DOT1L

What is DOT1L protein?

DOT1L (DOT1 like histone lysine methyltransferase) gene is a protein coding gene which situated on the short arm of chromosome 19 at locus 19p13. DOT1L's primary function is to catalyze the methylation of lysine 79 of histone H3 (H3K79me1, me2, and me3), which is the only known enzyme that catalyzes the methylation of H3K79. Dot1l-mediated H3K79 methylation plays an important role in the activation and maintenance of gene transcription. It affects the structure and accessibility of chromatin, thereby regulating the expression of specific genes. The DOT1L protein is consisted of 1537 amino acids and its molecular mass is approximately 164.9 kDa.

What is the function of DOT1L protein?

DOT1L is an enzyme with important biological functions that plays a role in a variety of cellular processes. DOT1L plays a role in maintaining the self-renewal of stem cells, especially spermatogonial stem cells. It is essential for the spermatogenic process and affects the development of germ cells. DOT1L also plays a role in protecting cartilage tissue and maintaining cartilage homeostasis, which has potential implications for the development of osteoarthritis. DOT1L acts synergistically with Npm1 to control the expression and deposition of the histone H1 variant to inhibit the expression of endogenous retrovirus MERVL.

Fig1. Model for the function of the DOT1L complex in transcription initiation and enhancement of H2Bub1 levels. (Aiwei Wu, 2021)

DOT1L Related Signaling Pathway

DOT1L plays a key role in multiple nucleosome modifications and gene expression regulation, mainly involved in the following signaling pathways:

Transcriptional regulation. DOT1L regulates transcriptional activity of genes by methylating H3K79, affecting chromatin status and recruitment of transcription factors. DNA damage response. DOT1L plays a role in DNA double-strand break repair, and its mediated H3K79 methylation helps promote repair protein localization and chromosome stability. Cell cycle and proliferation. DOT1L plays a role in cell cycle progression, particularly in the G2/M phase transition, where it regulates cell cycle progression by influencing the expression of related genes. Embryonic development. During embryonic development, DOT1L is essential for maintaining the pluripotency of embryonic stem cells and promoting proper differentiation.

DOT1L Related Diseases

The most notable is acute myeloid leukemia (AML), which is related to mixed leukemia (MLL). DOT1L is overexpressed in AML containing the MLL gene rearrangement, a type of leukemia that often does not respond well to traditional treatments. DOT1L is also upregulated in a variety of solid tumors, including breast cancer, prostate cancer, and colorectal cancer, and overexpression is associated with tumor progression and poor prognosis. Studies have shown that DOT1L may also play a role in brain development and cognitive function, so abnormal DOT1L function is associated with neurodevelopmental disorders. At the same time, it is also associated with some viral diseases and metabolic diseases.

Bioapplications of DOT1L

DOT1L has become an important target for cancer therapy due to its key role in a variety of leukemia and other cancers. The researchers developed and tested DOT1L inhibitors to explore their potential as anti-tumor agents. The leukemia associated with DOT1L did not respond well to traditional treatments. Therefore, small molecule inhibitors of DOT1L are being developed and used to treat this disease.

Case Study 1: Yanguang Hou, 2023

DOT1L, a histone methylase, is overexpression in renal cell cancer. However, the role and detailed molecular mechanism of DOT1L involved in renal cancer development remain unknown. The inhibition of DOT1L was used by SGC0946 and short hairpin RNA silencing. Monodansylcadaverine staining and transmission electron microscope were performed to detect autophagy changes as a result of the inhibition of DOT1L. MitoTracker Red assay was used to analyze mitochondrial morphology. The autophagy markers and mitochondria-related proteins were analyzed by Western blot, qPCR, or immunofluorescence. ChIP assay was performed to demonstrate H3K79me2 is involved in the direct regulation of Farnesoid X receptor transcription. DOT1L inhibition increased autophagy activity and promoted mito chondria fusion in cell lines of renal cancer. Inhibition of DOT1L upregulated levels of LC3α/β, P62, MFN1, and MFN2, which contributed to autophagy activity or mitochondria fusion. DOT1L knockdown showed a similar the above process. DOT1L inhibition or silencing resulted in AMP-activated protein kinase activation and mammalian target of rapamycin inhibition.

Fig1. Protein expression of DOT1L, H3K79me2, and H3K79me3 detected by Western blot in 786-O cells after DOT1L knockdown.

Fig2. GSEA indicated that DOT1L expression was closely related to mTOR signaling in renal cancer samples (TCGA-KIRC).

Case Study 2: Yaqi Zhang, 2023

Cancer stem cells (CSC) represent a population of cancer cells responsible for tumor initiation, chemoresistance, and metastasis. shRNA-mediated DOT1 L knockdown decreased the aldehyde dehydrogenase (ALDH)+ cell population, impaired the tumor initiation capacity (TIC) of ovarian CSCs, and blocked the expression of stemness-associated genes. Inhibition of DOT1L's methyltransferase activity by the small-molecule inhibitor (DOT1Li) EPZ-5676 also effectively targeted ovarian CSCs. Integrated RNA-sequencing analyses of ovarian cancer cells in which DOT1 L was knocked down versus control cells and of ovarian CSCs versus non-CSCs, identified Wnt signaling as a shared pathway deregulated in both CSCs and in DOT1L-deficient ovarian cancer cells. β-catenin, a key transcription factor regulated by Wnt, was downregulated in ovarian cancer cells in which DOT1 L was knocked down and upregulated in DOT1 L overexpressing ovarian cancer cells. Chromatin immunoprecipitation (ChIP) revealed enrichment of the H3K79Me3 mark at the β-catenin promoter, suggesting that its transcription is regulated by DOT1L.

Fig3. Western blot measured DOT1L and H3K79Me3 protein levels in ALDH+ ovarian CSCs and ALDH− cells sorted from OVCAR5 cells.

Fig4. Western blot analysis of H3K79Me3 and H3K79Me2 protein levels in OVCAR5 cells treated with different doses of DOT1L inhibitor EPZ-5676 (DOT1Li) or DMSO for 5 days.

DOT1L has several biochemical functions, for example, DNA binding,histone methyltransferase activity (H3-K79 specific),histone-lysine N-methyltransferase activity. Some of the functions are cooperated with other proteins, some of the functions could acted by DOT1L itself. We selected most functions DOT1L had, and list some proteins which have the same functions with DOT1L. You can find most of the proteins on our site.

DOT1L has direct interactions with proteins and molecules. Those interactions were detected by several methods such as yeast two hybrid, co-IP, pull-down and so on. We selected proteins and molecules interacted with DOT1L here. Most of them are supplied by our site. Hope this information will be useful for your research of DOT1L.

MLLT3;MLLT1;MLLT10;MLLT6;HIST2H3D