NCoR1 limits angiogenic capacity by altering Notch signaling
Authors:   , Teichmann Tom 
Published: December 2024

Eukaryotic gene expression is a well-regulated process, with the dynamics of chromatin state serving as a crucial control system. Chromatin condensation, known as heterochromatin formation, represses gene expression by limiting accessibility for the transcriptional machinery. Conversely, euchromatin represents a more accessible chromatin state allowing active gene expression. The transition between these chromatin states is facilitated by coregulatory proteins, which recruit specific enzymes. Coactivators recruit histone acetyltransferases (HATs) to acetylate lysine residues on histones, while corepressors recruit histone deacetylases (HDACs) to remove these acetyl groups. Decreased acetylation leads to increased positive charge on histones, thereby strengthening the interaction between DNA and histones.
The nuclear receptor corepressor 1 (NCoR1) stands out as one of the best-studied members within the family of nuclear corepressors. Its strong evolutionary conservation underscores its significance in vertebrate development and diverse cellular fate determinations. Research indicates that tissue-specific deletion yields unique effects that cannot be compensated for by another corepressor. Notably, NCoR1 showed the highest expression levels among all corepressors in single-cell sequencing data from Tabula Sapiens and Tabula Muris. These data also highlighted NCoR1 as the most abundant corepressor in endothelial cells (ECs). Despite its remarkably high expression, its exact role in ECs remained so far unclear.
This study aimed to investigate the role of NCoR1 in ECs, with a specific focus on its involvement in angiogenic processes. To this end, NCoR1 was depleted from ECs using a tamoxifen-inducible knockout mouse model, as well as knockdown approaches in HUVECs. The impact of NCoR1 loss on the endothelial phenotype was assessed by examining key angiogenic features such as sprouting, migration, and proliferation. Interestingly, although proliferation and undirected migration were decreased upon NCoR1 depletion, a significant increase in the angiogenic capacity of HUVECs was noted. This effect seemed to be driven by loss of growth factor sensing and a VEGF independent mechanism, which therefore appeared to cause random sprouting in a 3D environment. Notably, loss of NCoR1 promoted the number of tip cells. Similarly, increased angiogenic capacity was also observed in organ culture of mouse aortae of Ncor1 knockout mice.
Following the establishment of the phenotype, the underlying mechanism was explored using RNA- and ATAC-seq techniques. Depletion of NCoR1 revealed genome wide chromatin opening with a clear enrichment in gene regulatory regions, especially promotor regions. This finding, together with an overlay of the RNA-seq data indicated a strong gene regulatory relevance of NCoR1, especially on genes associated with angiogenesis. Since NCoR1 itself is incapable of direct DNA binding, a subsequent transcription factor analysis was performed. With the aid of a bioinformatic tool (TEPIC2), multiple transcription factors, especially from the Fos gene family and Jun family, were predicted to bind in regions of differentially expressed genes. Both families are known to be part of the AP-1 transcription factor complex, which was previously shown to interact with NCoR1. Interestingly, in ECs, AP-1 was shown to regulate vascular development and migration. This could potentially explain why the loss of NCoR1 promotes angiogenesis.
As the loss of NCoR1 particularly increased the number of tip cells during angiogenic sprouting, direct implications for this phenotype were investigated. Notch signalling is the main pathway, determining whether an EC differentiates into a highly migratory tip cell or highly proliferative stalk cell. Activation of Notch signalling involves the nuclear translocation of NICD, the intracellular domain of the transmembrane Notch receptor, with subsequent binding to RBPJκ. This interaction leads to a conformational change releasing an initially bound corepressor, followed by recruitment of a coactivator. In fact, NCoR1 was observed to bind RBPJκ, whereas depletion of NCoR1 – and hence reduced interaction between both proteins – directly increased RBPJκ promotor activity. Moreover, target gene expressions including DLL4 were upregulated. Hence, loss of NCoR1 leads to heightened Notch signaling by both derepressing the pathway and enhancing ligand expression, resulting in "superactive" Notch signaling. In ECs, the distinction between tip and stalk cells relies on competitive interaction and lateral inhibition. In NCoR1 depleted cells, uniform hyperactive Notch signaling potentially disrupts the reshuffling mechanism that tip and stalk cells usually undergo, thus locking in cell fate once a tip cell is established, promoting the overall number and thus angiogenesis.
In functional ECs, activation of Notch signaling suppresses angiogenesis. However, this creates a paradox response as NCoR1 depletion clearly enhances Notch activity, but at the same time elicits a pro-angiogenic effect. Interestingly, Notch4, when expressed in cis with Notch1, was shown to act as an endogenous inhibitor of Notch1, thus resulting in a contradictory Notch response. This was confirmed by a spheroid outgrowth assay, where only knocking down NOTCH4, but not NOTCH1, in NCoR1 depleted cells blocked the hypersprouting phenotype.
In summary, this study revealed that NCoR1 is crucial for maintaining precise endothelial gene expression programs. Loss of NCoR1 triggers an angiogenic response, but compromises the focused maintenance of gene expression programs, as highlighted by genome-wide chromatin opening. Consequently, prolonged depletion of NCoR1 may not be a favorable strategy for sustaining angiogenesis. However, in acute scenarios like diabetic wound healing, targeting NCoR1 could offer a potential therapeutic approach to restore angiogenesis.