and p53 transcriptional networks.

The master tumor suppressor p53 regulates multiple cell fate decisions, like cell cycle arrest and apoptosis, via transcriptional control of a broad gene network. Dysfunction in the p53 network is common in cancer, often through mutations that inactivate p53 or other members of the pathway. Induction of tumor-specific cell death by restoration of p53 activity without off-target effects has gained significant interest in the field. In this study, we explore the gene regulatory mechanisms underlying a putative anti-cancer strategy involving stimulation of the p53-independent Integrated Stress Response (ISR). Our data demonstrate the p53 and ISR pathways converge to independently regulate common metabolic and pro-apoptotic genes. We investigated the architecture of multiple gene regulatory elements bound by p53 and the ISR effector ATF4 controlling this shared regulation. We identified additional key transcription factors that control basal and stress-induced regulation of these shared p53 and ATF4 target genes. Thus, our results provide significant new molecular and genetic insight into gene regulatory networks and transcription factors that are the target of numerous antitumor therapies.

Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System.

BACKGROUND: Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS. RESULTS: Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC. CONCLUSIONS: Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.

Mental Health Service Provision During COVID-19: Results of the Third Survey of Licensed Psychologists.

A third survey of the practice of licensed psychologists during the pandemic conducted in June 2021 revealed that the rapid adoption of telepsychological service provision has continued approximately 15 months after a national public health emergency was declared. Most respondents intend to make telepsychology a permanent component of their practice going forward. Other notable findings from our survey revealed that after an initial decline in caseload reported in the early days of the pandemic, the majority of psychologists surveyed now report an increase in caseload, often necessitating the establishment of a waitlist. Respondents reported that their patients/clients are more accepting of telepsychology than in our previous survey. That said, a significant minority of psychologists expressed concerns that this technology will negatively affect their future practice. Results also indicated that psychologists are encountering greater symptom acuity among their patients associated with the pandemic, including an increase in reports of suicidal thinking or behavior. Supplementary Information: The online version contains supplementary material available at 10.1007/s42843-021-00044-3.

p63 and p53: Collaborative Partners or Dueling Rivals?

The tumor suppressor p53 and its oncogenic sibling p63 (ΔNp63) direct opposing fates in tumor development. These paralog proteins are transcription factors that elicit their tumor suppressive and oncogenic capacity through the regulation of both shared and unique target genes. Both proteins predominantly function as activators of transcription, leading to a paradigm shift away from ΔNp63 as a dominant negative to p53 activity. The discovery of p53 and p63 as pioneer transcription factors regulating chromatin structure revealed new insights into how these paralogs can both positively and negatively influence each other to direct cell fate. The previous view of a strict rivalry between the siblings needs to be revisited, as p53 and p63 can also work together toward a common goal.

Psychological Practice at Six Months of COVID-19: A Follow-Up to the First National Survey of Psychologists During the Pandemic.

We conducted a survey of licensed psychologists at two weeks and again at six months after the declaration of a national emergency related to the COVID-19 pandemic. This article describes the results of the second survey conducted approximately six months after the crisis began. The rapid shift to telepsychological services seen in the first survey in the pandemic has solidified in the second survey. More providers reported delivering a larger percentage of services via telepsychology than early in the pandemic. The majority of respondents do not anticipate resuming in-person services until after a vaccine is made available, although a consistent minority reports ongoing in-person service provision. A majority reported their patients had appropriate access to internet and telepsychological service platforms, although one-fifth of respondents reported their patients had difficulty accessing such services. Early concerns about technological or regulatory problems involved in telepsychology are no longer evident. Most respondents indicated they will continue to use telepsychological services for the delivery of some of their psychological services after the pandemic ends. Forty-five percent knew of individuals who contracted the disease, 13% knew someone who died of the disease, and 2% reported contracted the disease themselves.

Tumor suppressor p53: from engaging DNA to target gene regulation.

The p53 transcription factor confers its potent tumor suppressor functions primarily through the regulation of a large network of target genes. The recent explosion of next generation sequencing protocols has enabled the study of the p53 gene regulatory network (GRN) and underlying mechanisms at an unprecedented depth and scale, helping us to understand precisely how p53 controls gene regulation. Here, we discuss our current understanding of where and how p53 binds to DNA and chromatin, its pioneer-like role, and how this affects gene regulation. We provide an overview of the p53 GRN and the direct and indirect mechanisms through which p53 affects gene regulation. In particular, we focus on delineating the ubiquitous and cell type-specific network of regulatory elements that p53 engages; reviewing our understanding of how, where, and when p53 binds to DNA and the mechanisms through which these events regulate transcription. Finally, we discuss the evolution of the p53 GRN and how recent work has revealed remarkable differences between vertebrates, which are of particular importance to cancer researchers using mouse models.

