Clinical Utility of the OmniGraf Biomarker Panel in the Care of Kidney Transplant Recipients (CLARITY): Protocol for a Prospective, Multisite Observational Study.

BACKGROUND: Death with a functioning allograft has become the leading category of graft loss in kidney transplant recipients at all time points. Previous analyses have demonstrated that causes of death in kidney transplant recipients are predominated by comorbidities strongly associated with immunosuppressant medications. Adverse drug events (ADEs) have been strongly associated with nonadherence, health care utilization, and graft loss; clinicians face a difficult decision on whether making immunosuppressant adjustments in the face of ADEs will improve symptomology or simply increase the risk of acute rejection. Clinicians also face a treatment quandary in 50% of kidney transplant recipients with stage 3 or worse chronic kidney disease at 1 year post transplantation, as progressive decline in renal function has been strongly associated with inferior allograft survival. OBJECTIVE: The primary objective of the CLinical Utility of the omnigrAf biomarkeR Panel In The Care of kidneY Transplant Recipients (CLARITY) trial is to evaluate change in renal function over time in kidney transplant recipients who are undergoing OmniGraf monitoring in conjunction with monitoring of their medication-related symptom burden (MRSB). A secondary objective of this study is to identify the impact of OmniGraf use in conjunction with patient-reported MRSB as part of clinical care on patients' self-efficacy and quality of life. METHODS: CLARITY is a 3-year prospective, multisite, observational study of 2000 participants with a matched control, measuring the impact of real-time patients' MRSB and the OmniGraf biomarker panel on change in renal function over time. Secondary outcome measures include the Patient-Reported Outcomes Measurement Information System (PROMIS) Self-Efficacy for Managing Chronic Conditions-Managing Medications and Treatment-Short Form 4a; the PROMIS-29 Profile (version 2.1); the PROMIS Depression Scale, hospitalizations-subcategorized for hospitalizations owing to infections; treated rejections, MRSB, and proportion of participants with overall graft survival at year 3 post transplantation; graft loss or death during the 3-year study follow-up period; and change in provider satisfaction. RESULTS: The primary outcome measure of the study will be a comparison of the slope change in estimated glomerular filtration rate from baseline to the end of follow-up between study participants and a matched control group. Secondary outcome measures include changes over time in PROMIS Self-Efficacy for Managing Chronic Conditions-Managing Medications and Treatment-Short Form 4a, the PROMIS-29 Profile (version 2.1), and PROMIS Depression Scale in the study group, as well as a comparison of hospitalizations and causes, rejections, and graft and patient survival compared between participants and a matched cohort. The anticipated first enrollment in the study is October 2022 with data analysis and publication expected in October 2027. CONCLUSIONS: Through this report, we describe the study design, methods, and outcome measures that will be utilized in the ongoing CLARITY trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT05482100; https://clinicaltrials.gov/ct2/show/NCT05482100. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/41020.

UBAP2/UBAP2L regulate UV-induced ubiquitylation of RNA polymerase II and are the human orthologues of yeast Def1.

During transcription, RNA polymerase II (RNAPII) faces numerous obstacles, including DNA damage, which can lead to stalling or arrest. One mechanism to contend with this situation is ubiquitylation and degradation of the largest RNAPII subunit, RPB1 - the 'last resort' pathway. This conserved, multi-step pathway was first identified in yeast, and the functional human orthologues of all but one protein, RNAPII Degradation Factor 1 (Def1), have been discovered. Here we show that following UV-irradiation, human Ubiquitin-associated protein 2 (UBAP2) or its paralogue UBAP2-like (UBAP2L) are involved in the ubiquitylation and degradation of RNAPII through the recruitment of Elongin-Cul5 ubiquitin ligase. Together, our data indicate that UBAP2 and UBAP2L are the human orthologues of yeast Def1, and so identify the key missing proteins in the human last resort pathway.

Combining Blood Gene Expression and Cellfree DNA to Diagnose Subclinical Rejection in Kidney Transplant Recipients.

