O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis.

The addition of a single ß-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after ß-estradiol (E2) addition. We observed that during G1E-ER4 differentiation, overall O-GlcNAc levels decrease, and physical interactions of GATA-1 with both OGT and OGA increase. RNA-Seq-based transcriptome analysis of G1E-ER4 cells differentiated in the presence of the OGA inhibitor Thiamet-G (TMG) revealed changes in expression of 433 GATA-1 target genes. ChIP results indicated that the TMG treatment decreases the occupancy of GATA-1, OGT, and OGA at the GATA-binding site of the lysosomal protein transmembrane 5 (Laptm5) gene promoter. TMG also reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. Sustained treatment of G1E-ER4 cells with TMG before differentiation reduced hemoglobin-positive cells and increased stem/progenitor cell surface markers. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for O-GlcNAcylation in regulating hematopoietic cell fate.

The regulation of glycogenolysis in the brain.

The key regulatory enzymes of glycogenolysis are phosphorylase kinase, a hetero-oligomer with four different types of subunits, and glycogen phosphorylase, a homodimer. Both enzymes are activated by phosphorylation and small ligands, and both enzymes have distinct isoforms that are predominantly expressed in muscle, liver, or brain; however, whole-transcriptome high-throughput sequencing analyses show that in brain both of these enzymes are likely composed of subunit isoforms representing all three tissues. This Minireview examines the regulatory properties of the isoforms of these two enzymes expressed in the three tissues, focusing on their potential regulatory similarities and differences. Additionally, the activity, structure, and regulation of the remaining enzyme necessary for glycogenolysis, glycogen-debranching enzyme, are also reviewed.

Gap junctions and hemichannels: communicating cell death in neurodevelopment and disease.

Gap junctions are unique membrane channels that play a significant role in intercellular communication in the developing and mature central nervous system (CNS). These channels are composed of connexin proteins that oligomerize into hexamers to form connexons or hemichannels. Many different connexins are expressed in the CNS, with some specificity with regard to the cell types in which distinct connexins are found, as well as the timepoints when they are expressed in the developing and mature CNS. Both the main neuronal Cx36 and glial Cx43 play critical roles in neurodevelopment. These connexins also mediate distinct aspects of the CNS response to pathological conditions. An imbalance in the expression, translation, trafficking and turnover of connexins, as well as mutations of connexins, can impact their function in the context of cell death in neurodevelopment and disease. With the ever-increasing understanding of connexins in the brain, therapeutic strategies could be developed to target these membrane channels in various neurological disorders.

Medical Student Perspectives of Active Learning: A Focus Group Study.

Phenomenon: Medical student perspectives were sought about active learning, including concerns, challenges, perceived advantages and disadvantages, and appropriate role in the educational process. APPROACH: Focus groups were conducted with students from all years and campuses of a large U.S. state medical school. FINDINGS: Students had considerable experience with active learning prior to medical school and conveyed accurate understanding of the concept and its major strategies. They appreciated the potential of active learning to deepen and broaden learning and its value for long-term professional development but had significant concerns about the efficiency of the process, the clarity of expectations provided, and the importance of receiving preparatory materials. Most significantly, active learning experiences were perceived as disconnected from grading and even as impeding preparation for school and national examinations. Insights: Medical students understand the concepts of active learning and have considerable experience in several formats prior to medical school. They are generally supportive of active learning concepts but frustrated by perceived inefficiencies and lack of contribution to the urgencies of achieving optimal grades and passing United States Medical Licensing Examinations, especially Step 1.

On the Origins of Perceptions: Student Perceptions of Active Learning and Their Implications for Educational Reform.

This Conversation Starters article presents a selected research abstract from the 2016 Association of American Medical Colleges Central Region Group on Educational Affairs annual spring meeting. The abstract is paired with the integrative commentary of three experts who shared their thoughts stimulated by the study. These thoughts highlight the value of exploring what drives student perceptions of active learning in order to reform medical education.

Neuronal gap junction coupling as the primary determinant of the extent of glutamate-mediated excitotoxicity.

