Development and Applications of Shape-Based DNA Motifs

Transcriptional regulation is imperative for proper development of multicellular organisms, and disregulation of this process can lead to genetic disease. Due to technical limitations, the full human regulome has not been assayed. Computational methods provide resources to fill the gaps in our understanding of these processes. Sequence based representations of transcription factor DNA motifs have long been used for this purpose. We developed a model based on estimates of DNA shape known as Structural Motifs, extending the position weight matrix to accommodate multiple continuous shape parameters at each position. Using expectation maximization, Structural Motifs are discovered de novo from transcription factor binding data, and these motifs are specific to their cognate factors. When considered jointly with sequence motifs, Structural Motifs improve classification of transcription factor binding sites. Joint models also provide insight into the readout mechanisms utilized by transcription factors. DNA shape is an important component of the protein-DNA interaction to consider and improves the computational predictions of transcription factor binding, elevating our understanding of the regulatory landscape

MAGP2 Controls Notch via Interactions with RGD Binding Integrins: Identification of a Novel ECM-Integrin-Notch Signaling Axis

Canonical Notch signaling involves Notch receptor activation via interaction with cell surface bound Notch ligand. Recent findings also indicate that Notch signaling may be modulated by cross-talk with other signaling mechanisms. The ECM protein MAGP2 was previously shown to regulate Notch in a cell type dependent manner, although the molecular details of this interaction have not been dissected. Here, we report that MAGP2 cell type specific control of Notch is independent of individual Notch receptor-ligand combinations but dependent on interaction with RGD binding integrins. Overexpressed MAGP2 was found to suppress transcriptional activity from the Notch responsive Hes1 promoter activity in endothelial cells, while overexpression of a RGD→RGE MAGP2 mutant increased Notch signaling in the same cell type. This effect was not unique to MAGP2 since the RGD domain of the ECM protein EGFL7 was also found to be an important modulator of Hes1 promoter activity. Independently of MAGP2 or EGFL7, inhibition of RGD-binding integrins with soluble RGD peptides also increased accumulation of active N1ICD fragments and Notch responsive promoter activity independently of changes in Notch1, Jag1, or Dll4 expression. Finally, β1 or β3 integrin blocking antibodies also enhanced Notch signaling. Collectively, these results answer the question of how MAGP2 controls cell type dependent Notch signaling, but more importantly uncover a new mechanism to understand how extracellular matrices and cellular environments impact Notch signaling

Characterization of Basement Membrane Induced Endothelial Cell Quiescence in the Presence of Growth factors

The importance of the basement membrane (BM) in endothelial cells\u27 (ECs) response to growth factors (GF) during angiogenesis is not well characterized. This is in part due to the lack of laboratory models which accurately replicate the angiogenic conditions experienced inside of tissues. We hypothesize that the degradation of the BM is required for ECs to respond to GF signaling. We propose to address this hypothesis with two specific aims: to develop a model that accurately portrays angiogenic signals in capillaries, and to determine if ECs will respond to GF signaling in the presence of BM

The Role of Integrins in the Regulation of Angiogenesis by the Notch Signalling Pathway in Endothelial Cells

The Notch pathway is an important juxtacrine signaling mechanism that controls gene expression during cell development and angiogenesis. An excess of pro-angiogenic activity promotes the outgrowth of capillaries to tumors, providing nutrients and providing a pathway for metastasis. It has been demonstrated that microfibril-associated glycoprotein-2 (MAGP-2) inhibits Notch signaling in endothelial cells thereby promoting angiogenic cell sprouting. MAGP-2 contains and RGD domain which has been shown to associate with integrins. When the RGD domain is mutated to an RGE there was an increase in Notch activity subsequently decreasing angiogenic cell sprouting. Several other extra cellular matrix proteins have been identified which also associate with the Notch pathway, not all of which have apparent integrin binding activity. We have selected four to characterize, both with and without RGD domains. The key to better understanding the relationships between these proteins and their effect on angiogenesis via the notch signaling pathway is to distort or encourage their binding affinity to integrins via RGD domains similar to the one in MAGP-2

The Influence of Integrin Binding RGD Domains on Notch Signaling and Angiogenesis

The Notch pathway is an important juxtacrine signaling mechanism that controls gene expression during cell development and angiogenesis. Unbalanced Notch signaling can provoke an excess of angiogenesis and capillary growth to tumors, providing nutrients and a pathway for metastasis. Therefore understanding the basic mechanism of angiogenesis will lead to therapies targeting this mechanism, aiming to combat tumors and prevent their metastatic spread. It has been demonstrated that the extracellular matrix (ECM) protein microfibril-associated glycoprotein-2 (MAGP-2) inhibits Notch signaling in endothelial cells, thereby promoting angiogenic cell sprouting. MAGP-2 contains an RGD integrin-binding domain that, when mutated, converts MAGP-2 from an inhibitor of Notch to a signaling promoter. Further experiments have demonstrated that integrin ligation in general seems to be responsible for this observed regulation of Notch signaling. Thus we hypothesize that integrin signaling suppresses Notch signaling. To test our hypothesis, we will experimentally add or remove RGD integrin-binding domains to and from other ECM molecules with various Notch activities, and compare the ability of the different mutants to control Notch signaling. A Notch responsive luciferase assay and western blot for the Notch intracellular domain were used to assess Notch activity. Our preliminary data shows that CCN-3 and MAGP-1 significantly increase Notch signaling activity, while the RGD containing EGFL-7 and FBLN-5 do not. Results from these experiments may be useful to help design new molecules to disrupt integrin-Notch signaling, and suppress inappropriate angiogenesis critical for cancer and metastasis

COVID-19-related Genes in Sputum Cells in Asthma. Relationship to Demographic Features and Corticosteroids.

Rationale: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 (angiotensin-converting enzyme 2), and TMPRSS2 (transmembrane protease serine 2) mediate viral infection of host cells. We reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells among patients with asthma may identify subgroups at risk for COVID-19 morbidity.Objectives: To determine the relationship between demographic features and sputum ACE2 and TMPRSS2 gene expression in asthma.Methods: We analyzed gene expression for ACE2 and TMPRSS2, and for ICAM-1 (intercellular adhesion molecule 1) (rhinovirus receptor as a comparator) in sputum cells from 330 participants in SARP-3 (Severe Asthma Research Program-3) and 79 healthy control subjects.Measurements and Main Results: Gene expression of ACE2 was lower than TMPRSS2, and expression levels of both genes were similar in asthma and health. Among patients with asthma, male sex, African American race, and history of diabetes mellitus were associated with higher expression of ACE2 and TMPRSS2. Use of inhaled corticosteroids (ICS) was associated with lower expression of ACE2 and TMPRSS2, but treatment with triamcinolone acetonide did not decrease expression of either gene. These findings differed from those for ICAM-1, where gene expression was increased in asthma and less consistent differences were observed related to sex, race, and use of ICS.Conclusions: Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity