KMT2B as a Regulator of Developmental Genes

KMT2B (Lysine [K] Methyltransferase 2B) or Mixed Lineage Leukemia 2 protein (MLL2) is a mammalian histone H3 lysine 4 (H3K4) methyltransferase. It readily forms complexes with WRAD, host cell factors 1 and 2, and Menin. These complexes confer H3K4 trimethylation to specific gene promoters and some enhancers to regulate bivalent developmental genes and stem cell/germ cell differentiation. In essence, KMT2B and its partner proteins keep these genes primed for when they are needed in development.¹

KMT2B is required for proper embryonic development, hematopoiesis, neural development, memory, and muscle coordination. Dysfunctional KMT2B expression or gene mutation causes dystonia. Several cancer types link to dysregulation of KMT2B expression.¹

Structural Features and Functional Significance

Full-length KMT2B is 2715 amino acids long and weighs in at nearly 294 kDa. From front to back, it consists of N-terminal HMG-like AT hooks that enable binding to specific regions of AT rich DNA, a Menin binding site, a Zinc-finger CXXC domain that recognizes and binds non-methylated CpG DNA (present in most active promoters) to facilitate chromatin association, 4 plant homeotic domains (PHD) that coordinate binding to methylated histone H3, a bromodomain (BRD) between PHD3 and PHD4 to augment PHD3’s function, two phenylalanine-tyrosine rich (FYR) domains (FYRN and FYRC) separated by a threonine aspartase 1 (Taspase1) cleavage site, and, finally, the WRAD binding site embedded just inside the S-adenosylmethionine (SAM) dependent SET lysine methyltransferase domain. Cleavage of KMT2B by Taspase 1 is essential for proper methyltransferase function. After cleavage, the FYR domains reassociate to form a covalent bridge between the two fragments.²

The SET domain of KMT2B is instrumental in forming COMPASS complexes (complex of proteins associated with SET). These complexes contain four common subunits (WDR5, RbBP5, ASH2L, and DPY30) that form the WRAD module. This module regulates the enzyme activity and stability of the complex, in addition to its chromatin affinity. KMT2B’s COMPASS complex trimethylates H3K4 and interacts with transcription factors to recruit it to specific loci. KMT2B’s COMPASS complex is responsible for priming developmental differentiation of bivalent neural and germinal stem cells. Menin binds KMT2B and recruits it to the estrogen receptor a gene locus. Macrophages require KMT2B for proper cytokine signaling. In concert with b-catenin, KMT2B even promotes the expression of c-MYC via H3K4 trimethylation of its enhancer.³

Implications in Cancer, Neurology, and Inflammation

Nonsense, missense, or frameshift mutations in KMT2B’s PHD and SET domains are present in uterine corpus endometrial carcinoma, esophageal sarcomatoid carcinoma, and gastric cancer. Truncated KMT2B is found in neurofibromatosis 1-glioblastoma. KMT2B and its enhancement of c-MYC expression have been implicated in colorectal cancer. Inactivating mutations of KMT2B correlate to squamous cell cancer of the head and neck, suggesting its role as a growth suppressor in those tissues. KMT2B is significantly upregulated in cervical cancer cells, upregulating EGF expression, and facilitating migration and invasion. In contrast, KMT2B downregulation correlates to breast cancer.³

Heterozygous mutations in KMT2B lead to characteristic childhood and adult- onset dystonias. KMT2B is required for neuronal transdifferentiation of fibroblasts into neurons, activating the genes required for neuronal maturation, and repressing defective muscle innervation outcomes.⁴

KMT2B’s induction of riboflavin kinase (RFK) in response to stress leads to damaging inflammatory signaling. In response to myocardial ischemia and subsequent reperfusion, KMT2B’s enhanced expression of RFK induces the TNF-a/NOX2 inflammatory pathway, increasing ferroptosis and myocyte mortality.⁵ In microglia, KMT2B modulates RFK in a similar way to activate the TNFR1/NF-kb pathway, increasing cellular ROS, inflammatory cytokines, and neuroinflammation. This inflammatory phenomenon plays a key role in neurodegenerative diseases, such as Alzheimer’s Disease and Lewy body dementia.⁶

The AptaFluor SAH Methyltransferase Assay detects the enzymatic activity of histone methyltransferases, like KMT2B, via direct detection of SAH produced during methylation events. The assay relies on a naturally-occurring SAH-sensing RNA aptamer that binds SAH with nanomolar affinity and exquisite selectivity. The assay detects activity of several purified histone methyltransferases at concentrations well below the sensitivity limit for current assays. Our MLL4 Assay provides an excellent example of the applications of the AptaFluor Assay with various histone methyltransferases.

References

1. Klonou, A, et al. (2021) Structure, Activity and Function of the MLL2 (KMT2B) Protein Lysine Methyltransferase. Life, 11(8), 823. Review. https://doi.org/10.3390/life11080823

2. Antunes, E.T.B and Ottersbach K. (2020) The MLL/SET family and haematopoiesis. Biochimica et Biophysica Acta, 1863(8), 194579. Review. https://doi.org/10.1016/j.bbagrm.2020.194579

3. Poreba, E. et al. (2020) Aberrant Activity of Histone–Lysine N-Methyltransferase 2 (KMT2) Complexes in Oncogenesis. International Journal of Molecular Sciences, 21(24), 9340. Review. https://doi.org/10.3390/ijms21249340

4. Barbagiovanni, G. et al. (2018). KMT2B Is Selectively Required for Neuronal Transdifferentiation, and Its Loss Exposes Dystonia Candidate Genes. Cell reports, 25(4), 988–1001. https://doi.org/10.1016/j.celrep.2018.09.067

5. Cao, Y. et al (2022) KMT2B-dependent RFK transcription activates the TNF-α/NOX2 pathway and enhances ferroptosis caused by myocardial ischemia-reperfusion. Journal of Molecular and Cellular Cardiology, 173, 75-91. https://doi.org/10.1016/j.yjmcc.2022.09.003

6. Zhang, M. et al (2023) Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation. Advanced Science, 10(12), 2300180. https://doi.org/10.1002/advs.202300180