In the mammalian central nervous system (CNS), coupling of neurons by gap junctions (electrical synapses) increases during early postnatal development, then decreases, but increases in the mature CNS following neuronal injury, such as ischemia, traumatic brain injury and epilepsy. Glutamate-dependent neuronal death also occurs in the CNS during development and neuronal injury, i.e., at the time when neuronal gap junction coupling is increased. Here, we review our recent studies on regulation of neuronal gap junction coupling by glutamate in developing and injured neurons and on the role of gap junctions in neuronal cell death. A modified model of the mechanisms of glutamate-dependent neuronal death is discussed, which includes neuronal gap junction coupling as a critical part of these mechanisms.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies-which would also detect tuneability-used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: the ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. In this study, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITA's AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITA's function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behavior for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

Neuronal gap junction coupling is regulated by glutamate and plays critical role in cell death during neuronal injury.

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI), and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. We report here that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluRs). Specifically, using electrotonic coupling, Western blots, and siRNA in the mouse somatosensory cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36) (neuronal gap junction protein), and inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. We also show that the regulation is via cAMP/PKA (cAMP-dependent protein kinase)-dependent signaling and posttranscriptional control of Cx36 expression and that other glutamate receptors are not involved in these regulatory mechanisms. Furthermore, using the analysis of neuronal death, we show that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemia-mediated neuronal death in vitro and in vivo. Similar results are obtained using in vitro models of TBI and epilepsy. Our results indicate that neuronal gap junction coupling is a critical component of glutamate-dependent neuronal death. They also suggest that causal link among group II mGluR function, neuronal gap junction coupling, and neuronal death has a universal character and operates in different types of neuronal injuries.

Equivalent Performance of Exam Items Associated with Case-Based Learning, Flipped Classroom, and Lecture in a Pre-clerkship Medical Curriculum.

The purpose of our study was to determine if knowledge acquisition, as measured by exam item performance, differed for active or passive learning activities in our medical curriculum. Additionally, we looked for differences in exam item performance in one second-year course that varies the method of an active learning activity, case-based collaborative learning (CBCL). Finally, we assessed whether item performance was impacted when small group activities were conducted online due to the COVID-19 pandemic. Exam item difficulty values were collected for several years of lectures, flipped classroom, and CBCL. Statistical analysis and modeling of data were performed to identify differences in difficulty of exam items that assess content delivered by different learning activities. Our analysis revealed no differences in difficulty of exam items that assess content delivered by different learning activities. Similarly, we determined that varying the execution of CBCL in one course did not impact exam item performance. Finally, moving CBCL small group sessions online did not impact exam item difficulty. However, we did detect a minor reduction in overall exam scores for the period of online instruction. Our results indicate that knowledge acquisition, as assessed by our multiple-choice summative exams, was equivalent regardless of learning activity modality. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-023-01842-8.

Physician Perception of the Importance of Medical Genetics and Genomics in Medical Education and Clinical Practice.

PURPOSE: The objective of this study was to determine physician perceptions regarding the importance of and comfort with the use of medical genetics and genomics in medical education and practice, as well as physician expectations for medical trainees. METHODS: A retrospective survey was sent to physicians employed by a health system associated with a public medical school to assess their perceived training in medical genetics and genomics and their comfort level with ordering genetic testing. METHODS: Despite reporting formal genetics training in medical schools, clinicians' comfort with and knowledge in this content area does not meet personal expectations of competency. Though physicians report some discomfort with the use of medical genetics and genomics, the majority also believe that its impact on practice will increase in the next five years. Survey recipients were also asked about their expectations for preparation in the same domains for medical students and incoming residents. The surveyed physicians expect a high level of competency for medical students and incoming residents. METHODS: Our study revealed that practicing physicians feel current medical curricula do not produce physicians with the necessary competency in medical genetics and genomics. This is despite physicians' perceived importance of this domain in medical practice. Our findings suggest a need for re-evaluation of medical genetics and genomics education at all levels of training.

Interplay of chemical neurotransmitters regulates developmental increase in electrical synapses.