BACKGROUND AND OBJECTIVES: Subclinical acute rejection is associated with poor outcomes in kidney transplant recipients. As an alternative to surveillance biopsies, noninvasive screening has been established with a blood gene expression profile. Donor-derived cellfree DNA (cfDNA) has been used to detect rejection in patients with allograft dysfunction but not tested extensively in stable patients. We hypothesized that we could complement noninvasive diagnostic performance for subclinical rejection by combining a donor-derived cfDNA and a gene expression profile assay. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a post hoc analysis of simultaneous blood gene expression profile and donor-derived cfDNA assays in 428 samples paired with surveillance biopsies from 208 subjects enrolled in an observational clinical trial (Clinical Trials in Organ Transplantation-08). Assay results were analyzed as binary variables, and then, their continuous scores were combined using logistic regression. The performance of each assay alone and in combination was compared. RESULTS: For diagnosing subclinical rejection, the gene expression profile demonstrated a negative predictive value of 82%, a positive predictive value of 47%, a balanced accuracy of 64%, and an area under the receiver operating curve of 0.75. The donor-derived cfDNA assay showed similar negative predictive value (84%), positive predictive value (56%), balanced accuracy (68%), and area under the receiver operating curve (0.72). When both assays were negative, negative predictive value increased to 88%. When both assays were positive, positive predictive value increased to 81%. Combining assays using multivariable logistic regression, area under the receiver operating curve was 0.81, significantly higher than the gene expression profile (P<0.001) or donor-derived cfDNA alone (P=0.006). Notably, when cases were separated on the basis of rejection type, the gene expression profile was significantly better at detecting cellular rejection (area under the receiver operating curve, 0.80 versus 0.62; P=0.001), whereas the donor-derived cfDNA was significantly better at detecting antibody-mediated rejection (area under the receiver operating curve, 0.84 versus 0.71; P=0.003). CONCLUSIONS: A combination of blood-based biomarkers can improve detection and provide less invasive monitoring for subclinical rejection. In this study, the gene expression profile detected more cellular rejection, whereas donor-derived cfDNA detected more antibody-mediated rejection.

A role for the Cockayne Syndrome B (CSB)-Elongin ubiquitin ligase complex in signal-dependent RNA polymerase II transcription.

The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.

Regulation of the RNAPII Pool Is Integral to the DNA Damage Response.

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.

Imaging-based assays for investigating functions of the RNA polymerase II elongation factor Elongin and the Elongin ubiquitin ligase.

Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.

Cockayne syndrome B protein regulates recruitment of the Elongin A ubiquitin ligase to sites of DNA damage.

Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.

Multiomic Analysis of the UV-Induced DNA Damage Response.

In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response.

Assembly of the Elongin A Ubiquitin Ligase Is Regulated by Genotoxic and Other Stresses.

Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription.

Class II histone deacetylases downregulate GLUT4 transcription in response to increased cAMP signaling in cultured adipocytes and fasting mice.

Insulin-mediated glucose uptake is highly sensitive to the levels of the facilitative glucose transporter protein, GLUT4. Repression of GLUT4 expression is correlated with insulin resistance in adipose tissue. We have shown that differentiation-dependent GLUT4 transcription was under control of class II histone deacetylases (HDACs). We hypothesized that HDACs may regulate gene expression in adipocytes as a result of adrenergic activation. To test this hypothesis, we activated cAMP signaling in 3T3-L1 adipocytes and in mice after an overnight fast. Chromatin immunoprecipitation experiments showed the association of HDAC4/5 with the GLUT4 promoter in vivo and in vitro in response to elevated cAMP. Knockdown of HDACs by small interfering RNA in cultured adipocytes prevented the cAMP-dependent decrease in GLUT4 transcription. HDAC4/5 recruitment to the GLUT4 promoter was dependent on the GLUT4 liver X receptor (LXR) binding site. Treatment of cells with an LXR agonist prevented the cAMP-dependent decrease in GLUT4 transcription. A loss of function mutation in the LXR response element was required for cAMP-dependent downregulation of GLUT4 expression in vitro, in fasted mice, and in mice subjected to diet-induced obesity. This suggests that activation of LXR signaling can prevent loss of GLUT4 expression in diabetes and obesity.

Class II histone deacetylases limit GLUT4 gene expression during adipocyte differentiation.

