RBPJ / CBF 1 interacts with L3 MBTL 3/ MBT 1 to promote repression of Notch signaling via histone demethylase KDM 1A/ LSD 1

Abstract

Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.

Stilbenoid‐Mediated Epigenetic Activation of Semaphorin 3A in Breast Cancer Cells Involves Changes in Dynamic Interactions of DNA with DNMT3A and NF1C Transcription Factor

“…This would indicate their tumor suppressor role in cancer. Indeed, the genes are involved in inhibition of main pathways associated with oncogenic properties (Figure H,I) . Among genes that were the most robustly hypomethylated in invasive MCF10CA1a cells, we observed a progressive RSV‐mediated hypomethylation from lowly invasive to highly invasive stages (Table , Figure A).…”

“…Epigenetic regulators such as CDKN2BAS and METTL3 , and potential tumor suppressors SEMA3A and WFDC3 are also present among 113 “hypomethylated RSV targets.” RBPJ is another important candidate present among genes hypomethylated in both cancer cell lines. RBPJ acts as a transcriptional repressor by recruitment of chromatin remodeling complexes which consequently suppresses oncogenic NOTCH signaling …”

Structural and functional studies of the RBPJ-SHARP complex reveal conserved corepressor binding site

“…Whereas the CTD mutants L386A, L397A, and L466A had only a minor effect on SHARP binding (Table 3), consistent with these residues burying much less surface area at the CTD-SHARP interface (L386=39 Å 2 , L397=3 Å 2 , and L466=44 Å 2 ). Because SHARP binds the BTD of RBPJ in a structurally similar manner to the RAM domain of NICD, and the corepressors FHL1 and RITA1, we used a set of alanine mutants (F261A, V263A, A284V, and Q333A) that we have previously characterized for RAM, FHL1, L3MBTL3, and RITA1 binding to RBPJ (Figure 5G-H and Table 3) 12,13,19,36 . The BTD mutants F261A and A284V, which are more centrally located in the SHARP-BTD interface, have a stronger effect, significantly reducing binding by ~45 fold and ~50 fold, respectively (Figure 5G-H and Table 3).…”

“…This activity of CSL is required at all Notch target genes and is conserved in all metazoans. CSL can also function as a transcriptional repressor, by interacting with corepressors such as SHARP 8,9,27 , L3MBTL3 12 , FHL1 11,19 , and RITA1 13,14 in mammals, and Hairless in Drosophila 18,34,35 ; however, its role as a repressor, in particular in mammals, is not well understood. In order to begin to address this gap in our understanding, here we determine the X-ray structure of the RBPJ-SHARP corepressor complex bound to DNA and use a multitude of in vitro and cellular assays to characterize structure based RBPJ and SHARP mutants in order to better understand CSL-corepressor function.…”

“…Nuclear NICD directly binds the transcription factor CSL [CBF1/RBPJ, Su(H), Lag-1] and the CSL-NICD complex recruits a member of the Mastermind (MAM) family of transcriptional coactivators [Mastermind-like (MAML1-3) in mammals], resulting in the transcriptional activation of Notch target genes (Figure 1A) 7 . CSL can also function as a repressor by interacting with corepressor proteins, such as SHARP [SMRT/HDAC1-associated repressor protein, also known as MINT (Msx2-interacting nuclear target) or SPEN (split ends)] 8,9 , Hairless in Drosophila melanogaster 10 , FHL1 (Four and a half LIM domains 1, also known as KyoT2) 11 , L3MBTL3 (lethal 3 malignant brain tumor-like 3) 12 , and RITA1 (RBPJ-interacting and tubulin associated) 13,14 . Corepressors are part of large higher order transcriptional repression complexes that contain histone-modifying activity, e.g .…”