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development. We report here that the developmental increase in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) are regulated by an interplay between the activity of group II metabotropic glutamate receptors (mGluRs) and GABA(A) receptors. Specifically, using dye coupling, electrotonic coupling, Western blots and small interfering RNA in the rat and mouse hypothalamus and cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs augments, and inactivation prevents, the developmental increase in neuronal gap junction coupling and Cx36 expression. However, changes in GABA(A) receptor activity have the opposite effects. The regulation by group II mGluRs is via cAMP/PKA-dependent signaling, and regulation by GABA(A) receptors is via Ca(2+)/PKC-dependent signaling. Furthermore, the receptor-mediated upregulation of Cx36 requires a neuron-restrictive silencer element in the Cx36 gene promoter, and the downregulation involves the 3'-untranslated region of the Cx36 mRNA, as shown using reverse-transcription quantitative real-time PCR and luciferase reporter activity analysis. In addition, the methyl thiazolyl tetrazolium analysis indicates that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons. Together, the results suggest a multitiered strategy for chemical synapses in developmental regulation of electrical synapses.

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDAR-mediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

An N- and C-terminal truncated isoform of zinc finger X-linked duplicated C protein represses MHC class II transcription.

The zinc finger X-linked duplicated A (ZXDA) and ZXDC proteins are both required for robust transcription of major histocompatibility complex class II (MHC II) genes. Aside from the full length ZXDC mRNA transcript, at least one additional mRNA is produced by the ZXDC gene, in which transcription initiates within the first exon and terminates within the seventh intron. The protein product produced from this transcript, which we have named ZXDC2, is truncated on both the N- and C-terminus. We demonstrate here that ZXDC2 functions to repress MHC II transcription induced in HeLa cells treated with IFN-gamma. We further demonstrate that ZXDC2 interacts with both ZXDA and ZXDC, suggesting a mechanism by which ZXDC2 may inhibit MHC II transcription. These studies not only provide additional support for the role of ZXD proteins in regulating MHC II transcription, but also demonstrate a unique mechanism for the synthesis of a mRNA isoform.

BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

The zinc finger proteins ZXDA and ZXDC form a complex that binds CIITA and regulates MHC II gene transcription.

The transcription of major histocompatibility complex class II (MHC II) genes depends critically upon the activity of the class II trans-activator (CIITA) protein. We previously described a novel CIITA-binding protein named zinc finger X-linked duplicated family member C (ZXDC) that contributes to the activity of CIITA and the transcription of MHC II genes. Here, we examined the contribution of a closely related family member of ZXDC, the ZXDA protein, to MHC II gene transcription. ZXDA has a domain organization similar to ZXDC, containing ten zinc fingers and a transcriptional activation domain. Knockdown and overexpression of ZXDA demonstrated its importance in the transcriptional activation of MHC II genes. We found that ZXDA and ZXDC can self-associate, and also form a complex with each other. The regions of the two proteins that contain zinc fingers mediate these interactions. Importantly, we found that the ZXDA-ZXDC complex interacts with CIITA, rather than either protein alone. Given our additional finding that ZXDC is present at MHC II promoters in HeLa cells, prior to and after treatment with IFN-gamma, it appears that ZXDA and ZXDC form an important regulatory complex for MHC II gene transcription.

ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription.

The class II trans-activator (CIITA) is recognized as the master regulator of major histocompatibility complex (MHC) class II gene transcription and contributes to the transcription of MHC class I genes. To better understand the function of CIITA, we performed yeast two-hybrid with the C-terminal 807 amino acids of CIITA, and cloned a novel human cDNA named zinc finger, X-linked, duplicated family member C (ZXDC). The 858 amino acid ZXDC protein contains 10 zinc fingers and a transcriptional activation domain, and was found to interact with the region of CIITA containing leucine-rich repeats. Over-expression of ZXDC in human cell lines resulted in super-activation of MHC class I and class II promoters by CIITA. Conversely, silencing of ZXDC expression reduced the ability of CIITA to activate transcription of MHC class II genes. Given the specific interaction between the ZXDC and CIITA proteins, as well as the effect of ZXDC on MHC gene transcription, it appears that ZXDC is an important regulator of both MHC class I and class II transcription.

A potential role for neuronal connexin 36 in the pathogenesis of amyotrophic lateral sclerosis.