Insulin-dependent glucose homeostasis is highly sensitive to the levels of insulin-responsive glucose transporter 4 (GLUT4) expression in adipocytes. The level of GLUT4 protein expression is highly dependent on the rate of GLUT4 gene transcription. GLUT4 gene transcription is decreased in a variety of physiologic states of insulin resistance including type 2 diabetes, obesity, and prolonged fasting. GLUT4 gene expression in adipocytes is differentiation-dependent, with full expression delayed until late in the differentiation program. In this paper, we have tested the hypothesis that differentiation-dependent GLUT4 gene expression in 3T3-L1 adipocytes is dependent on the nuclear concentration of a class II histone deacetylase (HDAC) protein, HDAC5. We have tested this hypothesis by reducing the levels of class II HDACs in the nuclear compartment of 3T3-L1 preadipocytes using two experimental approaches. First, preadipocytes were treated with phenylephrine, an α-adrenergic receptor agonist, to drive HDACS out of the nuclear compartment. Also, the class II HDAC concentrations were reduced using siRNA knockdown. In each case, reduction of nuclear class II HDAC concentration resulted in increased expression of endogenous GLUT4 mRNA in preadipocytes. Together, our data indicate that class II HDAC expression is the major regulatory mechanism for inhibiting GLUT4 expression in the predifferentiated state.

Acute inhibition of fatty acid import inhibits GLUT4 transcription in adipose tissue, but not skeletal or cardiac muscle tissue, partly through liver X receptor (LXR) signaling.

OBJECTIVE: Insulin-mediated glucose uptake is highly sensitive to the levels of the facilitative GLUT protein GLUT4. Transcription of the GLUT4 gene is repressed in states of insulin deficiency and insulin resistance and can be induced by states of enhanced energy output, such as exercise. The cellular signals that regulate GLUT4 transcription are not well understood. We hypothesized that changes in energy substrate flux regulate GLUT4 transcription. RESEARCH DESIGN AND METHODS: To test this hypothesis, we used transgenic mice in which expression of the chloramphenicol acetyltransferase (CAT) gene is driven by a functional 895-bp fragment of the human GLUT4 promoter, thereby acting as a reporter for transcriptional activity. Mice were treated with a single dose of etomoxir, which inhibits the transport of long-chain fatty acids into mitochondria and increases basal, but not insulin-mediated, glucose flux. GLUT4 and transgenic CAT mRNA were measured. RESULTS: Etomoxir treatment significantly reduced CAT and GLUT4 mRNA transcription in adipose tissue, but did not change transcription in heart and skeletal muscle. Downregulation of GLUT4 transcription was cell autonomous, since etomoxir treatment of 3T3-L1 adipocytes resulted in a similar downregulation of GLUT4 mRNA. GLUT4 transcriptional downregulation required the putative liver X receptor (LXR) binding site in the human GLUT4 gene promoter in adipose tissue and 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the LXR agonist, TO901317, partially restored GLUT4 expression in etomoxir-treated cells. CONCLUSIONS: Our data suggest that long-chain fatty acid import into mitochondria in adipose tissue may produce ligands that regulate expression of metabolic genes.

GLUT4 enhancer factor (GEF) interacts with MEF2A and HDAC5 to regulate the GLUT4 promoter in adipocytes.

The insulin-responsive glucose transporter, GLUT4, is regulated in various physiologic states at the transcriptional level. When expressed in transgenic mice, the human GLUT4 promoter is governed by two cis-acting sequences: an MEF2 binding domain and Domain I, that function both as positive and negative regulators depending on the physiologic state. MEF2 proteins and GLUT4 enhancer factor (GEF) are known ligands for these cis-acting elements, but their mechanism of action is unclear. To begin to understand this important process, we have characterized GEF structural domains and its interactions with the MEF2A isoform. We find that the C terminus of GEF comprises its DNA-binding domain, but does not contribute to GEF homo-oligomerization. We also have found that GEF dimerizes with increased affinity to a hypophosphorylated form of MEF2A. Furthermore, we demonstrated that MEF2A binding to its cognate binding site can increase the DNA binding activity of GEF to Domain I, suggesting a novel mechanism for MEF2A transcriptional activation. Finally, we have demonstrated that the transcriptional co-repressor HDAC5 can interact with GEF in the absence of MEF2 proteins and specifically inhibit GLUT4 promoter activity. These findings lead to the hypothesis that GEF and the MEF2 proteins form a complex on the GLUT4 promoter that allows for recruitment of transcriptional co-regulators (repressors and/or activators) to control GLUT4 promoter activity.