Improved Zoom engagement by adopting flipped class strategies to the curriculum of a Biology of Aging course during the Covid-19 pandemic.

During the pandemic, a 4th year course: Biology of Aging was modified with extensive flipped classroom strategies to promote better student engagement. By utilizing the strength of the Zoom video conferencing platform students were able to have meaningful in-class experiences that promoted engagement and learning. This was further enhanced by shifting traditional lectures to a pre-recorded format to act as resources and promoting forum discussions outside of class times using the course management system (Brightspace). These changes influenced the satisfaction and benefited the student experience. Shifting to active student-centered learning and facilitation led to a dynamic well received teaching environment. The one trade off being that students needed to produce content every week, which was perceived as a heavy but manageable workload by many in the course. These changes can be used as a format for other online courses.

Complete Genome Sequence of Microbacterium Bacteriophage Erla.

We characterized the complete genome sequence of Siphoviridae bacteriophage Erla, an obligatory lytic subcluster EA1 bacteriophage infecting Microbacterium foliorum NRRL B-24224, with a capsid width of 65 nm and a tail length of 112 nm. The 41.5-kb genome, encompassing 62 predicted protein-coding genes, is highly similar (99.52% identity) to that of bacteriophage Calix.

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to γH2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.

UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119.

UbE2E1/UbcH6 is an E2 ubiquitin-conjugating enzyme that is regulated by USP7. We identified UbE2E1 as a novel component of Polycomb repressive complex 1 (PRC1), the E3 ligase complex responsible for histone H2A ubiquitination and gene silencing. We demonstrate that UbE2E1 is critical for the monoubiquitination of H2A at residue Lys-119 (uH2AK119) through its association with the PRC1 complex. UbE2E1 interacts with PRC1 subunits including Ring1A and Ring1B. Overexpression of UbE2E1 results in increased levels of uH2AK119, whereas overexpression of catalytically inactive UbE2E1_C131A or UbE2E1 knockdown results in decreased levels of uH2AK119. The down-regulation of H2A ubiquitination by loss of function of UbE2E1 is correlated with alleviated p16INK4a promoter repression and induced growth inhibition in HCT116 cells. These results are specific to UbE2E1 as knockdown of UbE2D E2s does not show any effect on uH2AK119. We extended the UbE2E1 regulation of uH2AK119 to USP7 and showed that USP7 is also a key regulator for monoubiquitination at H2A Lys-119 as both knockdown and deletion of USP7 results in decreased levels of uH2AK119. This study reveals that UbE2E1 is an in vivo E2 for the PRC1 ligase complex and thus plays an important role in the regulation of H2A Lys-119 monoubiquitination.

Ubiquitin-specific protease 7 is a regulator of ubiquitin-conjugating enzyme UbE2E1.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system.

ROS-mediated p53 induction of Lpin1 regulates fatty acid oxidation in response to nutritional stress.

The p53 protein is activated by stress signals and exhibits both protective and death-promoting functions that are considered important for its tumor suppressor function. Emerging evidence points toward an additional role for p53 in metabolism. Here, we identify Lpin1 as a p53-responsive gene that is induced in response to DNA damage and glucose deprivation. Lpin1 is essential for adipocyte development and fat metabolism, and mutation in this gene is responsible for the lypodystrophy phenotype in fld mice. We show that p53 and Lpin1 regulate fatty acid oxidation in mouse C2C12 myoblasts. p53 phosphorylation on Ser18 in response to low glucose is ROS and ATM dependent. Lpin1 expression in response to nutritional stress is controlled through the ROS-ATM-p53 pathway and is conserved in human cells. Lpin1 provides a critical link between p53 and metabolism that may be an important component in mediating the tumor suppressor function of p53.

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence.

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening-induced replicative senescence is dependent on the ATM-p53-p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53-responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti-proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum-dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late-passage cells, and ectopic Pin1 expression rescues cells from BTG2-induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.

Protein kinase C delta blocks immediate-early gene expression in senescent cells by inactivating serum response factor.

Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKC delta) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKC delta activity as cells age, production of the PKC delta catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKC delta in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKC delta inhibitor rottlerin and ectopic expression of a dominant negative form of PKC delta independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKC delta activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKC delta contributes to the senescent phenotype.