PHD and SET domains proteins are chromatin regulators and several of them are altered in cancer. Inactivation of MLL3 in mice results in epithelial tumor formation, suggesting that it functions as a tumor-suppressor gene. Also, MLL3 has been reported to be frequently deleted in myeloid leukemias. Moreover, other reports indicate somatic mutations in the MLL3 gene in glioblastoma and pancreatic ductal adenocarcinoma. However, subsequent reports have not yet confirmed MLL3 mutations in colon cancer. Thus, the role of MLL3 in the pathogenesis of colorectal neoplasia remains incompletely defined. In this paper, we investigated MLL3 alterations in colon cancer and found a two isoform of MLL3 of which the longer isoform has a previously unrecognized CpG island overlapping the promoter. Moreover, we found new genetic alterations in CRC cell lines and also primary tumors. In this study, we found frequent inactivation of MLL3 by frameshift mutations which had not been previously reported. We have shown that the 9 tract in MLL3 is mutated in mismatch repair deficient tumors. A previous study excluded mismatch repair deficient PJ34 hydrochloride tumors and still found mutations in 2.2% of cases. In primary tumors, however, we screened for mutations in the previously reported affected regions and found only polyA tract mutations. We have thus underestimated the precise mutation rate of the gene given that we did not sequence all 59 exons in all tumors. Nevertheless, it is clear that MLL3 mutations resemble those of other important tumor-suppressor gene in CRC �C TGFBRII. For both genes, most mutations seen in CRC are polyA tract mutations in mismatch repair deficient cases, but a few of the mutations are also found outside the polyA tract, including in cases without mismatch repair deficiency. Improvements in sequencing technologies and costs should allow the precise estimation of MLL3 mutations in primary CRCs in the near future. Pseudogenes are defunct relatives of known genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell. Although some do not have introns or promoters, most have some gene-like features, they are nonetheless considered nonfunctional, due to their lack of protein-coding ability resulting from various genetic disablements or their inability to encode RNA. Pseudogenes are characterized by a combination of homology to a known gene and nonfunctionality. That is, although every pseudogene has a DNA sequence that is similar to some functional gene, they are nonetheless unable to produce functional final products. Interestingly, Liang et al described that psiTPTE22-HERV is Nifedipine silenced by DNA methylation in not only GI cancers but also renal, liver and lung cancer. And HERV-related sequences in psiTPTE22-HERV are mostly spliced out as introns from the transcripts, and the amino acid sequence of the 15 kDa protein is not a homologue to any retroviral proteins. These make the HERV-related psiTPTE22-HERV gene an ordinary somatic gene. In summary, we report that MLL3 is inactivated in CRC by genetic alteration. In particular, we found that microsatellite unstable CRC cell liness have frequent frameshift mutations within an 9 tract coding region of MLL3 causing a loss of protein function, and a previous study reported on mutations outside this tract in microsatellite stable cancers. Moreover, the MLL3 promoter CpG island is highly homologous to a CpG island in the promoter region of a pseudogene psiTPTE22. psiTPTE22 was densely methylation in both primary CRCs and correlated with aging in normal epithelium but not MLL3. MLL3 loss of function may be a key feature of early CRC tumorigenesis.