Neuronal gap junctional protein connexin 36 (Cx36) contributes to neuronal death following a range of acute brain insults such as ischemia, traumatic brain injury and epilepsy. Whether Cx36 contributes to neuronal death and pathological outcomes in chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), is not known. We show here that the expression of Cx36 is significantly decreased in lumbar segments of the spinal cord of both human ALS subjects and SOD1G93A mice as compared to healthy human and wild-type mouse controls, respectively. In purified neuronal cultures prepared from the spinal cord of wild-type mice, knockdown of Cx36 reduces neuronal death caused by overexpression of the mutant human SOD1-G93A protein. Taken together, these data suggest a possible contribution of Cx36 to ALS pathogenesis. A perspective for the use of blockers of Cx36 gap junction channels for ALS therapy is discussed.

The class II transactivator requires brahma-related gene 1 to activate transcription of major histocompatibility complex class II genes.

The class II transactivator (CIITA) is the key regulator of major histocompatibility complex (MHC) class II gene transcription. We demonstrate here that CIITA requires the ATPase subunit of an hSWI/SNF complex, brahma-related gene 1 (BRG-1), to activate transcription. When introduced into a cell line lacking BRG-1, CIITA was unable to activate cellular MHC class II genes. Reexpression of the wild-type but not an ATP-binding-deficient BRG-1 protein in this cell line restored the ability of CIITA to transactivate transcription of MHC class II genes. Interestingly, when the activity of CIITA was assayed in the BRG-1-deficient cell line by using a plasmid-based reporter assay, BRG-1 was not required for transcriptional activation, suggesting that the chromatin structure on the plasmid is such that BRG-1 is not necessary. Coimmunoprecipitation experiments were performed to determine if BRG-1 and CIITA proteins associate with each other in cells. We found that the two proteins coimmunoprecipitate and that amino acids 1 to 140 of CIITA are sufficient for binding. Taken together, these data suggest that BRG-1 and, very likely, an hSWI/SNF complex are required for transcription of MHC class II genes. The complex is likely recruited to MHC class II promoters, at least in part, by interaction with CIITA.

Multiple interactions between BRG1 and MHC class II promoter binding proteins.

The class II transactivator (CIITA) interacts with the chromatin remodeling factor brahma related gene 1 (BRG1) as a necessary component of the transcriptional activation of human major histocompatibility complex (MHC) class II genes. We report here that RFXAP, a subunit of the DNA-binding RFX complex, also binds BRG1 and therefore provides a mechanism by which MHC class II gene chromatin can be remodeled in the absence of CIITA. RFXAP and CIITA bind to different regions of BRG1. The region of the RFXAP protein that binds BRG1 coincides with the minimally required fragment of RFXAP previously reported to be necessary to mediate MHC class II gene transcription. For CIITA, we found that BRG1 interacts with both the N-terminal acidic amino acid rich transactivation domain and a central region of CIITA that contains GTP-binding motifs. Analysis of chromatin structure of MHC class II genes in cell lines lacking CIITA or RFXAP, suggests that the RFXAP-BRG1 interaction may, in some cell types, produce chromatin remodeling.

Inhibition of class II trans-activator function by HIV-1 tat in mouse cells is independent of competition for binding to cyclin T1.

The Tat trans-activator protein from HIV-1 inhibits the function of the class II trans-activator protein (CIITA), resulting in reduced MHC class II gene transcription in human cells. Tat does so by competing with CIITA for binding to cyclin T1, a component of the transcriptional elongation complex PTEFb. Since Tat does not functionally interact with mouse cyclin T1, we decided to examine the ability of Tat to inhibit CIITA in mouse cells. We found that Tat inhibited CIITA activity in mouse cells though this inhibition was independent of cyclin T1. The inhibition required the transcriptional activation domain of CIITA, but did not involve alterations in MHC class II promoter occupancy. Although Tat blocked the interaction between CIITA protein and human cyclin T1, it had no effect on the binding between CIITA and mouse cyclin T1. Therefore, Tat can inhibit the ability of CIITA to activate transcription of MHC class II genes in mouse cells by a mechanism that appears to be distinct from that proposed for human cells.