Hiroshi Yuita^a,1,2, Isaac F. López-Moyado^a,b,1, Hyeongmin Jeong^c,1, Arthur Xiuyuan Cheng^a,b, James Scott-Browne^d,e, Jungeun An^f, Toshinori Nakayama^g,h, Atsushi Onodera^a,g,i,3, Myunggon Ko^c,j,3, and Anjana Rao^a,b,3

^aDivision  of  Signaling  and  Gene  Expression,  La  Jolla  Institute  for Immunology, La Jolla, CA 92037; 
^bSanford Consortium for Regenerative Medicine, La Jolla, CA  92093; 
^cDepartment  of  Biological  Sciences,  Ulsan  National  Institute  of  Science  and Technology, Ulsan 44919, Republic of Korea; 
^dDepartment of Immunology and Genomic Medicine, National Jewish Health, Denver, CO 80206; 
^eDepartment of Immunology and Microbiology,  University  of  Colorado,  Anschutz  Medical  Campus,  Aurora,  CO  80045; 
^fDepartment  of  Life  Sciences,  Jeonbuk  National  University,  Jeonju  54896,  Republic  of Korea; 
^gDepartment of Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670,  Japan; 
^hJapan  Agency  for  Medical  Research  and  Development  (AMED),  Core Research for Evolutional Science and Technology (CREST), Chiba 260-8670, Japan; 
ⁱInstitute for Advanced Academic Research, Chiba University, Inage-ku, Chiba 263-8522, Japan; 
^jCenter for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea

¹H.Y., I.F.L.-M., and H.J. contributed equally to this work.
²Present address: Shinagawa R&D Center, Daiichi Sankyo Co., Ltd, Tokyo 140-8710, Japan.
³To  whom  correspondence  may  be  addressed. : Atsushi Onodera, Myunggon Ko, and Anjana Rao

The three mammalian TET dioxygenases oxidize the methyl group of 5-methylcytosine in DNA, and the oxidized methylcytosines are essential intermediates in all known pathways of DNA demethylation. To define the in vivo consequences of complete TET deficiency, we inducibly deleted all three Tet genes in the mouse genome. Tet1/2/3-inducible TKO (iTKO) mice succumbed to acute myeloid leukemia (AML) by 4 to 5 wk. Single-cell RNA sequencing of Tet iTKO bone marrow cells revealed the appearance of new myeloid cell populations characterized by a striking increase in expression of all members of the stefin/cystatin gene cluster on mouse chromosome 16. In patients with AML, high stefin/cystatin gene expression correlates with poor clinical outcomes. Increased expression of the clustered stefin/cystatin genes was associated with a heterochromatin-to-euchromatin compartment switch with readthrough transcription downstream of the clustered stefin/cystatin genes as well as other highly expressed genes, but only minor changes in DNA methylation. Our data highlight roles for TET enzymes that are distinct from their established function in DNA demethylation and instead involve increased transcriptional readthrough and changes in three-dimensional genome organization.