Comparative gene expression profiling between optic nerve and spinal cord injury in Xenopus laevis reveals a core set of genes inherent in successful regeneration of vertebrate central nervous system axons.

BACKGROUND: The South African claw-toed frog, Xenopus laevis, is uniquely suited for studying differences between regenerative and non-regenerative responses to CNS injury within the same organism, because some CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs. Tissues from these CNS regions (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) were used in a three-way RNA-seq study of axotomized CNS axons to identify potential core gene expression programs for successful CNS axon regeneration. RESULTS: Despite tissue-specific changes in expression dominating the injury responses of each tissue, injury-induced changes in gene expression were nonetheless shared between the two axon-regenerative CNS regions that were not shared with the non-regenerative region. These included similar temporal patterns of gene expression and over 300 injury-responsive genes. Many of these genes and their associated cellular functions had previously been associated with injury responses of multiple tissues, both neural and non-neural, from different species, thereby demonstrating deep phylogenetically conserved commonalities between successful CNS axon regeneration and tissue regeneration in general. Further analyses implicated the KEGG adipocytokine signaling pathway, which links leptin with metabolic and gene regulatory pathways, and a novel gene regulatory network with genes regulating chromatin accessibility at its core, as important hubs in the larger network of injury response genes involved in successful CNS axon regeneration. CONCLUSIONS: This study identifies deep, phylogenetically conserved commonalities between CNS axon regeneration and other examples of successful tissue regeneration and provides new targets for studying the molecular underpinnings of successful CNS axon regeneration, as well as a guide for distinguishing pro-regenerative injury-induced changes in gene expression from detrimental ones in mammals.

Asian Zika Virus Isolate Significantly Changes the Transcriptional Profile and Alternative RNA Splicing Events in a Neuroblastoma Cell Line.

The alternative splicing of pre-mRNAs expands a single genetic blueprint to encode multiple, functionally diverse protein isoforms. Viruses have previously been shown to interact with, depend on, and alter host splicing machinery. The consequences, however, incited by viral infection on the global alternative slicing (AS) landscape are under-appreciated. Here, we investigated the transcriptional and alternative splicing profile of neuronal cells infected with a contemporary Puerto Rican Zika virus (ZIKVPR) isolate, an isolate of the prototypical Ugandan ZIKV (ZIKVMR), and dengue virus 2 (DENV2). Our analyses revealed that ZIKVPR induced significantly more differential changes in expressed genes compared to ZIKVMR or DENV2, despite all three viruses showing equivalent infectivity and viral RNA levels. Consistent with the transcriptional profile, ZIKVPR induced a higher number of alternative splicing events compared to ZIKVMR or DENV2, and gene ontology analyses highlighted alternative splicing changes in genes associated with mRNA splicing. In summary, we show that ZIKV affects cellular RNA homeostasis not only at the transcriptional levels but also through the alternative splicing of cellular transcripts. These findings could provide new molecular insights into the neuropathologies associated with this virus.

Psychological Practice and the COVID-19 Crisis: A Rapid Response Survey.

Psychological practice has changed dramatically over the past 125 years. The two world wars both served to stimulate and change the scope of practice for psychologists. We surveyed over 3,000 doctoral psychologists about the impact of the COVID-19 health crisis on their clinical practices. Practice changed from primarily in-office to mostly telepsychology practice over the course of 2 weeks in March of 2020. The long-term effect on professional practice in psychology is not known.

Smad4-dependent morphogenic signals control the maturation and axonal targeting of basal vomeronasal sensory neurons to the accessory olfactory bulb.

The vomeronasal organ (VNO) contains two main types of vomeronasal sensory neurons (VSNs) that express distinct vomeronasal receptor (VR) genes and localize to specific regions of the neuroepithelium. Morphogenic signals are crucial in defining neuronal identity and network formation; however, if and what signals control maturation and homeostasis of VSNs is largely unexplored. Here, we found transforming growth factor ß (TGFß) and bone morphogenetic protein (BMP) signal transduction in postnatal mice, with BMP signaling being restricted to basal VSNs and at the marginal zones of the VNO: the site of neurogenesis. Using different Smad4 conditional knockout mouse models, we disrupted canonical TGFß/BMP signaling in either maturing basal VSNs (bVSNs) or all mature VSNs. Smad4 loss of function in immature bVSNs compromises dendritic knob formation, pheromone induced activation, correct glomeruli formation in the accessory olfactory bulb (AOB) and survival. However, Smad4 loss of function in all mature VSNs only compromises correct glomeruli formation in the posterior AOB. Our results indicate that Smad4-mediated signaling drives the functional maturation and connectivity of basal VSNs.

Locally acting transcription factors regulate p53-dependent cis-regulatory element activity.

The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CREs), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors. Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. Our data suggest p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that global p53 activity is guarded against loss of any one regulatory partner, allowing for dynamic and redundant control of p53-mediated transcription.